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Simple Summary: This review aims to explore the role of the microRNA-204 (miR-204), a tumor suppressor gene, in the development of breast cancer, focusing on how it influences the hallmarks of cancer. By understanding the impact of miR-204 on these hallmarks, this review seeks to shed light on its potential as a molecular target for breast cancer therapy. microRNA-204-5p (miR-204) is a small noncoding RNA with diverse regulatory roles in breast cancer (BC) development and progression. miR-204 is implicated in the instauration of fundamental traits acquired during the multistep development of BC, known as the hallmarks of cancer. It may act as a potent tumor suppressor by inhibiting key cellular processes like angiogenesis, vasculogenic mimicry, invasion, migration, and metastasis. It achieves this by targeting multiple master genes involved in these processes, including HIF-1α, β-catenin, VEGFA, TGFBR2, FAK, FOXA1, among others. Additionally, miR-204 modulates signaling pathways like PI3K/AKT and interacts with HOTAIR and DSCAM-AS1 lncRNAs, further influencing tumor progression. Beyond its direct effects on tumor cells, miR-204 shapes the tumor microenvironment by regulating immune cell infiltration, suppressing pro-tumorigenic cytokine production, and potentially influencing immunotherapy response. Moreover, miR-204 plays a crucial role in metabolic reprogramming by directly suppressing metabolic genes within tumor cells, indirectly affecting metabolism through exosome signaling, and remodeling metabolic flux within the tumor microenvironment. This review aims to present an update on the current knowledge regarding the role of miR-204 in the hallmarks of BC. In conclusion, miR-204 is a potential therapeutic target and prognostic marker in BC, emphasizing the need for further research to fully elucidate its complex roles in orchestrating aggressive BC behavior. [ABSTRACT FROM AUTHOR]

Abstract Background Pulmonary arterial hypertension is most of the time diagnosed late in the course of the disease and necessitates right cardiac catheterization which is an invasive and costly tool. MicroRNAs have a role in the pathogenesis of pulmonary hypertension, systemic sclerosis, and schistosomiasis and their dosages are easy and non-expensive. Therefore, determining their levels in the blood may be helpful in detecting PAH and differentiating its idiopathic form from those caused by systemic sclerosis and schistosomiasis. Purpose of the study To evaluate the role of microRNA (miR) 204 and miR-210 in the diagnosis of PAH and to distinguish between idiopathic PAH (IPAH), systemic sclerosis-associated PAH (SSc-PAH), and schistosomiasis-associated PAH (Sch-PAH) and to identify patients who may benefit from simple non-expensive and non-invasive methods in diagnosis of PAH. Methods Sixty patients with PAH and 30 subjects as control were enrolled in the study. PAH was diagnosed by right heart catheterization, echocardiography, and laboratory tests. Blood samples were taken from all patients for measuring miR-204 and miR-210. Results MiR-204 was downregulated in PAH and there was a highly significant difference between PAH and control (p = 0.003) with cut-off predictive value ≤ 0.15 µM and 70% sensitivity, 85% specificity with AUC (0.749). However, miR-204 failed to distinguish between IPAH, SSc-PAH, and Sch-PAH. MiR-210 was upregulated in PAH with a highly significant difference between PAH and control (p

Objective: To explore the function and possible molecular mechanism of miR-204 and SOX4 in cutaneous squamous cell carcinoma (CSCC). Methods: A total of 70 patients with CSCC who were diagnosed and underwent surgical treatment in the dermatology department of the Second Affiliated Hospital of Air Force Military Medical University from May 2019 to March 2022 were selected. CSCC tissues and corresponding paracancer tissues of the patients were collected, and the mRNA levels of miR-204 and SOX4 in the tissue samples were detected by qRT-PCR. The protein level of SOX4 in tissue samples were detected by Western blot and immunohistochemical staining. The CSCC cell line SCL-1 was divided into control group, miR-NC group, miR-204 group, miR-204+pcDNA group and miR-204+SOX4 group. miR-NC, miR-204 mimics, miR-204 mimics and pcDNA3.1 empty vector, miR-204 mimics and pcDNA3.1+SOX4 vector were transfected into miR-NC group, miR-204 group, miR-204+pcDNA group and miR-204+SOX4 group by Lipofectamine 2000TM transfection reagents, respectively. And control group cells were not transfected. The targeting relationship between miR-204 and SOX4 was analyzed by dual luciferase reporter gene experiment. SCL-1 cell proliferation was detected by CCK-8 assay, cell cloning was detected by clonogenesis assay, cell apoptosis was detected by flow cytometry assay, and cell migration and invasion were detected by Transwell assay. The protein expression levels of SOX4, E-cadherin, N-cadherin and Vimentin were detected by Western blot. Results: The expression of miR-204 was low in CSCC, and the expression of SOX4 mRNA and protein was high. The online analysis results of TargetScan software showed that binding sites existed at 5' of miR-204 and 3' of SOX4. Compared with the control group or the NC group, the relative proliferation activity, number of clone formation, migration and invasion of SCL-1 cells in miR-204 group were decreased (P<0.05), and the apoptosis rate was increased (P<0.05). The protein levels of SOX4, N-cadherin and Vimentin in SCL-1 cells were decreased (P<0.05), while the protein level of E-cadherin was increased(P<0.05). Compared with miR-204 group or miR-204+pcDNA group, the relative proliferation activity, number of clone formation, migration and invasion of SCL-1 cells in miR-204+SOX4 group were increased (P<0.05), and the apoptosis rate was decreased (P<0.05). The protein levels of SOX4, N-cadherin and Vimentin in SCL-1 cells were increased(P<0.05), while the protein level of E-cadherin was decreased(P<0.05). Conclusion: miR-204 inhibits the proliferation, migration, invasion and epithelial mesenchymal transformation of CSCC cells by targeting SOX4, and promotes cell apoptosis, thus alleviating the development of CSCC. [ABSTRACT FROM AUTHOR]

Purpose: To characterize the phenotype and genotype of a Chinese family with autosomal‐dominant retinitis pigmentosa (RP) accompanied by iris coloboma. Methods: The proband, a 34‐year‐old male, was examined with his family by using fundus photography, optical coherence tomography (OCT), autofluorescence, and full‐field electroretinography (ffERG). Genetic analyses were conducted through whole‐exome sequencing (WES) to screen for variations. Results: Three members of this Chinese family were shown to be bilateral iris coloboma. The male proband and his mother exhibited typical RP feature. The proband's late grandfather had been documented manifestation of iris coloboma. The mode of inheritance was confirmed to be autosomal dominance. Through linkage analysis and WES, a heterozygous variation in the miR‐204 gene (n.37C>T), a noncoding RNA gene, was identified in these three members. Conclusions: In this third independent and the first Asian family, the existence of a miR‐204 variant associated with RP accompanied by iris coloboma was confirmed. Our findings reinforce the significance of miR‐204 as an important factor influencing visual function in the retina. When phenotypes like RP accompanied by iris coloboma in an autosomal‐dominant pattern, including in Chinese patients, miR‐204 aberrations should be considered. [ABSTRACT FROM AUTHOR]

Background: Pulmonary arterial hypertension is most of the time diagnosed late in the course of the disease and necessitates right cardiac catheterization which is an invasive and costly tool. MicroRNAs have a role in the pathogenesis of pulmonary hypertension, systemic sclerosis, and schistosomiasis and their dosages are easy and non-expensive. Therefore, determining their levels in the blood may be helpful in detecting PAH and differentiating its idiopathic form from those caused by systemic sclerosis and schistosomiasis. Purpose of the study: To evaluate the role of microRNA (miR) 204 and miR-210 in the diagnosis of PAH and to distinguish between idiopathic PAH (IPAH), systemic sclerosis-associated PAH (SSc-PAH), and schistosomiasis-associated PAH (Sch-PAH) and to identify patients who may benefit from simple non-expensive and non-invasive methods in diagnosis of PAH. Methods: Sixty patients with PAH and 30 subjects as control were enrolled in the study. PAH was diagnosed by right heart catheterization, echocardiography, and laboratory tests. Blood samples were taken from all patients for measuring miR-204 and miR-210. Results: MiR-204 was downregulated in PAH and there was a highly significant difference between PAH and control (p = 0.003) with cut-off predictive value ≤ 0.15 µM and 70% sensitivity, 85% specificity with AUC (0.749). However, miR-204 failed to distinguish between IPAH, SSc-PAH, and Sch-PAH. MiR-210 was upregulated in PAH with a highly significant difference between PAH and control (p < 0.001) with cut-off predictive value ≥ 1.16 µM and 93.33% sensitivity, 85% specificity with AUC (0.917). MiiR-210 showed a significant difference between SSc-PAH and idiopathic PAH (P = 0.012) and between SSc-PAH and Sch-PAH (P = 0.035). Conclusions: MiR-204 and miR-210 are useful non-invasive and non-expensive markers for the diagnosis of PAH, miR-210 is an excellent predictor in the diagnosis of PAH and also miR-210 might be used to distinguish SSc-PAH from idiopathic PAH and Sch-PAH. [ABSTRACT FROM AUTHOR]

Purpose: To study the regulatory impact of mir-204 on the biological behavior of childhood leukemia cells and to elucidate its mechanism of action. Methods: A total of 112 children with leukemia who were treated in Three Gorges University Yichang Central People's Hospital, Yichang, China from July 2018 to June 2020 were randomly divided into study and control groups (n = 56 each). The expression levels of mir-204 and hepatocyte growth factor (HGF) were determined using real-time polymerase chain chain reaction (RT-PCR) while the cell proliferation potential was determined using the CCK-8 method. Apoptosis and cell cycle progression of each group were evaluated using flow cytometry while changes in cell invasion and migration were assessed by Transwell assay. Luciferase reporter assay was used to determine the binding of mir-204 and HGF to 3'UTR. Results: The expression level of mir-204 in study group was significantly lower than that in control group, while expression level of HGF was significantly higher (p < 0.01) compared to control group. There was no significant change in cell proliferation capacity at 24 h. The expression level of mir-204 was significantly decreased, while the expression level of HGF was significantly increased (p < 0.05). Luciferase activity of wild-type HGF 3'UTR in mir-204 overexpression group was significantly decreased, relative to mir-204 negative control group (p < 0.01). There was no significant difference in luciferase activity in mutant HGF gene between the two groups (p > 0.05). Conclusion: Mir-204 reduces HGF expression level via HGF 3'UTR end, thereby inhibiting cell proliferation, invasion and migration, while promoting apoptosis, blocking cell cycle and regulating biological behavior of childhood leukemia cells. [ABSTRACT FROM AUTHOR]

Background and Purpose: Atherosclerosis induced by cyclosporine A (CsA), an inhibitor of the calcineurin/nuclear factor of activated T cells (NFAT) pathway, is a major concern after organ transplantation. However, the atherosclerotic mechanisms of CsA remain obscure. We previously demonstrated that calcineurin/NFAT signalling inhibition contributes to atherogenesis via suppressing microRNA‐204 (miR‐204) transcription. We therefore hypothesised that miR‐204 is involved in the development of CsA‐induced atherosclerosis. Experimental Approach: ApoE−/− mice with macrophage‐miR‐204 overexpression were generated to determine the effects of miR‐204 on CsA‐induced atherosclerosis. Luciferase reporter assays and chromatin immunoprecipitation sequencing were performed to explore the targets mediating miR‐204 effects. Key Results: CsA alone did not significantly affect atherosclerotic lesions or serum lipid levels. However, it exacerbated high‐fat diet‐induced atherosclerosis and hyperlipidemia in C57BL/6J and ApoE−/− mice, respectively. miR‐204 levels decreased in circulating monocytes and plaque lesions during CsA‐induced atherosclerosis. The upregulation of miR‐204 in macrophages inhibited CsA‐induced atherosclerotic plaque formation but did not affect serum lipid levels. miR‐204 limited the CsA‐induced foam cell formation by reducing the expression of the scavenger receptors SR‐BII and CD36. SR‐BII was post‐transcriptionally regulated by mature miR‐204‐5p via 3′‐UTR targeting. Additionally, nuclear‐localised miR‐204‐3p prevented the CsA‐induced binding of Ago2 to the CD36 promoter, suppressing CD36 transcription. SR‐BII or CD36 expression restoration dampened the beneficial effects of miR‐204 on CsA‐induced atherosclerosis. Conclusion and Implications: Macrophage miR‐204 ameliorates CsA‐induced atherosclerosis, suggesting that miR‐204 may be a potential target for the prevention and treatment of CsA‐related atherosclerotic side effects. [ABSTRACT FROM AUTHOR]

Abstract Purpose To characterize the phenotype and genotype of a Chinese family with autosomal‐dominant retinitis pigmentosa (RP) accompanied by iris coloboma. Methods The proband, a 34‐year‐old male, was examined with his family by using fundus photography, optical coherence tomography (OCT), autofluorescence, and full‐field electroretinography (ffERG). Genetic analyses were conducted through whole‐exome sequencing (WES) to screen for variations. Results Three members of this Chinese family were shown to be bilateral iris coloboma. The male proband and his mother exhibited typical RP feature. The proband's late grandfather had been documented manifestation of iris coloboma. The mode of inheritance was confirmed to be autosomal dominance. Through linkage analysis and WES, a heterozygous variation in the miR‐204 gene (n.37C>T), a noncoding RNA gene, was identified in these three members. Conclusions In this third independent and the first Asian family, the existence of a miR‐204 variant associated with RP accompanied by iris coloboma was confirmed. Our findings reinforce the significance of miR‐204 as an important factor influencing visual function in the retina. When phenotypes like RP accompanied by iris coloboma in an autosomal‐dominant pattern, including in Chinese patients, miR‐204 aberrations should be considered.

Osimertinib is an effective treatment option for patients with advanced non-small cell lung cancer (NSCLC) with EGFR activation or T790M resistance mutations; however, acquired resistance to osimertinib can still develop. This study explored novel miRNA–mRNA regulatory mechanisms that contribute to osimertinib resistance in lung cancer. We found that miR-204 expression in osimertinib-resistant lung cancer cells was markedly reduced compared to that in osimertinib-sensitive parental cells. miR-204 expression levels in cancer cells isolated from treatment-naive pleural effusions were significantly higher than those in cells with acquired resistance to osimertinib. miR-204 enhanced the sensitivity of lung cancer cells to osimertinib and suppressed spheroid formation, migration, and invasion of lung cancer cells. Increased miR-204 expression in osimertinib-resistant cells reversed resistance to osimertinib and enhanced osimertinib-induced apoptosis by upregulating BIM expression levels and activating caspases. Restoration of CD44 (the direct downstream target gene of miR-204) expression reversed the effects of miR-204 on osimertinib sensitivity, recovered cancer stem cell and mesenchymal markers, and suppressed E-cadherin expression. The study demonstrates that miR-204 reduced cancer stemness and epithelial-to-mesenchymal transition, thus overcoming osimertinib resistance in lung cancer by inhibiting the CD44 signaling pathway.

Osteoarthritis (OA) is a painful degenerative joint disease and is the leading cause of chronic disability among elderly individuals. To improve the quality of life for patients with OA, the primary goal for OA treatment is to relieve the pain. During OA progression, nerve ingrowth was observed in synovial tissue and articular cartilage. These abnormal neonatal nerves act as nociceptors to detect OA pain signals. The molecular mechanisms for transmitting OA pain in the joint tissues to the central nerve system (CNS) is currently unknown. MicroRNA miR-204 has been demonstrated to maintain the homeostasis of joint tissues and have chondro-protective effect on OA pathogenesis. However, the role of miR-204 in OA pain has not been determined. In this study, we investigated interactions between chondrocytes and neural cells and evaluated the effect and mechanism of miR-204 delivered by exosome in the treatment of OA pain in an experimental OA mouse model. Our findings demonstrated that miR-204 could protect OA pain by inhibition of SP1- LDL Receptor Related Protein 1 (LRP1) signaling and blocking neuro-cartilage interaction in the joint. Our studies defined novel molecular targets for the treatment of OA pain.

Uveal melanoma (UM), a frequently seen adulthood primary ocular malignancy, shows high aggressiveness. Accumulating studies have revealed the crucial effects of microRNAs (miRNAs) on tumorigenesis and development in various human tumors. miR-204, the cancer-associated miRNA, shows dysregulation and is related to several human malignancies, but its effect on UM remains unknown. The present work focused on exploring miR-204’s effect on UM and elucidating its possible molecular mechanisms. According to our results, miR-204 expression markedly increased within both UM tissues and cell lines. As revealed by functional analysis, miR-204 suppressed UM cell invasion and migration. Besides, RAB22A expression decreased through directly binding miR-204 into the corresponding 3′ untranslated region (3′UTR) in UM cells. Furthermore, the RAB22A mRNA level increased, which was negatively related to the miR-204 level within UM samples. As revealed by mechanical research, miR-204 exerted its inhibition on the invasion and migration of UM cells via RAB22A. Taken together, this study suggested the tumor-suppressing effect of miR-204 on UM through down-regulating RAB22A. Thus, miR-204 may serve as the new anti-UM therapeutic target. [ABSTRACT FROM AUTHOR]

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Abstract Objective Hypertensive disorder complicating pregnancy (HDCP) is a unique and common obstetrical complication in pregnancy. The current study sought to investigate the diagnostic value of serum miR-204 in HDCP patients. Methods A total of 196 HDCP patients were enrolled, with 54 healthy pregnant women as controls. The expression levels of miR-204 and inflammatory factors in the serum were determined. Receiver operating characteristic (ROC) curve was used to assess the diagnostic value of miR-204 in HDCP patients. Person coefficient was introduced to analyze the correlation between miR-204 and inflammatory indexes. Kaplan–Meier method was employed to analyze the effect of miR-204 expression on the incidence of adverse pregnancy outcomes. Logistic regression was adopted to assess the risk factors for adverse pregnancy outcomes. Results miR-204 expression was upregulated in the serum of HDCP patients. The serum miR-204 level > 1.432 could assist the diagnosis of HDCP. miR-204 level in the serum was positively correlated with TNF-α, IL-6, and hs-CRP concentrations in HDCP patients. The risk of adverse outcomes was higher in pregnant women with high miR-204 expression. High miR-204 expression was associated with an increased risk of adverse pregnancy outcomes after adjusting the family history of HDCP, systolic pressure, diastolic pressure, AST, ALT, LDH, 24-h urinary protein, TNF-α, IL-6, and hs-CRP. Conclusion The high expression of miR-204 assists the diagnosis of HDCP and is an independent risk factor for adverse pregnancy outcomes in HDCP patients.

Objective(s): Methylglyoxal (MG) provokes endoplasmic reticulum (ER) stress in β-cells and triggers pancreatic β-cell dysfunction. Crocin has anti-diabetic properties. The present study investigated whether crocin prevented pancreas damages induced by MG.Materials and Methods: Diabetes was induced by MG administration (600 mg/kg/day, PO). On the fourteenth day, after proving hyperglycemia, crocin (15, 30, and 60 mg/kg) and metformin (MT) (150 mg/kg) were used for detoxification of MG until the end of the experiment. The animals were divided into 6 groups: 1) control, 2) diabetic by MG, 3) MG + crocin 15 mg/kg, 4) MG + crocin 30 mg/kg, 5) MG + crocin 60 mg/kg, and 6) MG + MT. The data were analyzed by one-way analysis of variance and significant differences were compared by Tukey and Bonferroni tests (P

Background/aim: NLRP3 inflammasome activation has been known to be involved in the etiology and progression of Alzheimer's disease (AD). Furthermore, AD and diabetes mellitus have common pathomechanisms. It has been shown that P2X7R whose expression is increased in brain tissues with AD and plays a role in the activation of NLRP3 inflammasome is suppressed by miR-373 in patients with osteoarthritis. Therefore, the question of whether the suppressive effect of miR-373 on NLRP3 may have a role in the pathophysiology of AD comes to mind. On the other hand, it is known that the miR-204 level increases in response to TXNIP, another NLRP3 inflammasome inducer with high expression in AD. In primary human islets, miR-204 reduces the expression of GLP-1R. It has been discovered that in vivo deletion of miR-204 is protective against diabetes by increasing GLP-1R and insulin secretion. Considering the relationship between miR-204 and TXNIP and the relationship of miR-204 with diabetes suggests investigating the effect of miR-204 on the inflammatory pathway in AD. Based on the common pathophysiological mechanisms between AD and diabetes and the reported changes related to NLRP3 inflammasome, we analyzed miR-373 and miR-204 in neuron-derived serum exosomes in this study. Neuronderived exosomes in neurodegenerative diseases are considered to be better candidates for developing potential biomarkers. Materials and methods: The expression levels of miR-204 and miR-373 were investigated in neuron-derived serum exosomes obtained from 15 patients with mild AD, 18 with moderate AD, and 21 cognitively healthy individuals. Results and discussion: The miR-204 and miR-373 expressions were significantly decreased in both patient groups compared to the control group. Therefore, we suggest that miR-204 and miR-373 are potential biomarkers for AD. However, due to the preliminary nature of this study, further large-scale studies are needed to support our findings. [ABSTRACT FROM AUTHOR]

With increase in cancer burden worldwide and poor survival rates due to delayed diagnosis, it is pertinent to develop a device for early diagnosis. We report an electrochemical biosensor for quantification of miRNA-204 (miR-204) biomarker that is dysregulated in most of the cancers. The proposed methodology uses the gold nanoparticles-modified carbon screen-printed electrode for immobilization of single-stranded DNA probe against miR-204. Colloidal gold nanoparticles were synthesized using l-glutamic acid as reducing agent. Nanoparticles were characterized by UV–visible spectroscopy and transmission electron microscopy. Spherical gold nanoparticles were of 7–28 nm in size. Biosensor fabricated using these nanoparticles was characterized by cyclic voltammetry after spiking 0.1 fg/mL–0.1 µg/mL of miR-204 in fetal bovine serum. Response characteristics of the miR-204 biosensor displayed high sensitivity of 8.86 µA/µg/µL/cm2 with wide detection range of 15.5 aM to 15.5 nM. The low detection limit makes it suitable for early diagnosis and screening of cancer. [ABSTRACT FROM AUTHOR]

Objective: Hypertensive disorder complicating pregnancy (HDCP) is a unique and common obstetrical complication in pregnancy. The current study sought to investigate the diagnostic value of serum miR-204 in HDCP patients.Methods: A total of 196 HDCP patients were enrolled, with 54 healthy pregnant women as controls. The expression levels of miR-204 and inflammatory factors in the serum were determined. Receiver operating characteristic (ROC) curve was used to assess the diagnostic value of miR-204 in HDCP patients. Person coefficient was introduced to analyze the correlation between miR-204 and inflammatory indexes. Kaplan-Meier method was employed to analyze the effect of miR-204 expression on the incidence of adverse pregnancy outcomes. Logistic regression was adopted to assess the risk factors for adverse pregnancy outcomes.Results: miR-204 expression was upregulated in the serum of HDCP patients. The serum miR-204 level > 1.432 could assist the diagnosis of HDCP. miR-204 level in the serum was positively correlated with TNF-α, IL-6, and hs-CRP concentrations in HDCP patients. The risk of adverse outcomes was higher in pregnant women with high miR-204 expression. High miR-204 expression was associated with an increased risk of adverse pregnancy outcomes after adjusting the family history of HDCP, systolic pressure, diastolic pressure, AST, ALT, LDH, 24-h urinary protein, TNF-α, IL-6, and hs-CRP.Conclusion: The high expression of miR-204 assists the diagnosis of HDCP and is an independent risk factor for adverse pregnancy outcomes in HDCP patients. [ABSTRACT FROM AUTHOR]

Objective(s): Methylglyoxal (MG) provokes endoplasmic reticulum (ER) stress in ß-cells and triggers pancreatic ß-cell dysfunction. Crocin has anti-diabetic properties. The present study investigated whether crocin prevented pancreas damages induced by MG. Materials and Methods: Diabetes was induced by MG administration (600 mg/kg/day, PO). On the fourteenth day, after proving hyperglycemia, crocin (15, 30, and 60 mg/kg) and metformin (MT) (150 mg/kg) were used for detoxification of MG until the end of the experiment. The animals were divided into 6 groups: 1) control, 2) diabetic by MG, 3) MG + crocin 15 mg/kg, 4) MG + crocin 30 mg/kg, 5) MG + crocin 60 mg/kg, and 6) MG + MT. The data were analyzed by one-way analysis of variance and significant differences were compared by Tukey and Bonferroni tests (P<0.05). Biochemical assays, antioxidant evaluation, and microRNAs expression associated with ER stress were assessed. Results: MG induced hyperglycemia, insulin resistance, and dyslipidemia (P<0.001). Crocin and MT significantly ameliorated ß-cell function through reduction of fasting blood glucose, malondialdehyde levels (P<0.001), and significant elevation of anti-oxidant enzyme activity accompanied by regulation of glutathione and glyoxalase1-Nrf2 in MG induced diabetic mice. Crocin and MT significantly down-regulated microRNAs 204, 216b, 192, and 29a expression (P<0.001). Crocin (60 mg/kg) (P<0.01) and MT (P<0.001) could improve diameter of pancreatic islets in MG treated mice. Conclusion: Crocin prevents the progression of diabetes through modulating ER stress-associated microRNAs and GLO1 activity with the helpful effects of glutathione and Nrf2. [ABSTRACT FROM AUTHOR]

Ming-Ming Zhao, Lin-Yang Ge, Liang-Feng Yang, Hai-Xia Zheng, Gang Chen, Li-Zheng Wu, Shao-Ming Shi, Nan Wang, Yan-Ping Hang Department of Respiratory and Critical Care Medicine, People’s Hospital of Gaochun, Nanjing 211300, People’s Republic of ChinaCorrespondence: Yan-Ping HangDepartment of Respiratory and Critical Care Medicine, People’s Hospital of Gaochun, No. 53, Maoshan Road, Nanjing 211300, People’s Republic of ChinaTel +86 25-57311232Email hangyanping4444@163.comBackground: Long non-coding RNA (lncRNA) is a key part of non-coding RNA, and more and more evidence has revealed that it plays a vital role in tumors. NEAT1 is a lncRNA discovered in the early stage. However, it is still unclear whether NEAT1 and miR-204 play a regulatory role in lung cancer (LC). This research aimed to determine the biological function of NEAT1/miR-204 in non-small cell lung cancer (NSCLC).Materials and Methods: In order to research the function of NEAT1 in NSCLC, RT-PCR, Western blot, luciferase assay and RNA immunoprecipitation assay were used to determine the relationship between NEAT1, miR-204 and NUAK1. CCK8 test, cell migration and invasion test were used to explore the influence of NEAT1 on proliferation and metastasis of LC cells. Tumor allotransplantation was used to detect the influence of NEAT1 on the growth of LC.Results: The results revealed that NEAT1 was obviously enhanced in LC cell lines. Further functional analysis showed that low expression of NEAT1 obviously suppressed the growth, migration and invasion of NSCLC and facilitated cell apoptosis. Determination of luciferase reporter gene revealed that miR-204 was the direct target of NEAT1 in LC. In addition, NUAK1 was called the direct target of miR-204, and miR-204/NUAK1 had saved the role of NEAT1 in NSCLC cells. Tumor allotransplantation experiments showed that knocking down NEAT1 could inhibit the growth of LC.Conclusion: In summary, our results showed that the down-regulation of NEAT1 in NSCLC inhibited its growth, migration and invasion through the miR-204/NUAK1 axis.Keywords: NEAT1, miR-204, NUAK1, non-small cell lung cancer

Aims Our previous study demonstrated that Ca2+ influx through the Orai1 store-operated Ca2+ channel in macrophages contributes to foam cell formation and atherosclerosis via the calcineurin–ASK1 pathway, not the classical calcineurin–nuclear factor of activated T-cell (NFAT) pathway. Moreover, up-regulation of NFATc3 in macrophages inhibits foam cell formation, suggesting that macrophage NFATc3 is a negative regulator of atherogenesis. Hence, this study investigated the precise role of macrophage NFATc3 in atherogenesis. Methods and results Macrophage-specific NFATc3 knockout mice were generated to determine the effect of NFATc3 on atherosclerosis in a mouse model of adeno-associated virus-mutant PCSK9 -induced atherosclerosis. NFATc3 expression was decreased in macrophages within human and mouse atherosclerotic lesions. Moreover, NFATc3 levels in peripheral blood mononuclear cells from atherosclerotic patients were negatively associated with plaque instability. Furthermore, macrophage-specific ablation of NFATc3 in mice led to the atherosclerotic plaque formation, whereas macrophage-specific NFATc3 transgenic mice exhibited the opposite phenotype. NFATc3 deficiency in macrophages promoted foam cell formation by potentiating SR-A- and CD36-meditated lipid uptake. NFATc3 directly targeted and transcriptionally up-regulated miR-204 levels. Mature miR-204-5p suppressed SR-A expression via canonical regulation. Unexpectedly, miR-204-3p localized in the nucleus and inhibited CD36 transcription. Restoration of miR-204 abolished the proatherogenic phenotype observed in the macrophage-specific NFATc3 knockout mice, and blockade of miR-204 function reversed the beneficial effects of NFATc3 in macrophages. Conclusion Macrophage NFATc3 up-regulates miR-204 to reduce SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis, indicating that the NFATc3/miR-204 axis may be a potential therapeutic target against atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Several recent studies have suggested that the long non-coding RNA (lncRNA) DSCAM-AS1 (Down syndrome cell adhesion molecule - anti-sense 1) is aberrantly expressed in many malignancies. Purpose: In this study, we aimed to explore the the role of DSCAM-AS1 in gastric carcinoma. Research Design: Expression of DSCAM-AS1 mRNA, miR-204, and TPT1 (Tumor Protein, Translationally-Controlled 1) were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Proliferation and apoptosis of GC cells were determined using the CCK-8 cell counting assay and flow cytometry. The rate of cell migration and invasion was determined using a transwell assay. The relationships between DSCAM-AS1, miR-204, and TPT1 were predicted and confirmed using a dual-luciferase reporter assay. Expression of TPT1 protein was quantified by Western blot. Results: In this study, we found that DSCAM-AS1 was significantly overexpressed in GC tissues and cell lines. Functional experiments indicated that GC cells with DSCAM-AS1 silencing exhibited a dynamic reduction in proliferation and migration. We identified miR-204 as a target of DSCAM-AS1 and found that it targeted TPT1 in GC cells, which further led to decreased expression of miR-204 in GC tissues and cell lines. A rescue assay revealed that knocked-down DSCAM-AS1 hindered GC progression, which was reversed upon miR-204 downregulation or TPT1 overexpression. Conclusion: We conclude that DSCAM-AS1 is expressed as a tumor oncogene in GC progression, modulated via the miR-204/TPT1 axis. These findings indicate the potential of DSCAM-AS1 as a therapeutic target for GC prevention. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) was confirmed to be associated with hematopoietic insufficiency, as well as abnormal proliferation, differentiation or survival of myeloid progenitors. Multiple studies reported that microRNA-204 (miR-204) and Hepatocyte growth factor (HGF) played important roles in types of cancers. However, the potential molecular regulatory mechanism between miR-204 and HGF in AML remains to be further defined. Real-time PCR (RT-PCR) was adopted to detect the expression of miR-204 and HG. Relative protein levels were detected by western blot assay. The viability, cell cycle, apoptosis, migration, and invasion were analyzed by MTT, flow cytometry, and transwell assays. Moreover, the target relationship between miR-204 and HGF was predicted by MiRcode website and confirmed by luciferase reporter, RNA pull-down, and western blot assays. Our data suggested that miR-204 was downregulated in AML serum samples and cells. MiR-204 overexpression repressed cell proliferation, migration, invasion, and induced cell apoptosis in AML cells. HGF was upregulated in AML samples and cells, and HGF knockdown inhibited the malignancy of AML cells. In addition, HGF was directly targeted by miR-204. HGF overexpression reversed the effects of miR-204 mimic on AML cell proliferation, apoptosis, migration, and invasion. Besides, miR-204 regulated the c-Met signaling by targeting HGF, thereby regulating the downstream protein levels related to cell proliferation, apoptosis, migration, and invasion in AML cells. In conclusion, miR-204 could regulate AML progression through regulating the HGF/c-Met pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Pulmonary artery hypertension (PAH) is a vascular disease in the lung characterized by elevated pulmonary arterial pressure (PAP). Many miRNAs play a role in the pathophysiology of PAH. Perillyle alcohol (PA) and Quercetin (QS) are plant derivatives with antioxidant and anti-proliferative properties. We investigated the effect of PA and QS on PAP, expression of PARP1, miR-204, and their targets, HIF1α and NFATc2, in experimental PAH. Methods Thirty rats were divided into control, MCT, MCT + Veh, MCT + PA and MCT + QS groups. MCT (60 mg/kg) was injected subcutaneously to induce PAH. PA (50 mg/kg daily) and QS (30 mg/kg daily) were administered for 3 weeks after inducing PAH. PAP, lung pathology, expression of miRNA and mRNA, and target proteins were evaluated through right ventricle cannulation, H&E staining, real-time qPCR, and western blotting, respectively. Results Inflammation and lung arteriole thickness in the MCT group increased compared to control group. PA and QS ameliorated inflammation and reduced arteriole thickness significantly. miR-204 expression decreased in PAH rats (p

Objective To investigate the regulatory mechanism of miR-204 on proliferation, apoptosis and autophagy of cervical cancer cells.Methods The expression of miR-204 in cervical cancer tissues and cell lines was detected by real-time PCR. Cell viability and apoptosis were detected by MTT and flow cytometry, respectively. Protein expression of Bcl-2, Bax, Caspase-3 and LC3I/II in C33A cells after transfection was detected by Western blot assay. The interaction between miR-204 and ATF2 was verified by Targetscan online prediction, dual-luciferase assay and Western blot.Results The expression of miR-204 was significantly down-regulated in cervical cancer tissues and cell lines (p

Pancreatic β-cell dysfunction is a key link during the progression of type 2 diabetes (T2DM), and SIRT1 participates in the regulation of various physiological activities of islet β-cells. However, as a key link in signal transduction, it is not clear how SIRT1 is regulated. By TargetScan prediction, we found that miR-204, which is enriched in islets, has highly complementary binding sites with SIRT1. Therefore, we speculate that miR-204 may be the upstream regulatory target of SIRT1 in islets and thus participate in the occurrence of β-cell dysfunction. In this study, we explored the association between miR-204 and β-cell dysfunction, the therapeutic effects of berberine (BBR) on β-cell function and the possible mechanisms. We found that miR-204 increased and SIRT1 mRNA and protein levels decreased significantly in islets both in vivo and in vitro. MIN6 cells induced by palmitic acid exhibited increased apoptosis, and the accumulation of insulin and ATP in the supernatant decreased. Importantly, palmitic acid treatment combined with miR-204 silencing showed opposite changes. MiR-204 overexpression in MIN6 cells increased apoptosis and decreased insulin and ATP production and SIRT1 expression. SIRT1 overexpression reversed the damage to β-cells caused by miR-204. The BBR treatment effectively improved insulin synthesis, reduced miR-204 levels, and increased SIRT1 expression in islet tissue in diabetic mice. Overexpression of miR-204 reversed the protective effect of BBR on apoptosis and insulin secretion in MIN6 cells. Our study identifies a novel correlation between miR-204 and β-cell dysfunction in T2DM and shows that administration of BBR leads to remission of β-cell dysfunction by regulating the miR-204/SIRT1 pathway. [ABSTRACT FROM AUTHOR]

Skeletal muscle is a heterogeneous tissue that is essential for initiating movement and maintaining homeostasis. The genesis of skeletal muscle is an integrative process that lasts from embryonic development to postnatal stages, which is carried out under the modulation of many factors. Recent studies have shown that circular RNAs (circRNAs), a class of non-coding RNAs, are involved in myogenesis. However, more circRNAs and their mechanisms that may regulate skeletal muscle development remain to be explored. Through in-depth analysis of our previous RNA-Seq data, circFNDC3AL was found to be a potentially functional circRNA highly expressed during embryonic development of chicken skeletal muscle. Therefore, in this study, we investigated the effect of circFNDC3AL on skeletal muscle development in chickens and found that circFNDC3AL promoted chicken skeletal muscle satellite cell (SMSC) proliferation and differentiation. To gain a thorough understanding of the exact modulatory mechanisms of circFNDC3AL in chicken skeletal muscle development, we performed target miRNA analysis of circFNDC3AL and found that circFNDC3AL has a binding site for miR-204. Subsequently, we demonstrated that miR-204 inhibited chicken SMSC proliferation and differentiation, which showed the opposite functions of circFNDC3AL. Furthermore, we identified the miR-204 target gene B-cell CLL/lymphoma 9 (BCL9) and validated that miR-204 had an inhibitory effect on BCL9, while the negative effect could be relieved by circFNDC3AL. In addition, we verified that BCL9 performed the same positive functions on chicken SMSC proliferation and differentiation as circFNDC3AL, as opposed to miR-204. In conclusion, our study identified a circRNA circFNDC3AL that upregulates BCL9 expression to promote the proliferation and differentiation of chicken SMSCs by binding to miR-204.

Pancreatic β-cell dysfunction is a key link during the progression of type 2 diabetes (T2DM), and SIRT1 participates in the regulation of various physiological activities of islet β-cells. However, as a key link in signal transduction, it is not clear how SIRT1 is regulated. By TargetScan prediction, we found that miR-204, which is enriched in islets, has highly complementary binding sites with SIRT1. Therefore, we speculate that miR-204 may be the upstream regulatory target of SIRT1 in islets and thus participate in the occurrence of β-cell dysfunction. In this study, we explored the association between miR-204 and β-cell dysfunction, the therapeutic effects of berberine (BBR) on β-cell function and the possible mechanisms. We found that miR-204 increased and SIRT1 mRNA and protein levels decreased significantly in islets both in vivo and in vitro. MIN6 cells induced by palmitic acid exhibited increased apoptosis, and the accumulation of insulin and ATP in the supernatant decreased. Importantly, palmitic acid treatment combined with miR-204 silencing showed opposite changes. MiR-204 overexpression in MIN6 cells increased apoptosis and decreased insulin and ATP production and SIRT1 expression. SIRT1 overexpression reversed the damage to β-cells caused by miR-204. The BBR treatment effectively improved insulin synthesis, reduced miR-204 levels, and increased SIRT1 expression in islet tissue in diabetic mice. Overexpression of miR-204 reversed the protective effect of BBR on apoptosis and insulin secretion in MIN6 cells. Our study identifies a novel correlation between miR-204 and β-cell dysfunction in T2DM and shows that administration of BBR leads to remission of β-cell dysfunction by regulating the miR-204/SIRT1 pathway.

Objective: To explore the regulatory mechanism of long non-coding RNAs (lncRNAs) in the occurrence and development of epithelial-mesenchymal transition (EMT) in calcium oxalate crystal-induced kidney injury.Materials and Methods: Gene core technique was used to screen differentially expressed lncRNAs and mRNAs in HK-2 cells before and after calcium oxalate monohydrate (COM) stimulation; differentially expressed mRNAs were then analyzed using GO and pathway analysis. The role of target lncRNA in EMT in renal tubular epithelial cells induced by COM was further investigated by applying a series of in vitro experiments.Results: Four differentially expressed lncRNAs (ABCA9-AS1, SPANXA2-OT1, RP11-955H22.1, and RP11-748C4.1) were up-regulated after 48 h of COM stimulation compared to the control group, where up-regulated expression of lncRNA SPANXA2-OT1 was the most significant. Thus, lncRNA SPANXA2-OT1 was further examined. Interference lncRNA SPANXA2-OT1 reversed the down-regulation of E-cadherin and Pan-ck, and up-regulated Vimentin and α-SMA induced by COM stimulation. The application of miR204 inhibitor weakened the interference effect of interfering RNA on lncRNA SPANXA2-OT1 and promoted the occurrence of EMT. Moreover, the miR204 simulator alleviated the overexpression effect of lncRNA SPANXA2-OT1 on COM-stimulated renal tubular epithelial cells and inhibited the occurrence of EMT in renal tubular epithelial cells. Also, a dual-luciferase reporter assay showed that miR-204 could bind to lncRNA SPANXA2-OT1 and Smad5, while lncRNA SPANXA2-OT1 could inhibit cell proliferation and promote cell apoptosis.Conclusion: The lncRNA SPANXA2-OT1 is involved in the occurrence and development of EMT in renal tubular epithelial cells induced by crystalline kidney injury by adsorbing miR-204 and up-regulating Smad5.

Aim Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is the key of the development of diabetic retinopathy (DR), and lncRNA NEAT1 could accelerate EMT in diabetic nephropathy. Meanwhile, as a diabetes susceptibility gene, whether sex-determining region Y-related (SRY) high-mobility group box 4 (SOX4) has relationship with lncRNA NEAT1 in DR remains unclear. Methods Firstly, NEAT1, SOX4 and miR-204 were evaluated by qRT-PCR (quantitative reverse-transcriptase PCR) under high glucose condition. Then, cell viability, proliferation, migration and invasion were respectively detected by MTT, BrdU staining, wound healing and transwell assay after NEAT1 knockdown or miR-204 overexpression. Also, the EMT-related proteins were examined by western blot and cell immunofluorescence assay. In order to confirm the relationship between miR-204 and NEAT1 or SOX4, dual luciferase reporter gene assay was conducted. At the same time, the protein levels of SOX4 and EMT-related proteins were investigated by immunohistochemistry in vivo. Results High glucose upregulated NEAT1 and SOX4 and downregulated miR-204 in ARPE19 cells. NEAT1 knockdown or miR-204 overexpression inhibited the proliferation and EMT progression of ARPE19 cells induced by high glucose. NEAT1 was identified as a molecular sponge of miR-204 to increase the level of SOX4. The effect of NEAT1 knockdown on the progression of EMT under high glucose condition in ARPE19 cells could be reversed by miR-204 inhibitor. Also, NEAT1 knockdown inhibited retinal EMT in diabetic mice. Conclusion NEAT1 regulated the development of EMT in DR through miR-204/SOX4 pathway, which could provide reference for clinical prevention and treatment.

Vasculogenic mimicry (VM) is an angiogenesis-independent process that potentially contributes to the poor clinical outcome of anti-angiogenesis therapy in multiple malignant cancers, including pancreatic adenocarcinoma (PAAD). Several studies have shown that ginsenoside Rg3, a bioactive component of ginseng, holds considerable potential for cancer treatment. Our previous work has proved that Rg3 can inhibit VM formation in PAAD. However, its underlying mechanism remains unclear. To explore the underlying mechanism by which Rg3 affects VM formation in PAAD. We first investigated the effects of Rg3 on the cellular phenotypes of two PAAD cell lines (SW-1990 and PCI-35), and the expression of EMT- and stemness-related proteins. SW-1990 cells were adopted to construct xenograft models, and the anti-tumor effects of Rg3 in vivo were validated. Subsequently, we isolated the exosomes from the two PAAD cell lines with Rg3 treatment or not, and explored whether Rg3 regulated VM via PAAD cell-derived exosomes. MiRNA sequencing, clinical analysis, and rescue experiments were performed to investigate whether and which miRNA was involved. Subsequently, the target gene of miRNA was predicted using the miRDB website (https://mirdb.org/), and rescue experiments were further conducted to validate those in vitro and in vivo. Rg3 indeed exhibited excellent anti-tumor effects both in vitro and in vivo , with inhibitory effects on EMT and stemness of PAAD cells. More interestingly, Rg3-treated PAAD cell-derived exosomes suppressed the tube-forming ability of HUVEC and PAAD cells, with a decrease in stemness-related protein expression, indicating that Rg3 inhibited both angiogenesis and VM processes. Subsequently, we found that Rg3 induced the up-regulation of miR-204 in PAAD cell-derived exosomes, and miR-204 alone inhibited tube and sphere formation abilities of PAAD cells like exosomes. Specifically, miR-204 down-regulated DVL3 expression, which was involved in regulating cancer cell stemness, and ultimately affected VM. The in vivo experiments further indicated that Rg3-treated SW-1990 cell-derived exosome-inhibited tumor growth, VM formation, and stemness-related protein expression can be abrogated by DVL3 overexpression. Ginsenoside Rg3 increased the PAAD cell-derived exosomal miR-204 levels, which subsequently inhibited its target genes DVL3 expression in the receptor PAAD cells, and the down-regulated DVL3 broke stemness maintenance, ultimately suppressing VM formation of PAAD. Our findings revealed a novel mechanism by which Rg3 exerted its anti-tumor activity in PAAD via inhibiting VM, and provided a promising strategy to make up for the deficiency of anti-angiogenesis therapy in cancer. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background Ovarian cancer is often accompanied by the production of ascites, and patients with repeated ascites are associated with chemotherapy resistance. The previous study confirmed that the ovarian cancer patients who developed ascites after chemotherapy had elevated autophagy levels in the ascites and precipitated cells, which was positively correlated with MDR1 expression in the blood of patients. Methods In order to explore the correlation between autophagy and chemoresistant, we searched TCGA and GEO database to analyze the correlation between LC3B and MDR1, and identified the targeting miRNA of LC3B. It was verified by dual luciferase that miR-204 can target LC3B. The ovarian cancer cell line and the BALB/c nude mice tumor-bearing model were selected for in vitro and in vivo verification. In vitro studies confirmed that ovarian cancer cells were more sensitive to cisplatin by inhibiting LC3B. Results Overexpression of miR-204 reduced the expression of LC3B, Atg7, and MDR1, and promoted apoptosis. In vivo studies have also confirmed that reducing the level of autophagy in ovarian cancer cells increases the sensitivity to cisplatin. Conclusions It suggests that miR-204 can be used as a tumor suppressor gene and LC3B expression level can be used as a potential molecular marker to guide the diagnosis and treatment of patients with ovarian cancer.

Abstract Colorectal cancer (CRC) is still the third most common cancer in the world with a limited prognosis due to the chemoresistance of CRC cells to 5‐fluorouracil (5‐FU)‐based chemotherapy. In our previous study, we revealed that miR‐204 overexpression could sensitize CRC cell to 5‐FU treatment through targeting HMGA2/PI3K signaling pathway; however, miR‐204 expression in CRC tissues is abnormally downregulated. Long non‐coding RNAs (lncRNAs) dysregulation has been reported in human diseases, including cancer. Also, lncRNA can regulate cancer cell proliferation, invasion, migration, as well as chemoresistance. LncRNA prostate cancer‐associated transcript 6 (PCAT6) acts as an oncogene in many cancers; herein, PCAT6 expression was abnormally upregulated in CRC tissues and cell lines, suggesting its potential role in CRC. Further, we assessed the specific function and mechanism of PCAT6 in CRC. Furthermore, we revealed that PCAT6 knockdown attenuated CRC chemoresistance to 5‐FU through miR‐204/HMGA2/PI3K; miR‐204 inhibition could partially reverse the effect of PCAT6 knockdown. Taken together, we demonstrate that the abnormal PCAT6 overexpression inhibits miR‐204 expression in CRC, thereby promoting HMGA2/PI3K signaling activity, ultimately enhancing the chemoresistance of CRC cells to 5‐FU; PCAT6 represents a promising target for dealing with CRC chemoresistance.

Abstract Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastoma: WNT, SHH, Group 3 and Group 4. Molecular markers are necessary for accurate risk stratification in the non-WNT subgroups due to the underlying heterogeneity in genetic alterations and overall survival. MiR-204 expression was evaluated in molecularly classified 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC cohort, SickKids, Canada. Low expression of miR-204 in the Group 3 / Group 4 tumors identify a highly aggressive subset of tumors having poor overall survival, in the two independent cohorts of medulloblastomas. Downregulation of miR-204 expression correlates with poor survival within the Group 4 as well indicating it as a valuable risk-stratification marker in the subgroup. Restoration of miR-204 expression in multiple medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. IGF2R was identified as a novel target of miR-204. MiR-204 expression resulted in downregulation of both M6PR and IGF2R that transport lysosomal proteases from the Golgi apparatus to the lysosomes. Consistent with this finding, miR-204 expression resulted in reduction in the levels of the lysosomal proteases in medulloblastoma cells. MiR-204 expression also resulted in inhibition of autophagy that is known to be dependent on the lysosomal degradation pathway and LC3B, a known miR-204 target. Treatment with HDAC inhibitors resulted in upregulation of miR-204 expression in medulloblastoma cells, suggesting therapeutic role for these inhibitors in the treatment of medulloblastomas. In summary, miR-204 is not only a valuable risk stratification marker in the combined cohort of Group 3 / Group 4 medulloblastomas as well as in the Group 4 itself, that has paucity of good prognostication markers, but also has therapeutic potential as indicated by its tumor suppressive effect on medulloblastoma cells.

Abstract Background Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality. Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation. Methods Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis. Results A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset. Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity. We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures. We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2. By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein. Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression. Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression. Conclusion Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression.

Heterozygous mutations in the Paired like homeobox 2b (PHOX2B) gene are causative of congenital central hypoventilation syndrome (CCHS), a rare monogenic disorder belonging to the family of neurocristopathies and due to a defective development of the autonomic nervous system. Most patients manifest sudden symptoms within 1 year of birth, mainly represented by central apnea and cyanosis episodes. The sudden appearance of hypoxic manifestations in CCHS and their occurrence during sleep resemble two other unexplained perinatal disorders, apparent life-threatening event (ALTE) and sudden and unexpected infant death (SUID), among which the vast majority is represented by sudden infant death syndrome (SIDS). Differently from CCHS, characterized by Mendelian autosomal dominant inheritance, ALTE and SIDS are complex traits, where common genetic variants, together with external factors, may exert an additive effect with symptoms likely manifesting only over a “threshold.” Given the similarities observed among the three abovementioned perinatal disorders, in this work, we have analyzed the frequency of PHOX2B common variants in two groups of Italian idiopathic ALTE (IALTE) and SUIDs/SIDS patients. Here, we report that the c*161G>A (rs114290493) SNP of the 3′UTR PHOX2B (i) became overrepresented in the two sets of patients compared to population matched healthy controls, and (ii) associated with decreased PHOX2B gene expression, likely mediated by miR-204, a microRNA already known to bind the 3′UTR of the PHOX2B gene. Overall, these results suggest that, at least in the Italian population, the SNP c*161G>A (rs114290493) does contribute, presumably in association with others mutations or polymorphisms, to confer susceptibility to sudden unexplained perinatal life-threatening or fatal disorders by increasing the effect of miR-204 in inducing PHOX2B expression down-regulation. However, these are preliminary observations that need to be confirmed on larger cohorts to achieve a clinical relevance.

Objective: To detect the plasma expression of miR-17 and miR-204 in patients with non-small cell lung cancer (NSCLC), and analyze the relationship between their expression and clinicopathological parameters and their predictive value for the prognosis of NSCLC. Methods: 117 NSCLC patients (observation group) admitted to our hospital from June 2015 to June 2017 and 100 healthy people (control group) who underwent physical examination in our hospital during the same period were selected as the research objects, using real-time fluorescent quantitative PCR (QRT-PCR) The expression of miR-17 and miR-204 in plasma was detected, and the relationship between the expression of plasma miR-17 and miR-204 and the clinicopathological parameters of NSCLC patients was analyzed. The receiver operating characteristic (ROC) curve was used to analyze the predictive value of plasma miR-17 and miR-204 on the prognosis of NSCLC patients. Results: The expression of plasma miR-17 in the observation group was higher than that in the control group (P<0.05), and the expression of miR-204 was lower than that in the control group (P<0.05). The expression of miR-17 was related to lymph node metastasis and distant metastasis (P<0.05). ), the expression of miR-204 is related to TNM staging and distant metastasis (P<0.05). ROC curve analysis results showed that the area under the curve (AUC) of miR-17 and miR-204 predicting the prognosis of NSCLC patients were 0.821 and 0.836, respectively. The combined detection of miR-17 and miR-204 has higher predictive value for the prognosis of NSCLC patients: sensitivity and specificity are 89.36% (42/47) and 92.86% (65/70), respectively. Conclusion: There are abnormal expressions of miR-17 and miR-204 in plasma of NSCLC patients, which are related to the clinical pathological parameters of the patients, and may be used as potential biological indicators for the prognosis of NSCLC patients. [ABSTRACT FROM AUTHOR]

Heterozygous mutations in the Paired like homeobox 2b (PHOX2B) gene are causative of congenital central hypoventilation syndrome (CCHS), a rare monogenic disorder belonging to the family of neurocristopathies and due to a defective development of the autonomic nervous system. Most patients manifest sudden symptoms within 1 year of birth, mainly represented by central apnea and cyanosis episodes. The sudden appearance of hypoxic manifestations in CCHS and their occurrence during sleep resemble two other unexplained perinatal disorders, apparent life-threatening event (ALTE) and sudden and unexpected infant death (SUID), among which the vast majority is represented by sudden infant death syndrome (SIDS). Differently from CCHS, characterized by Mendelian autosomal dominant inheritance, ALTE and SIDS are complex traits, where common genetic variants, together with external factors, may exert an additive effect with symptoms likely manifesting only over a "threshold." Given the similarities observed among the three abovementioned perinatal disorders, in this work, we have analyzed the frequency of PHOX2B common variants in two groups of Italian idiopathic ALTE (IALTE) and SUIDs/SIDS patients. Here, we report that the c*161G>A (rs114290493) SNP of the 3′UTR PHOX2B (i) became overrepresented in the two sets of patients compared to population matched healthy controls, and (ii) associated with decreased PHOX2B gene expression, likely mediated by miR-204, a microRNA already known to bind the 3′UTR of the PHOX2B gene. Overall, these results suggest that, at least in the Italian population, the SNP c*161G>A (rs114290493) does contribute, presumably in association with others mutations or polymorphisms, to confer susceptibility to sudden unexplained perinatal life-threatening or fatal disorders by increasing the effect of miR-204 in inducing PHOX2B expression down-regulation. However, these are preliminary observations that need to be confirmed on larger cohorts to achieve a clinical relevance. [ABSTRACT FROM AUTHOR]

T cell acute lymphoblastic leukaemia (T‐ALL) is a highly aggressive haematological cancer of the bone marrow. The abnormal expression of microRNAs (miRNAs) is reportedly involved in T‐ALL development and progression. Thus, we aimed to decipher the involvement of miR‐204 silencing mediated by DNA methylation in the occurrence of T cell acute lymphoblastic leukaemia (T‐ALL). miR‐204 expression was determined in bone marrow and peripheral blood samples from T‐ALL patients by real‐time quantitative PCR (RT‐qPCR) with its effect on cell proliferation evaluated by functional assays. In addition, bisulphite sequencing PCR was employed to detect the DNA methylation level of the miR‐204 promoter region, and the binding site between miR‐204 and IRAK1 was detected by luciferase assay. We found that miR‐204 was down‐regulated in T cells of T‐ALL patients, which was caused by the increased DNA methylation in the promoter region of miR‐204. Moreover, overexpression of miR‐204 inhibited T‐ALL cell proliferation while enhancing their apoptosis through interleukin receptor‐associated kinase 1 (IRAK1), which enhanced the expression of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 through activation of p‐p65. Thus, miR‐204 modulated MMP‐2 and MMP‐9 through IRAK1/NF‐κB signalling pathway, which was confirmed by in vivo assay. Taken together, DNA methylation‐mediated miR‐204 silencing increased the transcription of IRAK1, thus activating the NF‐κB signalling pathway and up‐regulating the downstream targets MMP‐2/MMP‐9. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs), a class of non-coding RNAs, are essential key players in the control of biological processes in both physiological and pathological conditions. miRNAs play important roles in fine tuning the expression of many genes, which often have roles in common molecular networks. miRNA dysregulation thus renders cells vulnerable to aberrant fluctuations in genes, resulting in degenerative diseases. The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells that resides between the light-sensitive photoreceptors (PR) and the choriocapillaris. The demanding physiological functions of RPE cells require precise gene regulation for the maintenance of retinal homeostasis under stress conditions and the preservation of vision. Thus far, our understanding of how miRNAs function in the homeostasis and maintenance of the RPE has been poorly addressed, and advancing our knowledge is central to harnessing their potential as therapeutic agents to counteract visual impairment. This review focuses on the emerging roles of miRNAs in the function and health of the RPE and on the future exploration of miRNA-based therapeutic approaches to counteract blinding diseases.

Here, we present miniCoopR-I, an inducible upgrade of the constitutive miniCoopR vector. We developed miniCoopR-I-sponge-204 and miniCoopR-I-pre-miR-204 vectors and we successfully tested them for their ability to achieve time- (embryo/juvenile/adult) and space- (melanocytic lineage) restricted inhibition/overexpression of miR-204, a positive modulator of pigmentation previously discovered by us. Furthermore, melanoma-free survival curves performed on induced fish at the adult stage indicate that miR-204 overexpression accelerates the development of BRAFV600E-driven melanoma. miniCoopR-I allows study of the impact that coding and non-coding modulators of pigmentation exert on melanomagenesis in adult zebrafish, uncoupling it from the impact that they exert on melanogenesis during embryonic development. This article has an associated First Person interview with the first author of the paper.

Chicken atrophic ovaries have decreased volume and are indicative of ovarian failure, presence of a tumor, or interrupted ovarian blood supply. Ovarian tumor is accompanied by an increase in follicular atresia, granulosa cell (GC) apoptosis, and autophagy. In a previous study, we found using high throughput sequencing that miR-204 is highly expressed in chicken atrophic ovaries. Thus, in the present study, we further investigated its function in GC apoptosis and autophagy. We found that overexpression of miR-204 reduced mRNA and protein levels of proliferation-related genes and increased apoptosis-related genes. Cell counting kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), and flow cytometry assays revealed that miR-204 inhibited GC proliferation and promoted apoptosis. Furthermore, we confirmed with reporter gene assays that Forkhead box K2 (FOXK2) was directly targeted by miR-204. FOXK2, as a downstream regulator of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathways, promoted GC proliferation and inhibited apoptosis. Subsequently, we observed that miR-204 was involved in GC autophagy by targeting Transient Receptor Potential Melastatin 3 (TRPM3). The luciferase activities of the two binding sites of TRPM3 were decreased in response to treatment with a miR-204 mimic, and the autophagic flux was increased after miR-204 inhibition. However, overexpression of miR-204 had opposite results in autophagosomes and autolysosomes. miR-204 inhibits GC autophagy by suppressing the protein expression of TRPM3/AMP-activated protein kinase (AMPK)/ULK signaling pathway components. Inhibition of miR-204 enhanced autophagy by accumulating and degrading the protein levels of LC3-II (Microtubule Associated Protein Light Chain 3B) and p62 (Protein of 62 kDa), respectively, whereas miR-204 overexpression was associated with contrary results. Immunofluorescence staining showed that there was a significant reduction in the fluorescent intensity of LC3B, whereas p62 protein was increased after TRPM3 silencing. Collectively, our results indicate that miR-204 is highly expressed in chicken atrophic ovaries, which promotes GC apoptosis via repressing FOXK2 through the PI3K/AKT/mTOR pathway and inhibits autophagy by impeding the TRPM3/AMPK/ULK pathway.

RUNX2 and SOX9 are two pivotal transcriptional regulators of chondrogenesis. It has been demonstrated that RUNX2 and SOX9 physically interact; RUNX2 transactivation may be inhibited by SOX9. In addition, RUNX2 exerts reciprocal inhibition on SOX9 transactivity. Epigenetic control of gene expression plays a major role in the alternative differentiation fates of stem cells; in particular, it has been reported that SOX9 can promote the expression of miRNA (miR)-204. Our aim was therefore to investigate the miR-204-5p role during chondrogenesis and to identify the relationship between this miR and the transcription factors plus downstream genes involved in chondrogenic commitment and differentiation. To evaluate the role of miR-204 in chondrogenesis, we performed in vitro transfection experiments by using Mesenchymal Stem Cells (MSCs). We also evaluated miR-204-5p expression in zebrafish models (adults and larvae). By silencing miR-204 during the early differentiation phase, we observed the upregulation of SOX9 and chondrogenic related genes compared to controls. In addition, we observed the upregulation of COL1A1 (a RUNX2 downstream gene), whereas RUNX2 expression of RUNX2 was slightly affected compared to controls. However, RUNX2 protein levels increased in miR-204-silenced cells. The positive effects of miR204 silencing on osteogenic differentiation were also observed in the intermediate phase of osteogenic differentiation. On the contrary, chondrocytes’ maturation was considerably affected by miR-204 downregulation. In conclusion, our results suggest that miR-204 negatively regulates the osteochondrogenic commitment of MSCs, while it positively regulates chondrocytes’ maturation.

OBJECTIVE : The aim of this study was to investigate the influence of micro ribonucleic acid (miR)-204 on rats with myocardial infarction by targeting the silent information regulator 1 (SIRT1)/p53 signaling pathway. MATERIALS AND METHODS: A total of 36 Sprague-Dawley rats were randomly divided into three groups, including: sham-operation group (n=12), model group (n=12) and miR-204 mimics group (n=12). The rats in the sham-operation group only underwent thoracotomy, without myocardial infarction injury. Meanwhile, the rats in model group and miR-204 mimics group were utilized to establish the models of myocardial infarction, and then, intervened with normal saline and miR-204 mimics, respectively. The morphology of myocardial tissues was observed via hematoxylin-eosin (HE) staining. Immunofluorescence was performed to detect the expression of Caspase-3. Target genes of miR-204 were analyzed using bioanalysis software. Western blotting (WB) assay was applied to measure the relative protein expression of SIRT1. MiR-204 expression and the messenger RNA (mRNA) expressions of SIRT1 and p53 were measured via quantitative Polymerase Chain Reaction (qPCR). Furthermore, cell apoptosis was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: HE staining showed that the morphology of myocardial tissues was normal in sham-operation group. Severe myocardial tissue injury was visible in model group, and the injury was relieved in miR-204 mimics group when compared with model group. The results manifested that the positive expression of Caspase-3 in cardiac tissues increased remarkably in the model group and miR-204 mimics group in comparison with sham-operation group (p<0.05). Meanwhile, it was evidently lower in miR-204 mimics group than model group (p<0.05). Based on the analysis via bioanalysis software, SIRT1 was the target gene of miR-204. WB results revealed that the relative protein expression level of SIRT1 was elevated notably in the other two groups compared with the 2sham-operation group (p<0.05). However, it was markedly lowered in miR-204 mimics group in contrast with model group (p<0.05). QRT-PCR results demonstrated that the model group and miR-204 mimics group exhibited distinctly lower expression of miR-204 but higher mRNA expressions of SIRT1 and p53 than sham-operation group (p<0.05). However, miR-204 mimics group exhibited prominently higher expression of miR-204 but lower mRNA expressions of SIRT1 and p53 than model group (p<0.05). Finally, the results of TUNEL assay demonstrated that the apoptosis rate increased remarkably in the model group and miR-204 mimics group when compared with sham-operation group (p<0.05). However, it decreased notably in miR-204 mimics group in comparison with model group (p<0.05). CONCLUSIONS: MiR-204 reduces the apoptosis level in rats with myocardial infarction via targeted inhibition of the SIRT1/p53 signaling pathway. [ABSTRACT FROM AUTHOR]

Prevalence of obesity becomes an important health issue worldwide, but the management of obesity remains unsatisfied. This study aimed to explore the mechanism of long non-coding RNA TUG/miR-204/SIRT1 axis, which was involved in the pathogenesis of obesity. Obesity mouse model was induced by high-fat diet and treated with taurine upregulated gene1 (TUG1) virus via tail intravenous injection. Then, body weight, serum glucose, insulin tolerance, testicular fat weight were detected, as well as the expression of TUG1, microRNA-204 (miR-204), sirtuin1 (SIRT1), and inflammation and fatty accumulation associated proteins and cytokines. Regulatory relationship between TUG1/SIRT1 and miR-204 was confirmed by dual-luciferase reporter activity assay. A high-glucose-induced 3T3-L1 cell model was also constructed to explore the regulatory mechanism of TUG/miR-204/SIRT1 axis in the pathogenesis of obesity at cell level after altering the expression of TUG1, miR-204, and SIRT1. Overexpression of TUG1 could significantly attenuate the weight, serum glucose, glucose, insulin tolerance, fatty accumulation, and inflammation in obesity mice, as well as the elevation of miR-204, but increase the expression of SIRT1, phosphorylated AKT (p-AKT), glucose transporter4 (GLUT4), and peroxisome proliferator activated receptorγ (PPARγ). Both TUG1 and SIRT1 were targets of miR-204 and could be negatively regulated by miR-204. Overexpression of TUG1 could suppress the inflammation in adipocytes via downregulating miR-204 and promote GLUT4/PPARγ/AKT pathway high-glucose-induced inflammation in 3T3-L1 cells. miR-204 inhibitors could also suppress high-glucose-induced inflammation in 3T3-L1 cells via promoting SIRT1/ GLUT4/PPARγ/AKT pathway. LncRNA TUG1 could negatively regulate miR-204 to alleviate inflammation and insulin tolerance via promoting SIRT1/GLUT4/PPARγ/AKT pathway. [ABSTRACT FROM AUTHOR]

Acute myocardium infarction (AMI) is one of the main causes of cardiovascular death, and timely intervention and diagnosis are essential. Owing to the irreversible apoptosis and death of myocardial cells, which ultimately causes heart failure, the problem of myocardial repair after myocardial infarction needs to be urgently addressed. Exosomes can act as messengers between cells, delivering large amounts of proteins, RNA, and lipids to receptor cells, and regulating target cell functions. Studies have shown that exosomes can repair infarcted myocardium. We aimed to investigate the relationship between long non‐coding RNA NEAT1 in serum exosomes of patients and AMI and its underlying mechanism. Subjects were divided into control, UA, and STEMI groups. RNA was extracted from the serum exosomes, and the expressions of lncRNA NEAT1 and miR‐204 were detected by qRT‐PCR. MMP‐9 was detected by western blot, Spearman test was used to analyze the correlation among the three. Logistic regression and Receiver‐operating characteristic curve (ROC) were used to evaluate the prediction of acute myocardial infarction. The expressions of NEAT1 and MMP‐9 in serum exosomes of patients with acute ST‐segment elevation myocardial infarction were up‐regulated and positively correlated, miR‐204 expression was down‐regulated, there were no correlations between miR‐204 with NEAT1, or MMP‐9. Exosomal NEAT1, miR‐204, and MMP‐9 displayed potent biomarkers for diagnosis of acute ST‐segment elevation myocardial infarction. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is the most common ocular complication caused by diabetes mellitus and is the main cause of visual impairment in working‐age people. Reactive gliosis and pro‐inflammatory cytokine production by Müller cells contribute to the progression of DR. Melatonin is a strong anti‐inflammatory hormone, mediating the cytoprotective effect of a variety of retinal cells against hyperglycemia. In this study, melatonin inhibited the gliosis activation and inflammatory cytokine production of Müller cells in both in vitro and in vivo models of DR. The melatonin membrane blocker, Luzindole, invalidated the melatonin‐mediated protective effect on Müller cells. Furthermore, melatonin inhibited Müller cell activation and pro‐inflammatory cytokine production by upregulating the long noncoding RNA maternally expressed gene 3/miR‐204/sirtuin 1 axis. In conclusion, our study suggested that melatonin treatment could be a novel therapeutic strategy for DR. [ABSTRACT FROM AUTHOR]

Corneal neovascularization (NV) is the major sight-threatening pathology caused by angiogenic stimuli. Current drugs that directly target pro-angiogenic factors to inhibit or reverse the disease require multiple rounds of administration and have limited efficacies. Here, we identify potential anti-angiogenic corneal microRNAs (miRNAs) and demonstrate a framework that employs discovered miRNAs as biotherapies deliverable by recombinant adeno-associated viruses (rAAVs). By querying differentially expressed miRNAs in neovascularized mouse corneas induced by alkali burn, we have revealed 39 miRNAs that are predicted to target more than 5,500 differentially expressed corneal mRNAs. Among these, we selected miR-204 and assessed its efficacy and therapeutic benefit for treating injured corneas. Our results show that delivery of miR-204 by rAAV normalizes multiple novel target genes and biological pathways to attenuate vascularization of injured mouse cornea. Importantly, this gene therapy treatment alternative is efficacious and safe for mitigating corneal NV. Overall, our work demonstrates the discovery of potential therapeutic miRNAs in corneal disorders and their translation into viable treatment alternatives.

Abstract Background MicroRNAs (miRNAs or miRs) are small molecules known to be involved in post-transcriptional gene expression. Many of them have been shown to influence risk for various diseases. Recent studies suggest that lower expression of miR-204, a gene coding for miRNA-204, is correlated with shorter survival in patients with acute myeloid leukaemia (AML). This observation prompted us to analyse the effect of two polymorphisms of the miR-204 gene, one in the upstream flanking region (rs718447 A > G) and the other inside the gene itself (rs112062096 A > G), both also in intron 3 of the TRPM3 gene. Methods The study was conducted on DNA samples isolated from AML patients (n = 95) and healthy individuals (n = 148), who were genotyped using the Light SNiP assays. Results The miR-204 rs718447 GG homozygosity was found to constitute a risk factor associated with susceptibility to AML (73/95 vs 92/148, AML patients vs healthy controls, OR = 2.020, p = 0.017). Additionally, this genotype was more frequent in patients with subtypes M0-M1 in the French-American-British (FAB) classification as compared to patients with subtypes M2-M7 (23/25 vs 39/57, p = 0.026). We also found that presence of allele A was linked to longer survival of AML patients. Conclusions Our results show that polymorphism in miR-204 flanking region may constitute a risk and prognostic factor in AML.