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Background: Diabetic foot ulcer (DFU) is one of the most distressing complications in patients with type 2 diabetes mellitus (T2DM). Vascular endothelial growth factor (VEGF) is a strong angiogenic factor associated with wound healing and development. This study aimed to investigate the VEGF mRNA and miR-200b levels in patients with T2DM and to discover their relations with the clinicopathological and anatomical distribution of DFU. Methods: This case-control study enrolled 70 patients with T2DM and 70 healthy subjects as controls. Physical and neurological examination to assess Wagner classification. RT-PCR was done to assess VEGF mRNA and miR- 200b expression levels. Results: There were significantly higher values of VEGF mRNA levels in patients with DFU (3.13±1.87) compared to patients without DFU (2.41±0.197) and controls (1.07±0.363), with P value <0.001* Additionally, miR-200b levels were significantly higher in patients with DFU (3.48±1.47) compared to patients without DFU (2.94±0.187) and controls (1.13±0.37), with P value <0.001*. The power of VEGF mRNA and miR-200b levels as a diagnostic marker for DFU were projected (2.5 and 3.1, respectively), which yielded a sensitivity of (77.1% and 74.3, respectively) and a specificity of (77.3% and 75.2%), with the AUC at (0.886 and 0.845, respectively). The size of DFU in cm was 3.5±0.421 and the number of ulcers was 2.45±0.433. Conclusions: The VEGF mRNA and miR-200b levels were significantly higher in patients with T2DM, particularly those with DFU. Consequently, we believed that VEGF mRNA and miR-200b might serve as promising predictive biomarkers for diabetes and DFU. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the role of long non-coding RNA (lncRNA) small nucleolar RNA host gene 4 (SNHG4) in diabetic retinopathy (DR). We found that SNHG4 was downregulated in DR. SNHG4 could directly interact with miR-200b, while overexpression of miR-200b did not affect the expression of SNHG4 in human retinal pigment epithelial cells ARPE-19. In contrast, overexpression of SNHG4 led to the upregulation of oxidation resistance 1 (Oxr1), a target of miR-200b. Cell apoptosis analysis showed that overexpression of miR-200b increased the apoptotic rate of ARPE-19 cells under high glucose treatment. Oxr1 and SNHG4 played opposite roles and reduced the effects of overexpression of miR-200b. In conclusion, SNHG4 may sponge miR-200b to inhibit cell apoptosis in DR by upregulating Oxr1. [ABSTRACT FROM AUTHOR]

目的　探讨上皮性卵巢癌（EOC）患者血清miR-200a和miR-200b水平变化及意义。方法　选取EOC患者 80例（EOC 组）、卵巢良性病变患者 80例（良性对照组）、体检健康女性 80例（正常对照组），采集受试者血清，采用 qRT-PCR 法 检 测 miR-200a、miR-200b。比 较 三 组 血 清 miR-200a、miR-200b 水 平，分 析 EOC 患 者 血 清 miR-200a、 miR-200b水平与临床病理特征的关系，采用受试者工作特征（ROC）曲线评价二者对EOC的诊断价值。对EOC患者 行手术联合化疗方案进行治疗，术后随访2年，记录患者复发情况，根据miR-200a、miR-200b中位数将患者分为低水 平和高水平，比较二者的复发时间。结果 EOC组血清miR-200a和miR-200b水平较良性对照组和正常对照组升高 （P均<0. 01）。EOC晚期、合并淋巴结转移、腹水或腹腔灌洗液检出恶细胞患者的血清miR-200a、miR-200b水平分别 高于晚期、未合并淋巴结转移、腹水或腹腔灌洗液检出恶细胞患者（P 均<0. 05）。ROC 分析显示，血清 miR-200a （AUC=0. 890）和 miR-200b（AUC=0. 912）有助于 EOC 的诊断。在 EOC 患者中，血清 miR-200a（Log-Rank=4. 061，P= 0. 044）和 miR-200b（Log-Rank=5. 012，P=0. 025）高水平者的复发时间均短于低水平者。结论 EOC患者血清 miR200a和miR-200b水平升高，监测血清miR-200a和miR-200b水平有助于EOC的诊断和预后评估。 [ABSTRACT FROM AUTHOR]

Objective: To investigate the role of macrophage migration inhibition factor （MIF） in colon cancer and its possible regulatory mechanism. Methods: MIF expression in colon cancer tissues and para-carcinoma tissues was evaluated by qRT-PCR; expression of MIF in colon cancer cell lines HCT116 and a normal cell line HIEC-6 were detected by qRT-PCR and immunofluorescence. MIF expression was knocked down in vitro using si-RNA. Cell proliferative and migratory capacities were assessed by CCK-8, Transwell and scratch test, respectively. Long non-coding RNA, Zinc finger E-box binding homeobox 1 antisense 1 （lncRNA-ZEB1-AS1） expression was analyzed via qRT-PCR analysis. The interaction between lncRNA-ZEB1-AS1 and miR-200b were predicted by starBase software, and then verified by dual-luciferase reporter assay. Detection of HCT116 proliferation changes after overexpression of lncRNA-ZEB1-AS1 and silencing miR-200b. Results: MIF was significantly upregulated in colon cancer tissues. MIF has high expression in HCT116 cells while low expression in HIEC-6 cells. Furthermore, in vitro assays of silencing MIF inhibited HCT116 cell proliferation and migration ability, and MIF level were diminished in accordance with the MIF knockdown. starBase and dual-luciferase reporter assays suggested that lncRNA-ZEB1-AS1 inhibits the transcriptional activity of miR-200b promoter. Effects of decreased proliferation ability of colon cancer cells with knockdown of MIF could be significantly reversed by miR-200b silencing or lncRNAZEB1-AS1 overexpression. Conclusion: MIF may promote the proliferation and metastasis of colon cancer cells by activating the ZEB1-AS1/miR-200b signaling pathway. [ABSTRACT FROM AUTHOR]

Normal growth of granulosa cells (GCs) is essential for follicular development. miR-200b plays a vital role in litter size, estrous cycle, ovulation, and follicular development in sheep. However, it is unclear that the specific effect and regulatory mechanism of miR-200b on ovine GCs. miR-200b mimic inhibited GCs proliferation and induced cellular senescence through downregulating mitochondrial membrane potential (MMP), concentration of ATP and mitochondrial respiratory chain complex Ⅰ, and upregulating SA-β-gal positive rate and ROS production. A total of 597 differentially expressed genes were identified by RNA-Seq in GCs transfected with miR-200b mimic and mimic NC, and they were involved in cell cycle and cellular senescence. miR-200b directly targeted and downregulated MYBL2 and CDK1. Overexpression of MYBL2 promoted GCs proliferation and genes expression (CDK1 , CDC20 , MAD2L1 and FOXM1), which were suppressed by miR-200b mimic. Furthermore, MYBL2 negatively regulated miR-200b-induced GC senescence. In conclusion, miR-200b/MYBL2/CDK1 regulated proliferation and senescence through cell cycle pathway in ovine granulosa cells. Our study provides a novel insight that miR-200b regulates ovine follicular development. • miR-200b inhibits proliferation and induces cellular senescence of ovine granulosa cells. • miR-200b is involved in cell cycle and cellular senescence ovine granulosa cells through RNA-seq. • miR-200b directly targets MYBL2 and CDK1 by dual luciferase reporter assay. • miR-200b/MYBL2/CDK1 suppresses proliferation and induces senescence through cell cycle arrest in ovine GCs. [ABSTRACT FROM AUTHOR]

Each year, more than 790 000 people in the United States suffer from a stroke. Although progress has been made in diagnosis and treatment of ischemic stroke (IS), new therapeutic interventions to protect the brain during an ischemic insult is highly needed. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression post-transcriptionally. Growing evidence suggests that miRNAs have a profound impact on ischemic stroke progression and are potential targets of novel treatments. Notably, inflammatory pathways play an important role in the pathogenesis of ischemic stroke and its pathophysiologic progression. Experimental and clinical studies have illustrated that inflammatory molecular events collaboratively contribute to neuronal and glial cell survival, edema formation and regression, and vascular integrity. In the present review, we examine recent discoveries regarding miRNAs and their roles in post-ischemic stroke neuropathogenesis. [ABSTRACT FROM AUTHOR]

In this study, we conducted a systematic review and meta-analysis to summarize and evaluate the global research potential of different circulating miRNAs as an early diagnostic biomarker for OC. A systematic literature search for relevant studies was conducted in June 2020 and followed up in November 2021. The search was conducted in English databases (PubMed, ScienceDirect). The primary search resulted in a total of 1887 articles, which were screened according to the prior established inclusion and exclusion criteria. We identified 44 relevant studies, of which 22 were eligible for the quantitative meta-analysis. Statistical analysis was performed using the Meta-package in Rstudio. Standardized mean differences (SMD) of relative levels between control subjects and OC patients were used to evaluate the differential expression. All studies were quality evaluated using a Newcastle–Ottawa Scale. Based on the meta-analysis, nine miRNAs were identified as dysregulated in OC patients compared to controls. Nine were upregulated in OC patients compared to controls (miR-21, -125, -141, -145, -205, -328, -200a, -200b, -200c). Furthermore, miR-26, -93, -106 and -200a were analyzed, but did not present an overall significant difference between OC patients and controls. These observations should be considered when performing future studies of circulating miRNAs in relation to OC: sufficient size of clinical cohorts, development of consensus guidelines for circulating miRNA measurements, and coverage of previously reported miRNAs. [ABSTRACT FROM AUTHOR]

Abstract Background Long non-coding RNAs (lncRNAs) are implicated in the development of multiple cancers. In our previous study, we demonstrated that HDAC1/4-mediated silencing of microRNA-200b (miR-200b) enhances docetaxel (DTX)-resistance of human lung adenocarcinoma (LAD) cells. Methods and results Herein, we probed the function of LncRNA MARCKSL1–2 (MARCKSL1-transcript variant 2, NR_052852.1) in DTX resistance of LAD cells. It was found that MARCKSL1–2 expression was markedly reduced in DTX-resistant LAD cells. Through gain- or loss- of function assays, colony formation assay, EdU assay, TUNEL assay, and flow cytometry analysis, we found that MARCKSL1–2 suppressed the growth and DTX resistance of both parental and DTX-resistant LAD cells. Moreover, we found that MARCKSL1–2 functioned in LAD through increasing miR-200b expression and repressing HDAC1. Mechanistically, MARCKSL1–2 recruited the suppressor of zeste 12 (SUZ12) to the promoter of histone deacetylase 1 (HDAC1) to strengthen histone H3 lysine 27 trimethylation (H3K27me3) of HDAC1 promoter, thereby reducing HDAC1 expression. MARCKSL1–2 up-regulated miR-200b by blocking the suppressive effect of HDAC1 on the histone acetylation modification at miR-200b promoter. Furthermore, in vivo analysis using mouse xenograft tumor model supported that overexpression of MARCKSL1–2 attenuated the DTX resistance in LAD tumors. Conclusions We confirmed that MARCKSL1–2 alleviated DTX resistance in LAD cells by abolishing the inhibitory effect of HDAC1 on miR-200b via the recruitment of SUZ12. MARCKSL1–2 could be a promising target to improve the chemotherapy of LAD. Graphical abstract

Single-nucleotide polymorphisms (SNPs) of microRNAs (miRNAs) may alter miRNA transcription, maturation and target specificity, thus affecting stroke susceptibility. We aimed to investigate whether miR-200b and miR-495 SNPs may be associated with ischemic stroke (IS) risk and further explore underlying mechanisms including related genes and pathways. MiR-200b rs7549819 and miR-495 rs2281611 polymorphisms were genotyped among 712 large-artery atherosclerosis (LAA) stroke patients and 1,076 controls in a case–control study. Bioinformatic analyses were performed to explore potential association of miR-200b/495 with IS and to examine the effects of these two SNPs on miR-200b/495. Furthermore, we evaluated the association between these two SNPs and stroke using the public GWAS datasets. In our case–control study, rs7549819 was significantly associated with a decreased risk of LAA stroke (OR = 0.73, 95% CI = 0.58–0.92; p = 0.007), while rs2281611 had no significant association with LAA stroke risk. These results were consistent with the findings in East Asians from the GIGASTROKE study. Combined effects analysis revealed that individuals with 2–4 protective alleles (miR-200bC and miR-495 T) exhibited lower risk of LAA stroke than those with 0–1 variants (OR = 0.76, 95% CI = 0.61–0.96; p = 0.021). Bioinformatic analyses showed that miR-200b and miR-495 were significantly associated with genes and pathways related to IS pathogenesis, and rs7549819 and rs2281611 markedly influenced miRNA expression and structure. MiR-200b rs7549819 polymorphism and the combined genotypes of miR-200b rs7549819 and miR-495 rs2281611 polymorphisms were associated with decreased risk of LAA stroke in Chinese population. [ABSTRACT FROM AUTHOR]

Embryonic mortality is considered to be one of the main reasons for reduced conception rates in the cattle industry. Insufficient endometrial receptivity is a major factor resulting in embryo implantation failure and losses. Apoptosis of endometrial epithelial cells is an important process during establishment of uterine receptivity and embryo implantation. The aim of this study was to explore the role of bta-miR-200b on endometrial epithelial cell apoptosis in cattle. Overexpression of bta-miR-200b upregulated the expression of proapoptotic gene BCL2 associated X, apoptosis regulator (BAX) and endometrial receptivity marker gene osteopontin (OPN) at mRNA and protein level in bovine endometrial epithelial cells. Moreover, overexpression of bta-miR-200b was able to inhibit proliferation and promote apoptosis of bovine endometrial epithelial cells by arresting the cell cycle at the G0/G1 phase. MYB Proto-Oncogene (MYB) was verified to be a target of bta-miR-200b in bovine endometrial epithelial cells using dual-luciferase reporter assay. Transfection of bta-miR-200b mimics decreased the mRNA and protein expression of MYB. Overexpression of MYB decreased the effect of bta-miR-200b on apoptosis of bovine endometrial epithelial cells. Our findings suggest that bta-miR-200b can affect the apoptosis of endometrial epithelial cells in cattle by targeting the MYB gene. • Bta-miR-200b regulates the expression of proapoptotic gene BAX and endometrial receptivity marker gene OPN in bovine endometrial epithelial cells. • Bta-miR-200b inhibits proliferation and promote apoptosis of bovine endometrial epithelial cells by arresting the cell cycle at the G0/G1 phase. • MYB is a target gene of bta-miR-200b in bovine endometrial epithelial cells. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs) are implicated in the development of multiple cancers. In our previous study, we demonstrated that HDAC1/4-mediated silencing of microRNA-200b (miR-200b) enhances docetaxel (DTX)-resistance of human lung adenocarcinoma (LAD) cells. Methods and results: Herein, we probed the function of LncRNA MARCKSL1–2 (MARCKSL1-transcript variant 2, NR_052852.1) in DTX resistance of LAD cells. It was found that MARCKSL1–2 expression was markedly reduced in DTX-resistant LAD cells. Through gain- or loss- of function assays, colony formation assay, EdU assay, TUNEL assay, and flow cytometry analysis, we found that MARCKSL1–2 suppressed the growth and DTX resistance of both parental and DTX-resistant LAD cells. Moreover, we found that MARCKSL1–2 functioned in LAD through increasing miR-200b expression and repressing HDAC1. Mechanistically, MARCKSL1–2 recruited the suppressor of zeste 12 (SUZ12) to the promoter of histone deacetylase 1 (HDAC1) to strengthen histone H3 lysine 27 trimethylation (H3K27me3) of HDAC1 promoter, thereby reducing HDAC1 expression. MARCKSL1–2 up-regulated miR-200b by blocking the suppressive effect of HDAC1 on the histone acetylation modification at miR-200b promoter. Furthermore, in vivo analysis using mouse xenograft tumor model supported that overexpression of MARCKSL1–2 attenuated the DTX resistance in LAD tumors. Conclusions: We confirmed that MARCKSL1–2 alleviated DTX resistance in LAD cells by abolishing the inhibitory effect of HDAC1 on miR-200b via the recruitment of SUZ12. MARCKSL1–2 could be a promising target to improve the chemotherapy of LAD. [ABSTRACT FROM AUTHOR]

Abstract Nobiletin is a polymethoxylated flavone present in citrus fruits, which has been reported to have inhibitory effects on tumorigenesis of cancers. However, the biological function of nobiletin in breast cancer (BC) is largely unknown. To investigate the effect of nobiletin on growth of BC cells, the cell viability of BC was measured by MTT assay. In addition, gene and protein expressions were detected by qRT‐PCR and western blot, respectively. The apoptosis and pyroptosis of BC cells were tested by flow cytometry. Finally, the correlation between miR‐200b and JAZF1 was detected by dual luciferase report. The data indicated that nobiletin inhibited the proliferation of BC cells in a dose‐dependent manner. Moreover, miR‐200b mimics‐induced pyroptosis of BC cells was further increased by nobiletin. Meanwhile, JAZF1 was found to be the target of miR‐200b. Moreover, nobiletin induced apoptosis and pyroptosis of BC cells via miR‐200b/JAZF1/NF‐κB axis. In conclusion, nobiletin inhibited the tumorigenesis of BC via regulation of miR‐200b/JAZF1 axis. Thus, nobiletin might serve as a new agent for the treatment of BC.

Abstract Background Ovarian cancer is the seventh most common cancer in women and the second most reason of gynecologic cancer-related death. Growing evidence showed that exosomal miRNA plays a crucial role in the progression of ovarian cancer. Methods Exosomes were identified using nanoparticle tracking analysis, transmission electron microscopy and marker proteins detection. The levels of mRNA and proteins were ensured by qRT-PCR and western blot, respectively. Immunofluorescence, flow cytometry and ELISA assay were carried out to analyze macrophages polarization. CCK-8 and Transwell assay were used to measure the cell viability and invasion of ovarian cancer cells. The interaction of miR-200b and Kruppel like factor 6 (KLF6) was ensured by using luciferase reporter assay. Results Here, we obtained plasma-derived exosomes successfully, and proved that miR-200b was increased in the exosomes of ovarian cancer patients. Subsequently, our data showed that increasing of miR-200b could promote macrophage M2 polarization, but inhibit M1 polarization. miR-200b-overexpressed macrophages-conditioned medium notably enhanced the cell viability and invasion of ovarian cancer cells. Moreover, increasing of miR-200b inhibited KLF6 expression, while decreasing of miR-200b promoted KLF6 expression. Overexpression of KLF6 recused miR-200b-induced macrophage polarization toward M2, and the inhibitory effect of miR-200b on M1 polarization. Conclusions Overall, our results demonstrated that miR-200b was highly expressed in the plasma-derived exosome of ovarian cancer patients, and promoted the proliferation and invasion of ovarian cancer cells through inducing macrophage M2 polarization by suppressing KLF6 expression. Our results suggested that miR-200b might be a novel target for ovarian cancer treatment.

Changes in social preference of amphibian larvae result from sustained exposure to kinship odorants. To understand the molecular and cellular mechanisms of this neuroplasticity, we investigated the effects of olfactory system activation on neurotransmitter (NT) expression in accessory olfactory bulb (AOB) interneurons during development. We show that protracted exposure to kin or non-kin odorants changes the number of dopamine (DA)- or gamma aminobutyric acid (GABA)-expressing neurons, with corresponding changes in attraction/aversion behavior. Changing the relative number of dopaminergic and GABAergic AOB interneurons or locally introducing DA or GABA receptor antagonists alters kinship preference. We then isolate AOB microRNAs (miRs) differentially regulated across these conditions. Inhibition of miR-375 and miR-200b reveals that they target Pax6 and Bcl11b to regulate the dopaminergic and GABAergic phenotypes. The results illuminate the role of NT switching governing experience-dependent social preference. VIDEO ABSTRACT.

Purpose: We previously demonstrated that absence of miR-200b results in abnormal lung development in congenital diaphragmatic hernia due to imbalance between epithelial and mesenchymal cells. Tenascin C is a highly conserved extracellular matrix protein involved in epithelial to mesenchymal transition, tissue regeneration and lung development. Considering the involvement of Tenascin C and miR-200b and their potential interaction, we aimed to study Tenascin C during lung development in the absence of miR-200b. Methods: We collected lungs of miR-200b−/− mice (male, 8 weeks). We performed Western blot (WB) analysis (N = 6) and immunofluorescence (N = 5) for Tenascin C and alpha smooth muscle actin and RT-qPCR for Tenascin C gene expression (N = 4). Results: Using WB analysis, we observed a decreased total protein abundance of Tenascin C in miR-200b−/− lungs (miR-200b+/+: 3.8 × 107 ± 1 × 107; miR-200b−/−: 1.9 × 107 ± 5 × 106; p = 0.002). Immunofluorescence confirmed decreased total Tenascin C in miR-200b−/− lungs. Tenascin C was significantly decreased in the mesenchyme but relatively increased in the airways of mutant lungs. Total lung RNA expression of Tenascin C was higher in miR-200b−/− lungs. Conclusion: We report dysregulation of Tenascin C in lungs of miR-200b−/− mice. This suggests that absence of miR-200b results in abnormal Tenascin C abundance contributing to the lung hypoplasia observed in miR-200b−/− mice. [ABSTRACT FROM AUTHOR]

Abstract Human trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether trophoblast stem cell-derived exosomes (TSC-Exos) can protect cardiomyocytes from doxorubicin (Dox)-induced injury remains unclear. In the present study, TSC-Exos were isolated from the supernatants of human trophoblasts using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. In vitro, primary cardiomyocytes were subjected to Dox and treated with TSC-Exos, miR-200b mimic or miR-200b inhibitor. Cellular apoptosis was observed by flow cytometry and immunoblotting. In vivo, mice were intraperitoneally injected into Dox to establish a heart failure model. Then, different groups of mice were administered either PBS, adeno-associated virus (AAV)-vector, AAV-miR-200b-inhibitor or TSC-Exos via tail vein injection. Then, the cardiac function, cardiac fibrosis and cardiomyocyte apoptosis in each group were evaluated, and the downstream molecular mechanism was explored. TSC-Exos and miR-200b inhibitor both decreased primary cardiomyocyte apoptosis. Similarly, mice receiving TSC-Exos and AAV-miR-200b inhibitor exhibited improved cardiac function, accompanied by reduced apoptosis and inflammation. The bioinformatic prediction and luciferase reporter results confirmed that Zeb1 was a downstream target of miR-200b and had an antiapoptotic effect. TSC-Exos attenuated doxorubicin-induced cardiac injury by playing antiapoptotic and anti-inflammatory roles. The underlying mechanism could be an increase in Zeb1 expression by the inhibition of miR-200b expression. In summary, this study sheds new light on the application of TSC-Exos as a potential therapeutic tool for heart failure.

Abstract Background Recurrence and distant metastasis are still the main factors leading to treatment failure for malignant tumors including nasopharyngeal carcinoma (NPC). Therefore, elucidating the molecular mechanisms underlying nasopharyngeal carcinoma metastasis is of great clinical significance for targeted gene therapy and prognostic evaluation. PinX1, a tumor suppressor gene, was previously demonstrated to be a powerful tool for targeting telomerase in order to resist malignant tumor proliferation and migration. The aim of this study was to explore the mechanism through which PinX1 regulates epithelial–mesenchymal transition (EMT) and tumor metastasis in NPC and investigate its clinical significance and biological role with respect to disease progression. Methods Cell Counting Kit-8 (CCK8), Transwell assays, Colony formation analysis and Xenograft tumorigenicity assay were used to measure the nasopharyngeal CD133+ cancer stem cell proliferation, migration, and invasion abilities. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays were conducted to investigate the underlying mechanism that PinX1 inhibits cell proliferation, migration, and invasion via regulating EMT in nasopharyngeal CD133+ CSCs. Results We found that the overexpression of PinX1 and P53 inhibited cell proliferation, migration, and invasion, but that the inhibition of miR-200b blocked these effects, in nasopharyngeal CD133+ cancer stem cells (CSCs). Mechanistic investigations elucidated that PinX1 inhibits cell proliferation, migration, and invasion by regulating the P53/miR-200b-mediated transcriptional suppression of Snail1, Twist1, and Zeb1, consequently inhibiting EMT in nasopharyngeal CD133+ CSCs. Conclusions Our findings indicate that PinX1 inhibits cell proliferation, migration, and invasion via P53/miR-200b-regulated EMT in the malignant progression of human NPC, which might suggest novel clinical implications for disease treatment.

Objective To investigate the role of microRNA-200b (miR-200b) in corneal neovascularization (CNV) in rats and explore the possible mechanisms. Methods Twenty-four female SD rats were subjected to corneal suture to induce CNV in one eye, with the other eye as the normal control. On the 4th, 7th and 14th days after the suture, CNV was observed under a slit lamp, and the structure and inflammatory cell infiltration in the corneal tissue were observed using HE staining. On day 14 after the suture, the expression levels of miR-200b and vascular endothelial growth factor (VEGF) mRNA in the cornea were detected using qRT-PCR. Human umbilical vein endothelial cells (HUVECs) were transfected via liposome with a miR-200b mimic or a negative control sequence (miR-NC), and the cell proliferation, migration and tube formation abilities were evaluated using CCK-8 assay, Transwell chamber assay and Matrigel assay, respectively. The expression levels of VEGF and PIK3CA/AKT proteins in the cells were detected by Western blotting. Results CNV models were successfully established by corneal suture in the rats. HE staining showed that corneal suture resulted in disordered alignment of the corneal tissue and progressively increased inflammatory cell infiltration on days 4, 7 and 14 after the suture (P < 0.05). RT-PCR showed that miR-200b expression decreased and VEGF expression increased significantly in the corneal tissue on day 14 after the suture (P < 0.01). Compared with the cells without transfection or transfected with miR-NC, the HUVECs transfected with the miR-200b mimic exhibited significantly attenuated proliferation, migration and lumen formation abilities (P < 0.05) with also significantly lowered expression of VEGF, PIK3CA and p-AKT proteins (P < 0.05). Conclusion miRNA-200b can significantly inhibit the proliferation, migration and tube formation of HUVECs, and may participate in the occurrence and development of CNV by inhibiting the PIK3CA/AKT pathway.

Smoking is an important factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), which is commonly characterised by cellular senescence and inflammation. Recently, miR-200b has emerged as an important target to cure lung disease; however, the function of miR-200b in reducing cellular senescence and inflammatory responses has not been reported. In this study, we found that miR-200b was downregulated in the lungs of COPD model mice, and its expression is correlated with cellular senescence and inflammatory responses. We hypothesised that miR-200b may be a potential novel therapy for treating COPD. We performed senescence-Associated-β-galactosidase (SA-β-GAL) staining, western blot, qRT-PCR and ELISA; our data suggested that miR-200b is an anti-aging factor in the lungs that is involved in inflammatory responses. We also confirmed that ZEB2 (Zinc finger E-box binding homeobox 2) is a target gene of miR-200b using luciferase reporter assay. In addition, we verified the function of ZEB2 in cellular senescence and inflammatory responses through transfection experiments. Moreover, we found that the protective effects of miR-200b are inhibited when cells overexpress the ZEB2 protein. In conclusion, our results suggest that miR-200b may attenuate cellular senescence and inflammatory responses by targeting ZEB2 in pulmonary emphysema.

OBJECTIVE: The aim of this study is to investigate the clinical value of low-dose spiral CT (LDCT), plasma miR-200b, and miR-200c combined screening for lung cancer screening in the physical examination population. PATIENTS AND METHODS: From January 2016 to December 2018, the Physical Examination Center of our hospital underwent lowdose spiral CT lung cancer screening for 10,823 people aged =40 years. The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the relative expressions of miR-200b and miR-200c in plasma, analyze the imaging characteristics of suspicious nodules in the lung and the relative expressions of miR-200b and miR-200c in plasma. RESULTS: A total of 2,919 pulmonary nodules were detected in the 10823 physical examination population, with a total detection rate of 26.97%, including 1523 males and 1396 females. 1081 positive nodules were detected with a detection rate of 9.99%. According to the Lung-RADS classification, the number of type 2 nodules was the highest, with a detection rate of 22.13%. Meanwhile, the rate of type 3 nodules was 3.15%, and the rate of type 4 nodules was 1.69%. The sensitivity, accuracy, and negative predictive value of LDCT, miR-200b, and miR-200c in the diagnosis of lung cancer were significantly improved compared with the individual tests, which were 94.74%, 90.16%, and 95.88%, respectively. CONCLUSIONS: Low-dose spiral CT combined with plasma miR-200b and miR-200c for lung cancer screening in the physical examination population can help to detect lung cancer patients with early symptoms that are not significant, and achieve early diagnosis and early treatment. [ABSTRACT FROM AUTHOR]

Nobiletin is a polymethoxylated flavone present in citrus fruits, which has been reported to have inhibitory effects on tumorigenesis of cancers. However, the biological function of nobiletin in breast cancer (BC) is largely unknown. To investigate the effect of nobiletin on growth of BC cells, the cell viability of BC was measured by MTT assay. In addition, gene and protein expressions were detected by qRT‐PCR and western blot, respectively. The apoptosis and pyroptosis of BC cells were tested by flow cytometry. Finally, the correlation between miR‐200b and JAZF1 was detected by dual luciferase report. The data indicated that nobiletin inhibited the proliferation of BC cells in a dose‐dependent manner. Moreover, miR‐200b mimics‐induced pyroptosis of BC cells was further increased by nobiletin. Meanwhile, JAZF1 was found to be the target of miR‐200b. Moreover, nobiletin induced apoptosis and pyroptosis of BC cells via miR‐200b/JAZF1/NF‐κB axis. In conclusion, nobiletin inhibited the tumorigenesis of BC via regulation of miR‐200b/JAZF1 axis. Thus, nobiletin might serve as a new agent for the treatment of BC. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer is the seventh most common cancer in women and the second most reason of gynecologic cancer-related death. Growing evidence showed that exosomal miRNA plays a crucial role in the progression of ovarian cancer. Methods: Exosomes were identified using nanoparticle tracking analysis, transmission electron microscopy and marker proteins detection. The levels of mRNA and proteins were ensured by qRT-PCR and western blot, respectively. Immunofluorescence, flow cytometry and ELISA assay were carried out to analyze macrophages polarization. CCK-8 and Transwell assay were used to measure the cell viability and invasion of ovarian cancer cells. The interaction of miR-200b and Kruppel like factor 6 (KLF6) was ensured by using luciferase reporter assay. Results: Here, we obtained plasma-derived exosomes successfully, and proved that miR-200b was increased in the exosomes of ovarian cancer patients. Subsequently, our data showed that increasing of miR-200b could promote macrophage M2 polarization, but inhibit M1 polarization. miR-200b-overexpressed macrophages-conditioned medium notably enhanced the cell viability and invasion of ovarian cancer cells. Moreover, increasing of miR-200b inhibited KLF6 expression, while decreasing of miR-200b promoted KLF6 expression. Overexpression of KLF6 recused miR-200b-induced macrophage polarization toward M2, and the inhibitory effect of miR-200b on M1 polarization. Conclusions: Overall, our results demonstrated that miR-200b was highly expressed in the plasma-derived exosome of ovarian cancer patients, and promoted the proliferation and invasion of ovarian cancer cells through inducing macrophage M2 polarization by suppressing KLF6 expression. Our results suggested that miR-200b might be a novel target for ovarian cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Hypoxia is a key feature of breast cancer, which affects cancer development, metastasis and metabolism. Previous studies suggested that circular RNAs (circRNAs) could participate in cancer progression and hypoxia regulation. This study aimed to investigate the role of circRNA differentially expressed in normal cells and neoplasia domain containing 4C (circDENND4C) in breast cancer progression under hypoxia. Methods Forty-three patients with breast cancer were involved in this study. Breast cancer cell lines MDA-MB-453 and SK-BR-3 were cultured under hypoxia (1% O2) for experiments in vitro. The expression levels of circDENND4C, microRNA-200b (miR-200b) and miR-200c were measured by quantitative real-time polymerase chain reaction. Glycolysis was investigated by glucose consumption, lactate production and hexokinase II (HK2) protein level. Migration and invasion were evaluated via trans-well assay and protein levels of matrix metallopeptidase 9 (MMP9) and MMP2. The interaction between circDENND4C and miR-200b or miR-200c was explored by bioinformatics analysis, luciferase assay and RNA immunoprecipitation. Murine xenograft model was established to investigate the anti-cancer role of circDENND4C in vivo. Results circDENND4C highly expressed in breast cancer was up-regulated in response to hypoxia. Knockdown of circDENND4C decreased glycolysis, migration and invasion in breast cancer cells under hypoxia. circDENND4C was validated as a sponge of miR-200b and miR-200c. Deficiency of miR-200b or miR-200c reversed the suppressive effect of circDENND4C knockdown on breast cancer progression. Moreover, silence of circDENND4C reduced xenograft tumor growth by increasing miR-200b and miR-200c. Conclusion circDENND4C silence suppresses glycolysis, migration and invasion in breast cancer cells under hypoxia by increasing miR-200b and miR-200c.

Benign esophageal strictures (BESs) frequently results from esophageal fibrosis. The transformation of fibroblasts into fibrocyte is an important cause of fibrosis. The treatment of fibrosis is challenging. Some previous studies have indicated the antifibrotic effect of mitomycin C (MMC). However, the mechanism of action of MMC and its optimal dose for treatment remains unclear. In the present study, the role of MMC in fighting fibrosis and its mechanism was investigated. Human esophageal fibroblast cells (HEFs)were treated without or with MMC, at 2, 5, 10 μg/ml, combining with mimic lncRNA-ATB, miR-200b inhibitor, rapamycin (RAPA), and 3-Methyladenine (3-MA). The cell viability, and cell apoptosis were evaluated. In addition, expression of apoptosis related proteins (caspase8 and caspase3), autophagy related proteins (LC3II and ATG5) and fibrosis related proteins (α-SMA collagen-1 and TGF-β) were also evaluated. Furthermore, autophagosome was observed by transmission electron microscope. Results showed that the expression of lncRNA-ATB was down-regulated and miR-200b was up-regulated after treated with MMC. And MMC induced cell apoptosis and inhibited cell autophagy. On the other hand, RAPA, mimic lncRNA-ATB and miR-200b inhibitor reduced fibrogenic effect of MMC on HEFs. Collectively, this study suggests that MMC inhibited esophageal fibrosis by regulating cell apoptosis and autophagy via downregulating lncRNA-ATB and upregulating miR-200b.

Objective: Patients with diabetes mellitus frequently have chronic wounds or diabetic ulcers as a result of impaired wound healing, which may lead to limb amputation. Human umbilical vein endothelial cell (HUVEC) dysfunction also delays wound healing. Here, we investigated the mechanism of miR-200b in HUVECs under high glucose conditions and the potential of miR-200b as a therapeutic target. Materials and Methods: In this experimental study, HUVECs were cultured with 5 or 30 mM glucose for 48 hours. Cell proliferation was evaluated by CCK-8 assays. Cell mobility was tested by wound healing and Transwell assays. Angiogenesis was analyzed in vitro Matrigel tube formation assays. Luciferase reporter assays were used to test the binding of miR-200b with Notch1. Results: miR-200b expression was induced by high glucose treatment of HUVECs (P<0.01), and it significantly repressed cell proliferation, migration, and tube formation (P<0.05). Notch1 was directly targeted and repressed by miR-200b at both the mRNA and protein levels. Inhibition of miR-200b restored Notch1 expression (P<0.05) and reactivated the Notch pathway. The effects of miR-200b inhibition in HUVECs could be reversed by treatment with a Notch pathway inhibitor (P<0.05), indicating that the miR-200b/Notch axis modulates the proliferation, migration, and tube formation ability of HUVECs. Conclusion: Inhibition of miR-200b activated the angiogenic ability of endothelial cells and promoted wound healing through reactivation of the Notch pathway in vitro. miR-200b could be a promising therapeutic target for treating HUVEC dysfunction. [ABSTRACT FROM AUTHOR]

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3′-untranslated region (3′-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10 ng/ml, 12 h). AMTN and inhibitor of kappa-B kinase beta (IKKβ) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3′-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3′-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKβ mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3′-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKβ inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKβ mRNAs in the human gingival epithelial cells. [ABSTRACT FROM AUTHOR]

Epithelial to mesenchymal transition (EMT) is a process associated with cancer malignancy and metastases. Cells undergoing EMT lose their epithelial phenotype and acquire mesenchymal phenotype. This process is accompanied by several molecular changes such as decrease of E-cadherin and increase of N-cadherin which is called the “cadherin swich”.MicroRNAs (miRNAs, miRs) are small non-coding RNAs having ability to regulate genes post-transcriptionally. Nowadays they are believed to take part in multiple physiological and pathological processes including cancer development.Comparison between TargetScan7 (www.targetscan.org) results for miR-200b and metanalysis of genes involved in EMT showed that miR-200b has a potential binding site in 60 genes that are involved in EMT (the majority of them were associated with mesenchymal phenotype). Our review summarizes literature findings contributing to experimentally proven interactions between miR-200b and genes involved in EMT process including cell receptors, signaling pathways, cell cycle or cell adhesion. The results of those interactions indicate that miR-200b may have an inhibitory impact on EMT or even in selected cases is able to restore epithelial phenotype.

Smoking is an important factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), which is commonly characterised by cellular senescence and inflammation. Recently, miR-200b has emerged as an important target to cure lung disease; however, the function of miR-200b in reducing cellular senescence and inflammatory responses has not been reported. In this study, we found that miR-200b was downregulated in the lungs of COPD model mice, and its expression is correlated with cellular senescence and inflammatory responses. We hypothesised that miR-200b may be a potential novel therapy for treating COPD. We performed senescence-Associated-β-galactosidase (SA-β-GAL) staining, western blot, qRT-PCR and ELISA; our data suggested that miR-200b is an anti-aging factor in the lungs that is involved in inflammatory responses. We also confirmed that ZEB2 (Zinc finger E-box binding homeobox 2) is a target gene of miR-200b using luciferase reporter assay. In addition, we verified the function of ZEB2 in cellular senescence and inflammatory responses through transfection experiments. Moreover, we found that the protective effects of miR-200b are inhibited when cells overexpress the ZEB2 protein. In conclusion, our results suggest that miR-200b may attenuate cellular senescence and inflammatory responses by targeting ZEB2 in pulmonary emphysema. [ABSTRACT FROM AUTHOR]

Downregulation of microRNA-200b (miR-200b) has been identified in a range of cancers, yet the specific mechanisms whereby it influences lung cancer growth require further exploration. We determined that lung cancer patient tumor samples exhibit decreased miR-200b expression, and we further found this miRNA to inhibit tumor growth via interfering with ERK1/2 and AKT signaling, targeting p70S6K1 to suppress HIF-1α expression. This miRNA further rendered H1299 cells more sensitive to cisplatin while impairing their proliferative and invasive potential through its ability to target and inhibit the activity of p70S6K1. These results were further confirmed in a murine xenograft model in which miR-200b also inhibited the growth of tumor and suppressed p70S6K1, p-AKT, p-ERK1/2, and HIF-1α expression. These findings clearly demonstrate a role for miR-200b in suppressing lung cancer development, making it a potentially relevant target for future diagnostic and therapeutic interventions.

Secreted microRNAs (miRNAs) reside in a complex regulatory network with extracellular matrix (ECM) macromolecules, which affect cell-cell communication, therefore miRNA expression highlights its significance in several aspects of human diseases, including cancer. miRNA-mediated regulation of breast cancer has received considerable attention due to evidence that shows miRNAs to mediate estrogen receptor (ER) status, metastasis, chemoresistance and epithelial-to-mesenchymal transition (EMT). miR-200b is a pluripotent miRNA, which is inversely regulated by ERα and ERβ in mammary cancer. It has been identified as tumor suppressor and EMT inhibitor serving as a critical biomarker, as its expression in breast tumor determines the disease-free survival, thus highlighting its roles in breast cancer invasion and metastasis. The main goal of this study was to investigate the role of miR-200b in modulating the behavior of breast cancer cells with different ER status. We demonstrate that estrogen signaling through ERs reduces miR-200b expression levels in ERα-positive breast cancer cells. Moreover, miR-200b upregulation reduces the aggressive phenotype of ERβ-positive breast cancer cells by inhibiting cell invasiveness and motility, followed by ECM reorganization as well as cytoskeletal and morphological changes concluded from deep inspection of cell topography. Future investigation towards the mechanistic perspective of miR-200b effects in the behavior of aggressive mammary cancer cells appears rewarding in order to expand our understanding of miR-200b as a novel mediator beyond breast cancer diagnosis and pharmaceutical targeting.

Abstract Background Our previous studies indicated that miR-200b inhibits the growth of androgen-independent prostate cancer (AIPC) cells. In this study, we employed quantitative proteomics techniques to unravel the role of miR-200b in AIPC. Methods miR-200b was over-expressed or inhibited by transfection with miR-200b mimics or miR-200b inhibitor in PC3 cells. Total proteins were collected and the profiles of different groups were analyzed by label-free proteomics. PANTHER was applied to analyze biological processes, molecular functions and pathways of proteins regulated by miR-200b. miRBase was used to evaluate target genes of miR-200b in proteins regulated by miR-200b. Results Thirteen proteins were up-regulated in miR-200b mimics/mimics NC (negative miRNA control) and 14 proteins were down-regulated; 67 proteins were up-regulated in miR-200b inhibitor/inhibitor NC and 98 proteins were down-regulated. There were seven proteins which were both down-regulated by miR-200b mimics and up-regulated by miR-200b inhibitor, TM4SF1, YAP1, PPP1R2, MARCKS, RTN4, GLIPR2 and SUCLG1. Among these, TM4SF1, YAP1, PPP1R2, MARCKS, RTN4 were predicted as target genes of miR-200b by miRBase, while GLIPR2 and SUCLG1 were not. Conclusion This work identified several target genes of miR-200b by label free proteomics method, i.e., TM4SF1, YAP1, PPP1R2, MARCKS, RTN4, GLIPR2, and SUCLG1. The signaling pathways regulated by these proteins such as Hippo signaling may contribute to the phenotype resulting from miR-200b.

Asthma is one of the most frequent and serious diseases worldwide. Inflammation has been reported to correlate with airway remodeling, which is critical for the progression of asthma. Better understanding of novel molecules modulating asthma and the underlying mechanism will benefit explorations of new treatments. Method: To explore the role of miR-200a and miR-200b in asthma, miR-200a mimics/inhibitor and miR-200b mimics/inhibitor were employed in A549 cells, respectively. Expression levels of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1β, were measured by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). A dual luciferase reporter assay was performed to identify whether miR-200a/200b directly bound to Orosomucoid 1-like 3 (ORMDL3). ERK, p-ERK and MMP-9, involved in downstream pathways of ORMDL3, were detected using qRT-PCR and western blotting. Results: MiR-200a/200b silencing significantly increased the expression of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1β, in A549 cells. ORMDL3 was the target gene of miR-200a/200b, with high expression levels in miR-200a inhibitor and miR-200b inhibitor groups. MiR-200a and miR-200b played synergistic roles in the regulation of the inflammatory effect in A549 cells. Expression levels of p-ERK and MMP-9 were significantly increased in miR-200a inhibitor and miR-200b inhibitor groups and were rescued by ERK inhibitor and MMP-9 inhibitor, respectively. Conclusion: These findings suggest that miR-200a and miR-200b are required to regulate asthma inflammation. Reduction in miR-200a/200b promotes the development of asthma inflammation by targeting ORMDL3 to activate the ERK/MMP-9 pathway. Therefore, elevating miR-200a and miR-200b and decreasing ORMDL3 might be potential strategies for inhibition of the asthma process. [ABSTRACT FROM AUTHOR]

This study aimed to investigate OIP5-AS1 effects on microangiopathy in diabetic mouse. The expression levels of OIP5-AS1, miR-200b, and ACE2 expression were measured by RT-qPCR. Western blot was conducted to detect The ACE2 and Ang-(1-7) expression. Luciferase reporter assays were used to identify the interaction between miR-200b and OIP5-AS1 or ACE2. Morris water maze test was performed for detecting cognitive function. Our results indicated that diabetic mice exhibited much lower OIP5-AS1 expression in the hippocampus than normal mice. Diabetic mice of OIP5-AS1 KO group showed remarkably lower OIP5-AS1 expression in the hippocampus, longer escape latency and lower percentage of CD31+ cells in the hippocampusthan those of WT group. OIP5-AS1 knockdown directly up-regulated miR-200b expression and ACE2 was directly inhibited by miR-200b. Relative to normal mice, diabetic mice had markedly higher miR-200b expression and lower ACE2 expression in the hippocampus. Diabetic mice of OIP5-AS1 KO group were with obviously higher miR-200b expression and lower ACE2 expression in the hippocampus than those of WT group. Compared with diabetic mice of OIP5-AS1 KO group, those of WT group, OIP5-AS1 KO + miR-200b inhibitor group and OIP5-AS1 KO + ACE2 group had obviously shorter escape latency and higher percentage of CD31+ cells and more caspase-3 protein expression in the hippocampus. OIP5-AS1 attenuated microangiopathy in diabetic mouse by regulating miR-200b/ACE2. [ABSTRACT FROM AUTHOR]

The right ventricle (RV) enlargement and pulmonary fibrosis are involved in cor pulmonale. The role of miR‐200b in cor pulmonale is less well understood. This study was designed to evaluate the regulatory roles of miR‐200b in cor pulmonale. Cor pulmonary mouse model was built via monocrotaline injection of monocrotaline (MCT). The expression of miR‐200b in the lungs, RV and left ventricle (LV) are using real‐time polymerase chain reaction. The transthoracic echocardiography was employed to determine the effects of miR‐200b mimics and Gö6976 injection on MCT mice. The protein levels of protein kinase C α (PKCα), collagen, and fibronectin in the lung, RV, and LV in the mice with and without miR‐200b mimics and Gö6976 injection were evaluated using western blot. The expression of miR‐200b decreased in MCT mice, while there was no difference in LV. Both the miR‐200b mimics and Gö6976 injection reversed the muscularization in the pulmonary artery, reversed RV hypertrophy, reduced RV systolic pressure, wall thickness and pulmonary fibrosis. The injection of miR‐200b can reduce the PKCα expression in the lung, RV, and LV. This study confirmed the down‐regulation of miR‐200b in cor pulmonale. The reverse effects of miR‐200b in the present study may provide a potential tool for cor pulmonary treatment. [ABSTRACT FROM AUTHOR]

Background: Recurrence and distant metastasis are still the main factors leading to treatment failure for malignant tumors including nasopharyngeal carcinoma (NPC). Therefore, elucidating the molecular mechanisms underlying nasopharyngeal carcinoma metastasis is of great clinical significance for targeted gene therapy and prognostic evaluation. PinX1, a tumor suppressor gene, was previously demonstrated to be a powerful tool for targeting telomerase in order to resist malignant tumor proliferation and migration. The aim of this study was to explore the mechanism through which PinX1 regulates epithelial–mesenchymal transition (EMT) and tumor metastasis in NPC and investigate its clinical significance and biological role with respect to disease progression. Methods: Cell Counting Kit-8 (CCK8), Transwell assays, Colony formation analysis and Xenograft tumorigenicity assay were used to measure the nasopharyngeal CD133+ cancer stem cell proliferation, migration, and invasion abilities. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays were conducted to investigate the underlying mechanism that PinX1 inhibits cell proliferation, migration, and invasion via regulating EMT in nasopharyngeal CD133+ CSCs. Results: We found that the overexpression of PinX1 and P53 inhibited cell proliferation, migration, and invasion, but that the inhibition of miR-200b blocked these effects, in nasopharyngeal CD133+ cancer stem cells (CSCs). Mechanistic investigations elucidated that PinX1 inhibits cell proliferation, migration, and invasion by regulating the P53/miR-200b-mediated transcriptional suppression of Snail1, Twist1, and Zeb1, consequently inhibiting EMT in nasopharyngeal CD133+ CSCs. Conclusions: Our findings indicate that PinX1 inhibits cell proliferation, migration, and invasion via P53/miR-200b-regulated EMT in the malignant progression of human NPC, which might suggest novel clinical implications for disease treatment. [ABSTRACT FROM AUTHOR]

Background/aim: Colon cancer-associated transcript 2 (CCAT2) is a new lncRNA, which is closely associated with risk of several cancers. The aim of this study was to explore the regulatory mechanism of CCAT2 in osteosarcoma (OSA). Methods: Cells were transfected with si-CCAT2, microRNA (miR)-200b inhibitor and the corresponding controls. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the expression of CCAT2 and miR-200b in OSA tissues and cell lines. CCK8 and bromodeoxyuridine (BrdU) were conducted to examine cell proliferation. Apoptosis was detected by PI/FITC-Annexin V combining with flow cytometric analysis. Migration and invasion were respectively measured through transwell chambers assays. Western blot was used to examine expressions of relative proteins. Results: CCAT2 was highly expressed and miR-200b was lowly expressed in OSA tissues and cell lines. Knockdown of CCAT2 suppressed cell proliferation, migration and invasion but induced apoptosis and up-regulation of miR-200b. miR-200b inhibitor weakened the effect of si-CCAT2 on cell progression and cell mobility. Besides, knockdown of CCAT2 blocked the PI3K/Akt and AMPK pathways through up-regulating miR-200b. Conclusions: The CCAT2/miR-200b/vascular endothelial growth factor (VEGF) axis plays important regulating effect in OSA through the PI3K/Akt and AMPK pathways. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are crucial regulatory molecules in lipid deposition and metabolism. However, the effect of miR-200b on the regulation of proliferation and adipogenesis of ovine preadipocytes is unknown in the sheep (Ovis aries). In this study, the expression profiles of miR-200b were investigated in the seven tissues of Tibetan ewes and differentiated preadipocytes. The effect of miR-200b, as well as its target genes p27 and KLF9, on the proliferation of ovine preadipocytes and adipogenesis was also investigated, using cell viability analysis, EdU staining, Oil Red O staining and reverse transcription-quantitative PCR (RT-qRCR). The miR-200b was expressed in all the tissues investigated, and it was highly expressed in lung, liver, subcutaneous adipose and spleen tissues. The expression of miR-200b continuously decreased when the differentiation of ovine preadipocytes initiated. The miR-200b mimic dramatically accelerated the proliferation but inhibited differentiation of ovine preadipocytes. The miR-200b inhibitor resulted in an opposite effect on the proliferation and differentiation of ovine preadipocytes. The dual luciferase reporter assay results showed that miR-200b mimic significantly decreased the luciferase activity of p27 and KLF9 in HEK293 cells transfected with wild-type dual luciferase reporter vectors. This suggests that p27 and KLF9 are the target genes of miR-200b. In over-expressed-p27 preadipocytes, the number of EdU-labeled preadipocytes and the expression levels of proliferation marker genes CDK2, CDK4, CCND1 and PCNA significantly decreased. In addition, the transfection of over-expressed-KLF9 vector into adipocytes remarkably increased the accumulation of lipid droplets and the expression levels of differentiation marker genes aP2, PPARγ, LPL and GLUT4. These results suggest that miR-200b accelerated the proliferation but inhibited the adipogenic differentiation of ovine preadipocytes by targeting p27 and KLF9, respectively.

Abstract Background Cholangiocarcinoma (CCA) is characterized by early lymphatic, metastasis, and low survival rate. Epithelial-mesenchymal transition (EMT) is able to induce tumor metastasis. Although the TGF-β/miR-200 signals promote EMT in various types of cancer, the regulatory mechanism in CCA is still unclear. Methods Expression of miR-200b, TGF-β, and EMT markers were measured in tumor samples and cell lines by qRT-PCR and western blot. CCK8 assay was performed to measure the cell viability. Transwell assay was used to evaluate migration and invasion. The target genes of miR-200b and transcription factor of TGF-β were analyzed using dual-luciferase reporter system. Results We have demonstrated that CCA exhibited remarkable EMT phenotype and miR-200b was reduced in CCA patients (n = 20) and negatively correlated to TGF-β. Moreover, two CCA cells, HCCC, and RBE, with epithelial appearances treated with TGF-β, showed fibroblastic-like cell morphology with downregulated miR-200b expression. Forced expression of miR-200b abrogated TGF-β-induced EMT initiation, with decreased cell proliferation, migration, and invasion in vitro. Also, TFAP2A (encode AP-2α) and MAPK7 were found to be targeted by miR-200b to downregulate EMT and AP-2α inhibited miR-200b by directly promoting transcription of TGFB1. Overexpression of MAPK7 significantly reversed miR-200b-induced inhibition of EMT, migration, and proliferation by increasing the expression of TGF-β, cyclin D1, and Cdk2. Further, the administration of miR-200b induced a remarkably tumor regression in vivo and reduced the effect of TGF-β-related EMT in AP-2α and MAPK7-dependent manner. Conclusions Our study highlights that miR-200b-based gene therapy is effective in the treatment of CCA.

Background: MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. Methods: CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. Results: ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. Conclusions: Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b.

Abstract Background Recent studies have verified that long noncoding RNAs (lncRNAs) involved in many biological functions and play crucial roles in human cancers progression, the study aimed to detect the association between long non-coding RNA HOXA11-AS and epithelial–mesenchymal transition (EMT) process in non-small cell lung cancer (NSCLC). Methods The lncRNA HOXA11-AS expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assays in 78 paired of tumor tissue and adjacent normal tissue samples in NSCLC patients. Kaplan–Meier survival curves and log-rank test was used to examine the association between lncRNA HOXA11-AS expression and the over survival time in NSCLC patients. Transwell invasion assay was performed to detect the cell invasion ability. QRT-PCR and western-blot analysis detected the mRNA and protein expression of EMT related transcription factors ZEB1/ZEB2, Snail1/2 and EMT marker E-cadherin and N-cadherin in NSCLC cells. RIP and Chromatin immunoprecipitation assays were performed to analyze the association between lncRNA HOXA11-AS and miR-200b expression in NSCLC cells. Results The lncRNA HOXA11-AS expression levels were significantly higher in NSCLC tissues compared with adjacent normal tissues and higher HOXA11-AS expression levels had a poor prognosis in NSCLC patients. Furthermore, knockdown of lncRNA HOXA11-AS in A549 and H1299 cells dramatically inhibited cell invasive abilities. Besides, the transcription levels and protein levels of EMT related transcription factors ZEB1/ZEB2, Snail1/2, and EMT maker N-cadherin were down-regulated after lncRNA HOXA11-AS was knocked down, but the mRNA and protein expression levels of EMT maker E-cadherin was increasing in A549 and H1299 cells. The mechanistic findings showed demonstrated that HOXA11-AS interacted with EZH2 and DNMT1 and recruited them to the miR-200b promoter regions to repress miR-200b expression in NSCLC cells, which promoted cell EMT in NSCLC. Conclusions Our results showed that up-regulation of lncRNA HOXA11-AS predicted a poor prognosis and lncRNA HOXA11-AS promoted cell epithelial–mesenchymal transition (EMT) by inhibiting miR-200b expression in NSCLC.

Background/Aims: Renal tubular epithelial-mesenchymal transition (EMT) is regarded as an important factor leading to renal interstitial fibrosis. Erythropoietin (EPO) has been reported to attenuate renal fibrosis. The mechanism underlying this protective effect of EPO remains unclear. In this study, we aim to identify possible mechanisms of the EPO renoprotective effect. Methods: Hypoxia was induced in vitro by incubating human proximal tubular epithelial cell line HK-2 cells in 1% O2 and 5% CO2. Western blotting and reverse transcription polymerase chain reaction analyses were used to evaluate the expression of epithelial and mesenchymal markers in the cell samples. The expression of miR-200b in the HK-2 cells under hypoxia or treatment with EPO was examined. Results: EPO represses hypoxia-induced EMT by upregulating miR-200b in HK-2 cells. Overexpression of miR-200b represses the effect of ETS proto-oncogene 1 (Ets-1)-induced EMT in HK-2 cells. Conclusion: miR-200 mediates the protective effects of EPO on EMT in hypoxic HK-2 cells. EPO attenuated hypoxia-induced EMT by increasing miR-200 expression via the repression of Ets-1.

Aim: To investigate the diagnostic potential of the miR-200 family for early detection in non-small cell lung cancer (NSCLC). Materials & methods: A systematic search was conducted of PubMed, Embase and Web of Science databases to identify studies of the miR-200 family in NSCLC. Sixteen studies meeting the inclusion criteria were included in the analysis with a total of 20 cohorts. Results: The combined sensitivity and specificity reached 73% and 85%, with an area under the curve of 0.83. Notably, miR-200b introduced heterogeneity. Subgroup analysis highlighted miR-200a and miR-141 as more sensitive, while blood-derived miRNAs showed slightly lower accuracy. Conclusion: The miR-200 family, predominantly assessed in blood, exhibits significant diagnostic potential for NSCLC, especially in distinguishing it from benign diseases.

Supplemental Digital Content is available in the text Objective: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). Background: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. Methods: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. Results: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-β signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. Conclusions: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH. [ABSTRACT FROM AUTHOR]

Background: To determine the involvement of MicroRNA-200b (miR-200b) in human Tenon's fibroblasts (HTFs) fibrosis. Methods: HTFs were treated with 10 ng/ml TGF-β1 for 24 h. Expression of α-smooth muscle actin (α-SMA), Fibronectin and Collagen1A1 (COL1A1) was assessed by western blot and immunofluorescence. MiR-200b was detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and its potential target genes were validated by luciferase assay. The effects of miR-200b and PTEN (the target gene) on HTFs fibrogenesis were evaluated using miR-200b mimics or inhibitor transfected HTFs, and confirmed with PTEN-specific small interfering RNA (siRNA) and PTEN inhibitor. Results: TGF-β1 up-regulated miR-200b and promoted the expression of α-SMA, Fibronectin and COL1A1 in HTFs, and the effects could be strengthened or hampered by adding miR-200b mimics or inhibitor, respectively. Luciferase assay identified PTEN as the target gene of miR-200b, and decreased PTEN level due to up-regulation of miR-200b was found to correlate with intensified expression of α-SMA, Fibronectin and COL1A1. Determination of PTEN's role in fibrosis was carried out by adding PTEN specific siRNA or PTEN inhibitor into HTFs, which led to intensified expression of fibrosis-related proteins. Conclusion: TGF-β1-mediated miR-200b induces HTFs fibrosis through suppressing PTEN signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract MicroRNAs (miRNAs) may regulate diverse biological processes and play an important role in cancer. And MiRNAs have been proposed as a useful tool for lung cancer diagnosis and therapeutics in cancer. The purpose of the present study was to investigate the association among the expression level of mature miR-200b-5p in peripheral blood and the risk of lung cancer and clinic pathological characteristics. This case-control study included 24 patients with lung cancer and 12 healthy controls. MiR-200b expression was deleted using real-time PCR. and the miR-200b expression of normal controls was significantly higher than that in lung cancer patients (1732.13 pg/mL vs 881.67 pg/mL, P < 0.05), no difference with age, sex, tissue type and clinical stage of lung cancer patients (P > 0.05). Furthermore, miR-200b expression level fluctuated with tumor progression in lung cancer, and there was highly significant for clinical stage II compared with the clinical stage III (P < 0.05). In addition, the down-regulation of miR-200b showed a highly discriminative receiver operating characteristic (ROC) curve profile, clearly distinguishing cancer patients from cancer-free subjects with an area under the ROC curve (AUROC) of 0.87. The detection of miR-200b expression yielded 83.30% sensitivity and 100.00% specificity in the diagnosis of lung cancer. Therefore, these findings suggested that miR-200b may be used as a marker for the detection and diagnosis of lung cancer in peripheral blood. [ABSTRACT FROM AUTHOR]

Objective: MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).Materials and methods: Total RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.Results: IL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.Conclusions: These results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue. [ABSTRACT FROM AUTHOR]

Triple‐negative breast cancer (TNBC) is the most aggressive breast cancer subtype with a poor prognosis. The microRNA‐200 (miR‐200) family has been associated with breast cancer metastasis. However, the epigenetic mechanisms underlying miR‐200b repression in TNBC are not fully elucidated. In this study, we found that MYC proto‐oncogene, bHLH transcription factor (MYC) and DNA methyltransferase 3A (DNMT3A) were highly expressed in TNBC tissues compared with other breast cancer subtypes, while miR‐200b expression was inhibited significantly. We demonstrated that MYC physically interacted with DNMT3A in MDA‐MB‐231 cells. Furthermore, we demonstrated that MYC recruited DNMT3A to the miR‐200b promoter, resulting in proximal CpG island hypermethylation and subsequent miR‐200b repression. MiR‐200b directly inhibited DNMT3A expression and formed a feedback loop in TNBC cells. MiR‐200b overexpression synergistically repressed target genes including zinc‐finger E‐box‐binding homeobox factor 1, Sex determining region Y‐box 2 (SOX2), and CD133, and inhibited the migration, invasion and mammosphere formation of TNBC cells. Our findings reveal that MYC can collaborate with DNMT3A on inducing promoter methylation and miR‐200b silencing, and thereby promotes the epithelial to mesenchymal transition and mammosphere formation of TNBC cells. [ABSTRACT FROM AUTHOR]