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Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) transport and transmit intercellular information and play an essential role in physiological and pathological processes. MSC-EVs, MSC-EVs-microRNA, and genetically modified MSC-EVs are involved in the onset and progression of different liver diseases and play a role in reducing liver cell damage, promoting liver cell regeneration, inhibiting liver fibrosis, regulating liver immunity, alleviating liver oxidative stress, inhibiting liver cancer occurrence, and others. Hence, it will replace MSCs as a research hotspot for cell-free therapy. This article reviews the research progress of MSC-EVs in liver diseases and provides a new basis for cell-free therapy of clinical liver diseases.

Objective: Acute myocardial infarction (AMI) is a serious cardiovascular disease with a high incidence worldwide and the main cause of sudden cardiac death. The aim of this article was to study the protective role of miR-335 in myocardial infarction (MI) and the underlying molecular mechanism., Materials and Methods: Thirty Sprague Dawley (SD) rats were randomly divided into sham group, MI + NC group and MI + agomiR-335 group. The expression of miR-335 in rat myocardium was detected by quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Western blot was performed to detect the expression of TNF-α, IL-6, IL-1β, Caspase-3, Cleaved Caspase-3 (C-Caspase-3) and MAP3K2 in rat myocardium. On the 7th day of the establishment of the rat MI model, a high-resolution small animal ultrasound system was utilized to detect the cardiac function of the rats, and the heart tissues and blood samples of the rats were collected. The corresponding kits were purchased to detect the contents of LDH, CK-MB, MDA and SOD in rat serum, and HE staining was employed to observe the morphology of rat myocardial tissue., Results: The expression of miR-335 in myocardial infarcted zones and border zones of MI rats decreased significantly. The upregulation of miR-335 remarkably inhibited myocardial inflammation and apoptosis after MI, thus improving the cardiac function of MI rats. Compared with the sham group, the MAP3K2 expression in the MI + NC group was observably increased, while the overexpression of miR-335 could inhibit the expression of this protein. Through the Luciferase reporter gene experiment, we found that miR-335 could directly target MAP3K2., Conclusions: The expression of miR-335 decreased in myocardial tissue after MI, and the overexpression of miR-335 reduced myocardial damage by inhibiting oxidative stress, inflammation, and apoptosis via targeting MAP3K2, thereby improving the cardiac function of MI rats.

Objective: This study aimed to explore the effect of long non-coding RNA (LncRNA) LncOGD-1006 to ischemic stroke and the possible mechanism., Materials and Methods: The primary brain microvascular endothelial cells (bEnd.3) of oxygen-glucose deprivation (OGD) was used as a mimic of ischemic stroke in vitro., Results: The results showed that LncOGD-1006 was upregulated in bEnd.3 after OGD-induced., Conclusions: LncOGD-1006 might act as a ceRNA to inhibit apoptosis in bEnd.3 cells by targeting miR-184-5p/CAAP1 pathway.

1. The Tibetan chicken, which is an indigenous breed living on the Tibetan Plateau, exhibits hypoxic adaptations to its high-altitude environment. However, the molecular mechanism behind this hypoxic adaptation is still unclear. This study aimed to investigate differentially expressed miRNAs involved in hypoxic adaptation through high-throughput RNA sequencing. 2. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the differentially expressed miRNAs and their target genes in chicken embryonic heart tissues and fibroblasts. Luciferase reporter assays were performed to confirm the relationship between miRNAs and target genes. 3. The study identified 37 differentially expressed miRNAs in Tibetan chicken embryonic heart tissues, including 20 up- and 17 down-regulated miRNAs, compared to those found in lowland chickens. Differentially expressed miRNAs were mainly involved in biological processes, such as cell cycle arrest, toll-like receptor signalling pathways, and I-kappa B kinase/NF-kappa B signalling. The data showed that gga-miR-34 c-5p was significantly upregulated in Tibetan chicken tissues and hypoxic fibroblasts, while EHHADH , a target gene of gga-miR-34 c-5p, was downregulated. Moreover, gga-miR-34 c-5p dramatically decreased the luciferase activity of the wild EHHADH , whereas no effect on the mutational EHHADH was found. 4. This study identified miRNA expression profiles in the Tibetan chicken and suggested that miR-34 c-5p acts as a novel miRNA associated with hypoxic adaptation. This facilitates the understanding of molecular mechanisms that underlie long-term exposure to hypoxia.

Objective: Small nucleolus RNA Host Gene 8 (SNHG8) belongs to a subgroup of long non-coding RNAs. SNHG8 is upregulated in many cancers, such as gastric cancer, liver cancer, and esophageal squamous cell cancer. However, whether SNHG8 is abnormally expressed in breast cancer and its biological functions remain unclear. Therefore, our research intended to determine the expression status of SNHG8 in breast cancer, explore the effects of SNHG8 on the development of breast cancer, and investigate the potential molecular mechanisms in cancer progression., Patients and Methods: The expression levels of SNHG8 were detected in tissue samples and cell lines via qRT-PCR. The effects of SNHG8 on viability of breast cancer cells were detected via CCK-8, EdU, transwell, and flow cytometry analyses., Results: qRT-PCR results showed that the expression level of SNHG8 was significantly upregulated in tumor tissues and cell lines. Gene functional studies showed that the downregulation of the expression level of SNHG8 significantly inhibited the breast cancer cells migration and invasion, and induced apoptosis. Meanwhile, we found that SNHG8 served as an inhibitor of miR-634 in tumor tissues. SNHG8 may participate in the malignancy of breast cancer by sponging the miR-634 to increase the expression level of ZBTB20., Conclusions: The SNHG8-miR-634-ZBTB20 pathway may be a potential target for the treatment of breast cancers.

Background: Quercetin exerts anti-inflammatory effects, but whether it can benefit patients with the chronic inflammatory disease of oral lichen planus (OLP), which is a common chronic mucocutaneous disorder with an immune-mediated pathogenesis, is unclear. The present research examined the impacts of quercetin in a cell-based OLP model in which human oral keratinocytes (HOKs) were treated with lipopolysaccharide (LPS)., Methods: Effects of quercetin on viability, proliferation, and apoptosis of HOKs were assessed using the Cell Counting Kit-8 assay, Western blotting, and flow cytometry, respectively. Effects of treatment on levels of microRNA-22 (miR-22) were measured using stem-loop reverse transcription polymerase chain reaction, while levels of proteins and phosphorylation in the PI3K/AKT and JAK1/STAT3 cascades were analyzed by Western blot., Results: Quercetin mitigated LPS-induced reduction in HOK viability and elevation of apoptosis. It also weakened LPS-induced upregulation of miR-22. Quercetin treatment led to significantly higher levels of p-PI3K, p-AKT, p-JAK1, and p-STAT3. These effects of quercetin were enhanced when miR-22 was knocked down and partly reversed when miR-22 was overexpressed., Conclusion: Quercetin can mitigate LPS-induced injury in HOKs by downregulating miR-22, thereby activating PI3K/AKT and JAK1/STAT3 cascades.

Objective: To investigate the significance of microRNA (miR)-216a, miR-324-5p, miR-29a expression in peripheral blood in patients with acute pancreatitis (AP) and their correlation with liver injury. Methods: It was a case-control study design. To select 130 AP patients admitted from June 2017 to May 2019 in the First People's Hospital of Shangqiu, and the patients were divided into mild AP group (MAP group) and moderately severe AP group (SAP group) according to the disease severity, or 54 patients in the liver injury group (20 were MAP and 34 were SAP) and 76 in the non-liver injury group(all were MAP) according to liver injury. And another 40 healthy volunteers were selected as the healthy group. The expressions of miR-216a, miR-324-5p and miR-29a in peripheral blood of MAP group, SAP group, healthy group and liver injury group, non-liver injury group were compared, and the correlation between the miRNA levels and clinical indexes was analyzed. The predictive value of miRNA levels in peripheral blood for AP complicated with liver injury was analyzed by receiver operating characteristic (ROC) curve. Results: The levels of miR-216a and miR-29a in MAP group and SAP group were higher than those in healthy group, and the level of miR-324-5p was lower than that in healthy group (all P< 0.01). The levels of miR-216a and miR-29a in SAP group were higher than those in MAP group, and the level of miR-324-5p was lower than that in healthy group (all P< 0.01). Balthazar CT Score, acute physiology and chronic health evaluations (APACHE Ⅱ) score, C-reactive protein level, length of hospital stay were positively correlated with the levels of miR-216a and miR-29a in peripheral blood (all P< 0.05), and negatively correlated with the levels of miR-324-5p ( P< 0.05). The levels of miR-216a and miR-29a in the peripheral blood in the liver injury group were higher than those in the non-liver injury group, and they were higher inSAP patients than those in MAP patients in the liver injury group (all P< 0.05). The level of miR-324-5p in the peripheral blood in the liver injury group was lower than that in the non-liver injury group, and it was lower in SAP patients than that in MAP patientsin the liver injury group (all P< 0.05). The area under ROC curve of miR-216a, miR-324-5p, and miR-29a in peripheral blood to predicate the AP complicated with liver damage was 0.694, 0.750 and 0.814, respectively. Conclusions: The levels of miR-216a and miR-29a increase in peripheral blood and the level of miR-324-5p decreases in patients with AP, and they are closely related to Balthazar CT score, APACHEⅡ score, C-reactive protein and length of hospital stay. The levels of miR-216a, miR-324-5p, miR-29a has certain predictive value for AP with liver injury, of which miR-29a has the highest predictive value.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with a rising incidence around the world. MicroRNAs (miRNAs) have been reported to play essential roles in the progression of HCC. However, the precise mechanism of miR-3662 in the HCC process remains poorly understood. This study was aimed to determine the regulatory network of miR-3662 and hexokinase 2 (HK2) in HCC. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect miR-3662 expression. Cell proliferation and invasion were measured by Cell Counting Kit-8 (CCK-8) assay and Transwell assay, respectively. Glucose consumption and lactate production assays were used to detect glucose metabolism activity in HCC cells. The potential binding sites between miR-3662 and HK2 were predicted by TargetScan online software and the relationship between miR-3662 and HK2 was verified by luciferase report assay. The protein expression of HK2 was measured by western blot analysis. A xenograft tumor model was established to confirm the role of miR-3662 and HK2 in vivo. miR-3662 expression was downregulated in HCC tissues and cells, and it was reduced in hypoxia-induced HCC cells in a time-dependent manner. Overexpression of miR-3662 or knockdown of HK2 inhibited cell proliferation, invasion, and glucose metabolism in HCC cells, which could be reversed by upregulating HK2. Besides, HK2 was a direct target of miR-3662 in HCC cells, and hypoxia upregulated the expression of HK2. In addition, the upregulation of HK2 could abolish miR-3662 overexpression-induced inhibitory effects on tumor growth and glucose metabolism in vivo.

Objective: Retinoblastoma (RB) is the most prevalent intraocular malignancy in childhood. Long non-coding RNAs (lncRNAs) have been found as critical oncogenic drivers and tumor suppressor in RB. The aim of the present work was to investigate the impact and mechanism of XIST on RB cell autophagy and vincristine (VCR) sensitivity., Materials and Methods: The levels of XIST and miR-204-5p were assessed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Western blot analysis was used for the determination of related protein levels. Cell proliferation and IC50 value of VCR were detected using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Flow cytometry was performed to evaluate cell apoptosis. The activities of caspase-3 and caspase-9 were identified using a corresponding assay kit. The direct interaction between XIST and miR-204-5p was confirmed using Dual-Luciferase reporter assay. Xenograft model was established to observe the effect of XIST on RB in vivo., Results: Our data indicated that XIST was highly expressed in RB tissues and cell lines. XIST knockdown weakened the proliferation and autophagy and enhanced VCR sensitivity in RB cells. XIST acted as a molecular sponge of miR-204-5p. Moreover, the regulatory effects of XIST silencing on RB cell proliferation, autophagy and VCR sensitivity were mediated by miR-204-5p. Additionally, XIST silencing weakened tumor growth and enhanced VCR sensitivity in vivo through up-regulating miR-204-5p., Conclusions: Our current study suggested that XIST silencing suppressed RB progression and promoted VCR sensitivity in vitro and in vivo at least partially by acting as a miR-204-5p sponge, highlighting a powerful therapeutic strategy for RB treatment.

Objective: The aim of the study was to detect the expression level of microRNA-584 (miRNA-584) in ovarian cancer (OCa) and to elucidate its regulatory effect on OCa progression by regulating LPIN1., Patients and Methods: Expression levels of miRNA-584 and LPIN1 in 31 matched OCa tissues and paracancerous tissues were detected. The relationship of miRNA-584 level with clinical indicators and prognosis of OCa patients was analyzed. Influences of miRNA-584/LPIN1 regulatory loop on malignant phenotypes of OVCAR3 and PEO1 cells were assessed. In addition, the interaction between miRNA-584 and LPIN1 was confirmed by Dual-Luciferase reporter gene assay and rescue experiments., Results: MiRNA-584 was lowly expressed in OCa tissues, while LPIN1 was highly expressed. OCa patients expressing a low level of miRNA-584 suffered from higher rates of lymphatic metastasis and distant metastasis, as well as worse survival. The overexpression of miRNA-584 in OVCAR3 cells attenuated proliferative and migratory abilities, while the knockdown of miRNA-584 in PEO1 cells yielded the opposite results. LPIN1 was verified to be the target binding to miRNA-584 and its level was negatively regulated by miRNA-584. The overexpression of LPIN1 accelerated OCa cells to proliferate and migrate. Importantly, LPIN1 was responsible for OCa progression regulated by miRNA-584., Conclusions: MiRNA-584 is downregulated in OCa tissues and cell lines. MiRNA-584 level is correlated with lymphatic metastasis, distant metastasis, and poor prognosis in OCa patients. By negatively regulating LPIN1, miRNA-584 suppresses the malignant progression of OCa.

Objective: The aim of this study was to explore the influence of micro ribonucleic acid (miR)-29 on neuronal apoptosis in rats with cerebral infarction by regulating the protein kinase B (Akt) signaling pathway., Materials and Methods: A total of 36 Sprague-Dawley rats were randomly divided into three groups, including: Sham group (n=12), Model group (n=12), and Inhibitor group (n=12). Common carotid artery, external carotid artery, and internal carotid artery were only exposed in the Sham group. However, the ischemia-reperfusion model was established by the suture method in the other two groups. After modeling, artificial cerebrospinal fluid was injected into the lateral ventricle in the rats of the Sham and Model groups. Similarly, miR-29 inhibitor was injected into the lateral ventricle in the rats of the Inhibitor group. At 24 h postoperatively, the sampling was performed. Zea-Longa score was used to evaluate the neurological deficit of rats. Meanwhile, the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) in cerebral tissues were detected via immunohistochemistry. The protein expression levels of Akt and phosphorylated Akt (p-Akt) were determined using Western blotting. Furthermore, the expression of miR-29 and cell apoptosis were detected via quantitative Polymerase Chain Reaction (qPCR) and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, respectively., Results: Compared with Sham group, Model, and Inhibitor groups had substantially raised the Zea-Longa scores (p<0.05). The Zea-Longa score in the Model group was markedly lower than that of the Inhibitor group (p<0.05). The positive expression level of Bax was remarkably upregulated (p<0.05). However, the positive expression level of Bcl-2 declined dramatically in both Model group and Inhibitor group when compared with the Sham group (p<0.05). Besides, the Model group exhibited significantly lower positive expression level of Bax and higher positive expression level of Bcl-2 than the Inhibitor group (p<0.05). The relative protein expression level of p-Akt markedly increased in the Model and Inhibitor groups when compared with the Sham group (p<0.05). However, it was considerably higher in the Model group than that of the Inhibitor group (p<0.05). In comparison with the Sham group, both Model group and Inhibitor group exerted substantially elevated expression level of miR-29 (p<0.05). The relative expression level of miR-29 in the Model group was significantly upregulated when compared with the Inhibitor group (p<0.05). The apoptosis rate of cells in both Model group and Inhibitor group was markedly higher than that of the Sham group (p<0.05). Furthermore, the Model group showed remarkably lower apoptosis rate than the Inhibitor group (p<0.05)., Conclusions: MiR-29 inhibits neuronal apoptosis in cerebral infarction rats by upregulating the Akt signaling pathway, thereby serving as a protector.

Objective: To uncover the role of microRNA-127-5p (miRNA-127-5p) in aggravating motor dysfunction following spinal cord injury (SCI) by regulating the mitogen-activated protein kinase 1 (MAPK1) level., Materials and Methods: In vivo SCI model in mice was established by constructing spinal cord hitting injury. Mice were classified into sham group, SCI group, SCI+miRNA-127-5p mimics group, and SCI+miRNA-127-5p mimics+MAPK1 group, respectively. Grip strengths of mouse pair forepaws, right forepaw, and left forepaw at different time points were determined. Expression levels of miRNA-127-5p and MAPK1 in mice of each group at post-SCI were detected. Potential binding sites in promoter regions of miRNA-127-5p and MAPK1 were predicted by bioinformatics and further confirmed by Dual-Luciferase reporter gene assay and Western blot., Results: Grip strengths of SCI mice were much lower than those in sham group at different time points after SCI procedures. MiRNA-127-5p was markedly downregulated on the postoperative 3rd day in SCI group, and its level time-dependently decreased since after. In vivo overexpression of miRNA-127-5p in SCI mice improved their grip strengths from the postoperative 7th day. MAPK1 was the direct target of miRNA-127-5p. Transfection of miRNA-127-5p mimics downregulated protein level of MAPK1 in 293T cells. Overexpression of MAPK1 abolished the protective effect of miRNA-127-5p on motor function recovery following SCI., Conclusions: Downregulation of miRNA-127-5p aggravates SCI-induced motor dysfunction through negatively regulating MAPK1 level.

Objective: This study aims to investigate whether circ-VANGL1 can promote the progression of osteoporosis (OP) by absorbing miRNA-217 to regulate RUNX2 expression., Patients and Methods: The serum levels of circ-VANGL1, miRNA-217 and RUNX2 in OP patients and non-OP patients were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Their expression levels in human bone marrow mesenchymal stem cells (hBMSCs) at different time points of osteogenesis differentiation were determined as well. The expression levels of RUNX2 and osteogenic proteins (BSP, OCN, OPN) in hBMSCs were detected by Western blot. Dual-Luciferase reporter gene assay was performed to verify the relationship among circ-VANGL1, miRNA-217 and RUNX2. Alkaline phosphatase (ALP) staining was conducted to evaluate the degree of osteogenic differentiation influenced by circ-VANGL1 and miRNA-217., Results: OP patients presented a higher serum level of miRNA-217 and lower serum levels of circ-VANGL1 and RUNX2 relative to non-OP patients. Circ-VANGL1 accelerated osteogenic differentiation by absorbing miRNA-217 to regulate RUNX2 expression. Moreover, miRNA-217 inhibited osteogenic differentiation by degrading RUNX2 by targeting to RUNX2 3'UTR. The overexpression of circ-VANGL1 upregulated expressions of RUNX2, BSP, OCN, and OPN. Meanwhile, ALP activity increased in hBMSCs overexpressing circ-VANGL1. However, co-overexpression of circ-VANGL1 and miRNA-217 did not alter RUNX2 expression. ALP activity in hBMSCs co-overexpressing circ-VANGL1 and miRNA-217 slightly increased, but had no difference with controls., Conclusions: Circ-VANGL1 promotes the development of OP via binding to miRNA-217 to downregulate RUNX2 expression.

Objective: It was the aim of this study to investigate whether miR-339 may affect osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) by targeting DLX5, thereby alleviating osteoporosis., Materials and Methods: BMSCs were isolated from the bone marrow of mice. The expression levels of miR-339 and DLX5 during the process of osteogenesis was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Meanwhile, the expression of downstream osteogenesis-associated proteins, such as runt-related transcription factor 2 (RUNX2) and osteopontin (OPN), were also detected after overexpression or inhibition of miR-339. The alkaline phosphatase (ALP) activity was measured in cells by ALP activity assay kit. Alizarin red staining was performed to reveal the cell mineralization ability. The luciferase reporter gene assay was used to identify the targeted pairings of miR-339 and DLX5 genes. In addition, the expression of DLX5 was detected by Western blot analysis after overexpression or knockdown of miR-339. Rescue test was applied to evaluate whether miR-339 could affect the differentiation of BMSCs by inhibiting the expression of DLX5., Results: QRT-PCR showed that miR-339 expression gradually decreased while the expression of DLX5 increased during the induction culture of BMSCs. After overexpression of miR-339 in BMSCs, the expression levels of ALP, RUNX2, and OPN were reduced. Besides, ALP activity assay showed a decreased cell ALP activity. RUNX2 protein expression was also decreased. In addition, Alizarin red staining detected a significant increase in cell mineralization, whereas silencing miR-339 resulted in an opposite result. These results indicated that miR-339 could regulate the osteogenic differentiation of BMSCs. Subsequently, we predicted using bioinformatics software that miR-339 might target DLX5, and validated this hypothesis by luciferase reporter assay. Finally, Western blot and ALP activity assay revealed that DLX5 could reverse the inhibitory effect of overexpression of miR-339 on osteogenic differentiation of BMSCs., Conclusions: Down-regulation of miR-339 can promote osteogenic differentiation of BMSCs by targeting DLX5, thereby relieving osteoporosis.

Objective: The aim of this study was to elucidate the regulatory role of lncSNHG16 in the progression of osteosarcoma (OS) and its underlying mechanism., Materials and Methods: Expressions of lncSNHG16, microRNA-146a-5p and NOVA1 in OS tissues and adjacent normal tissues were determined by quantitative Real-time polymerase chain reaction (qRT-PCR). Their expressions in OS cell lines were detected by qRT-PCR as well. We analyzed the relationship between lncSNHG16 expression and tumor stage, diagnosis and survival prognosis of OS patients, respectively. Cell counting kit-8 (CCK-8) and transwell experiments were conducted to explore proliferative and migratory changes of OS cells. Dual-luciferase reporter assay was used to verify the binding relationship of lncSNHG16 to microRNA-146a-5p, and microRNA-146a-5p to NOVA1. Finally, rescue experiments were performed to elucidate the regulatory effect of lncSNHG16 on the cellular behaviors of OS cells., Results: LncSNHG16 was highly expressed in OS tissues and cell lines. Its expression was positively correlated with the tumor stage of OS patients. Receiver operating characteristic (ROC) curves suggested that lncSNHG16 can be used as a clinical indicator to distinguish OS patients from healthy controls. Survival analysis indicated a negative correlation between lncSNHG16 expression and survival of OS patients. Overexpression of lncSNHG16 enhanced the proliferative and migratory potentials of OS cell lines 143B and MNNG/HOS. MicroRNA-146a-5p was predicted to be the target gene of lncSNHG16, which was lowly expressed in OS tissues and cell lines. Overexpression of lncSNHG16 downregulated the expression of microRNA-146a-5p in 143B and MNNG/HOS cells. Furthermore, we verified that lncSNHG16 could bind to microRNA-146a-5p. The promotive role of lncSNHG16 in proliferative and migratory potentials of OS cells was reversed by microRNA-146a-5p. Subsequently, NOVA1 was predicted to be the target gene of microRNA-146a-5p, and was further verified by dual-luciferase reporter gene assay. Correlation analysis showed that microRNA-146a-5p expression was negatively correlated with NOVA1 expression in OS. More importantly, NOVA1 reversed the inhibitory effect of microRNA-146a-5p on the proliferative and migratory capacities of 143B and MNNG/HOS cells., Conclusions: LncSNHG16 is highly expressed in OS tissues and cell lines, participating in the development of OS by downregulating microRNA-146a-5p to upregulate NOVA1 expression.

Objective: MicroRNAs (miRNAs) as a new class of biomarkers have been explored in recent studies. We investigate whether miR-1256 could be considered as powerful biomarkers for predicting the prognosis of colorectal cancer (CRC)., Patients and Methods: The expression of miR-1256 in CRC was compared with matched normal tissue and using qRT-PCR. The correlation of miR-1256 expression with clinicopathological factors was statistically analyzed. Survival rate was determined with Kaplan-Meier and statistically analyzed with the log-rank method between groups. Univariate and multivariate Cox regression analysis was used to identify the independent risk factors for CRC., Results: We found that miR-1256 level in CRC tissues is notably reduced compared to matched non-cancerous specimens (p < 0.01), and the expression of miR-1256 was significantly correlated with TNM stage (p = 0.000) and lymph node metastasis (p < 0.07). Kaplan-Meier analysis showed that CRC patients with low miR-1256 expression level had distinctly shorter overall survival (p = 0.004) and disease-free survival (p < 0.001) than patients with high miR-1256 expression level. Finally, Cox regression analyses showed that low miR-1256 expression might be an independent prognostic parameter to predict poor prognosis of CRC patients., Conclusions: We firstly provided evidence that low miR-1256 expression was associated with the progression of CRC and could be used as a prognostic biomarker for breast cancer. Further studies are needed.

Objective: MicroRNAs (miRNAs) are increasingly recognized as oncogenes or tumor suppressors in colorectal cancer (CRC). The aim of this study was to explore the expression and functions of miR-340-5p in CRC., Patients and Methods: The expression of miR-340-5p in CRC tissues and cell lines was detected by quantitative RT-PCR. Associations of miR-340-5p expression with clinicopathological factors and overall survival (OS) and progression-free survival (PFS) were statistically evaluated. Luciferase assay, RT-PCR, and Western blot were performed to verify the precise target of miR-340-5p. MTT assay, colony formation and transwell assay were performed to determine the proliferation, migration and invasion, respectively., Results: Our results showed that miR-340-5p was significantly down-regulated in CRC tissues and cell lines, and was associated with histological grade (p=0.020), lymph nodes metastasis (p=0.003) and TNM stage (p=0.007). Furthermore, Kaplan-Meier and log-rank tests revealed that patients with low expression of miR-340-5p had a shorter OS (p=0.0110) and PFS (p=0.0032) than those with high expression of miR-340-5p. We further validated Annexin A3 (ANXA3) was a direct target of miR-340-5p in CRC. The functional assay showed that up-regulation of miR-340-5p or down-regulation of ANXA3 can both inhibit CRC cell proliferation, migration, and invasion. Besides, the re-expression of ANXA3 reversed the miR-340-5p induced suppression of cell proliferation, migration and invasion., Conclusions: Our data demonstrated that miR-340-5p exerted its tumor-suppressive function by directly targeting ANXA3 in CRC, suggesting that miR-340-5p might represent a novel prognostic biomarker and therapeutic target for CRC.

Objective: To investigate the clinical value of combined detection of serum miR-378 and miR-21 in gastric cancer (GC). Methods: Eighty-seven patients with GC and 78 patients with colorectal cancer(CRC) from National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences were selected, 83 individuals undergoing healthy physical examination were selected as the healthy controls. The levels of serum miR-378 and miR-21 were detected by quantitative real-time PCR (RT-qPCR) (result data were transformed as log2 for analysis). Results: Relative expression levels of miR-378 in the serum were -1.24, -3.25 and -2.73 in healthy controls, GC and CRC patients, respectively. Compared with the healthy controls, the levels of serum miR-378 were significantly decreased in GC and CRC patients (both P <0.05). Relative expression levels of miR-21 in the serum were 0.11, 2.34 and 2.47 in healthy controls, GC and CRC patients, respectively. Compared with the healthy controls, the levels of serum miR-21 were significantly up-regulated in GC and CRC patients (both P <0.05). Moreover, the serum level of miR-378 in GC patients was inversely associated with tumor clinical stage ( P <0.05). However, the level of miR-21 showed no significant differences among patients with different clinical and pathological characteristics (all P >0.05). The area under the receiver operating characteristic curve (AUC), sensitivity and specificity of miRNA-378 to diagnose GC was 0.770, 82.0% and 66.0%, respectively, and were 0.900, 85.0%, and 88.0% of miR-21, respectively. The AUC, sensitivity and specificity of combined detection of serum miR-378 and miR-21 to diagnose GC were 0.930, 92.0% and 87.0%, respectively, while the AUC of combined detection of serum CEA and CA-199 was 0.767, the AUC of combined all of the four factors was 0.946. Conclusion: The combined detection of serum miR-378 and miR-21 have a certain effect on diagnosis of GC.

Objective: To investigate the role of microRNAs (miRNAs) and its mechanism in osteoblast mineralization., Materials and Methods: Real-time polymerase chain reaction (PCR), Northern Blot, and Western Blot were used to identify the expression mode of regulators. Overexpression and down-regulation experiments were carried out to study the role of miR-98 and interactions between regulators. Bioinformatics calculation and luciferase reporter assay were used to prove the target gene. Electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (CHIP), and promoter luciferase reporter assay confirmed the relationship between the regulator and the promoter of miR-98., Results: MiR-98 was up-regulated during osteoblast mineralization. Overexpression of miR-98 promoted osteoblast mineralization. Factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a negative regulator of osteoblast differentiation, was confirmed to be a target of miR-98. As a motivator in osteoblast mineralization, Sp7 transcription factor 7 (Sp7) promoted miR-98 transcription by a combination on the promoter region., Conclusions: Our study showed that miR-98 was an important regulator in osteoblast mineralization and miR-98 carried out its function through a novel miR-98-FIAT/Sp7 regulatory loop. It provides new insights into the roles of miRNAs in osteoblast mineralization.

Objective: To investigate the effect of miR-524 on the proliferation of thyroid cancer and its underlying mechanism., Patients and Methods: MiR-524 expression levels in thyroid cancer samples and para-cancer tissues were tested by quantitative Real-time polymerase chain reaction (qRT-PCR). Cells proliferative ability and apoptosis were evaluated through methyl thiazolyl tetrazolium (MTT) and apoptosis assays, respectively. Luciferase reporter assay was used to confirm the regulatory mechanism., Results: MiR-524 expression was reduced in thyroid cancer specimen (p<0.05). Up-regulated miR-524 expression inhibited the proliferative ability and enhanced cell apoptosis of thyroid cancer cells. SPAG9 was a target gene of miR-524, and was reversely regulated by miR-524., Conclusions: MiR-524 represses thyroid cancer cell proliferation and induces cell apoptosis via targeting SPAG9.

Objective: MiR-381-3p plays an essential role in the progression of a variety of cancers, but its expression and role in papillary thyroid carcinoma (PTC) progression have not been investigated. The aim of this study was to investigate the expression of miR-381-3p and its function in PTC., Patients and Methods: The expression levels of miR-381-3p and low-density lipoprotein receptor‑related protein 6 (LRP6) mRNA in PTC tissues and cell lines were measured using RT-PCR. Cell proliferation, migration and invasion were assessed by cell viability assay and transwell assay. Luciferase assays and Western blotting were performed to demonstrate miR-381-3p target gene., Results: We found that miR-381-3p was significantly down-regulated in PTC tissues and cell lines. In vitro assay indicated that up-regulation of miR-381-3p significantly suppressed PTC cell proliferation, migration and invasion. Moreover, luciferase reporter gene assay demonstrated that miR-381-3p could target LRP6 by binding to the 3' UTR. Western blot and Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) showed that miR-381-3p overexpression suppressed the expression of LRP6 at both mRNA and proteins levels. In addition, functional experiment confirmed that LRP6 was involved in the suppressive effect of miR-381-3p-mediated PTC on cell proliferation, migration and invasion., Conclusions: Our findings suggested, for the first time, that miR-381-3p was lowly expressed in PTC tissues, and its up-regulation inhibited tumorigenesis of PTC by targeting LRP6.

Occupational noise-induced hearing loss (ONIHL) is a prevalent occupational disorder that impairs auditory function in workers exposed to prolonged noise. However, serum microRNA expression in ONIHL subjects has not yet been studied. We aimed to compare the serum microRNA expression profiles in male workers of ONIHL subjects and controls. MicroRNA microarray analysis revealed that four serum microRNAs were differentially expressed between controls (n=3) and ONIHL subjects (n=3). Among these microRNAs, three were upregulated (hsa-miR-3162-5p, hsa-miR-4484, hsa-miR-1229-5p) and one was downregulated (hsa-miR-4652-3p) in the ONIHL group (fold change >1.5 and Pbon value <0.05). Real time quantitative PCR was conducted for validation of the microRNA expression. Significantly increased serum levels of miR-1229-5p were found in ONIHL subjects compared to controls (n=10 for each group; P<0.05). A total of 659 (27.0%) genes were predicted as the target genes of miR-1229-5p. These genes were involved in various pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of miR-1229-5p dramatically inhibited the luciferase activity of 3' UTR segment of MAPK1 (P<0.01). Compared to the negative control, HEK293T cells expressing miR-1229-5p mimics showed a significant decline in mRNA levels of MAPK1 (P<0.05). This preliminary study indicated that serum miR-1229-5p was significantly elevated in ONIHL subjects. Increased miR-1229-5p may participate in the pathogenesis of ONIHL through repressing MAPK1 signaling.

Objective: To explore the role of miR-133b in ovarian cancer and to preliminarily elucidate the mechanism of miR-133b in epithelial-mesenchymal transition (EMT) of ovarian cancer., Patients and Methods: MiR-133b was detected in ovarian cancer specimens, and the relationship of miR-133b with each pathological index and clinical index of ovarian cancer was analyzed. The action targets of miR-133b in ovarian cancer were analyzed systematically and studied deeply via the target validation and cell function validation. Finally, the possible reasons of ovarian cancer metastasis were analyzed through the molecular regulation mechanism in EMT of ovarian cancer., Results: The miR-133b level in ovarian cancer was significantly lower than in normal ovarian tissues and benign ovarian tumors (p<0.05). The level of miR-133b in ovarian cancer was related to differentiated degree and lymphatic metastasis. Dual-luciferase assay indicated that connective tissue growth factor (CTGF) was the target gene regulated by miR-133b. Reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Western blot results proved that the expression level of E-cadherin representing the epithelial cell phenotype was increased, while the expression level of vimentin representing the mesenchymal cell phenotype was decreased. Transwell assay confirmed that the migration and invasion abilities of ovarian cancer cells declined after transfection with miR-133b plasmid. After co-transfection with miR-133b and CTGF overexpression plasmids, RT-PCR and Western blotting proved that the expression level of E-cadherin representing the epithelial cell phenotype was decreased, while the expression level of vimentin representing the mesenchymal cell phenotype was increased; transwell assay confirmed that the cell migration and invasion abilities were increased after co-transfection., Conclusions: The results of this study showed that miR-133b may serve as a new molecular marker of EMT of ovarian cancer, and act as a molecular marker of differentiated degree and lymphatic metastasis of ovarian cancer.

Objective: Postmenopausal osteoporosis (POMP) is a serious disorder with significant physical, psychosocial, and financial consequences, which greatly reduce the postmenopausal women's life quality. The related issues of postmenopausal osteoporosis are increasingly concerned by society. Past researches have shown that miRNAs play an important role in the occurrence and development of postmenopausal osteoporosis. However, the role of miR-218 and miR-618 in the osteoporosis regulation is still unclear., Materials and Methods: First of all, we investigated the alteration of miR-218 and miR-618 during osteoclastogenesis of RAW264.7 cells. Next, we transfected RAW264.7 cells with miR-218 or miR-618 mimics and inhibitors to explore the influences of miR-218 and miR-618 on osteoclast differentiation. Then, we conducted bioinformatics analysis and luciferase reporter assay to identify and test the target gene of miR-218 and miR-618., Results: MiR-218 and miR-618 were down-regulated when RAW264.7 cells differentiated into osteoclasts. In addition, overexpression of miR-218 or miR-618 attenuated RAW264.7 cells differentiated into osteoclasts in vitro, whereas inhibition of miR-218 or miR-618 promoted this progress. This was demonstrated by increased expression of osteoclast-specific genes and TRAP staining. TLR-4 was confirmed to be the direct target of miR-218 and miR-618 by bioinformatics and luciferase reporter assay., Conclusions: These results suggested that miR-218 and miR-618 play an important role in osteoclastogenesis via TLR-4/MyD88/NF-κB signaling pathway. Thus, targeting miR-218 and miR-618 promise a therapeutic potential in the treatment of osteoporosis.

Osteoporosis (OP) is a kind of disease with a 25% incidence, characterized by the bone mass loss, bone microstructure damage, increased bone fragility, and easy fracture. miRNA plays an important regulatory role in the process of bone remodeling, especially in the regulation of differentiation and function of osteoblasts and osteoclasts, and the development and progression of OP and other bone diseases. In the future, it is expected to delay the bone loss and promote the bone remodeling via the overexpression or inhibition of specific miRNAs in specific tissues, thereby treating OP.

Introduction: MicroRNA-34c (miR-34c) has been found to play important roles in tumorigenesis. However, little is known about miR-34c expression and the impact on prognosis in acute myeloid leukemia (AML)., Methods: Real-time quantitative PCR (qRT-PCR) was performed to analyze the status of miR-34c expression in 122 patients with de novo AML and 62 normal controls., Results: MiR-34c expression in AML was significantly downregulated compared to controls (P < 0.001). Receiver operating characteristic curves (ROC) indicated the distinguishing value of miR-34c for discriminating whole-cohort AML, non-M3 AML, and cytogenetically normal AML (CN-AML) patients from healthy controls. No significant differences were found between low miR-34c-expressing and high miR-34c-expressing patients in age, sex, hemoglobin, platelet count, percentage of blasts in bone marrow (BM), WHO classifications, karyotypes, and eight gene mutations, but low miR-34c cases had higher white blood cells (WBC) than high miR-34c cases (P = 0.035). MiR-34c low-expressed patients had similar rates of complete remission (CR) as miR-34c high-expressed patients in whole-cohort AML, non-M3 AML, and CN-AML patients (P = 0.347, 0.314 and 0.167, respectively). Kaplan-Meier analysis indicated that patients with low miR-34c level had markedly shorter overall survival (OS) time in whole-cohort AML, non-M3 AML, and CN-AML patients (P = 0.033, 0.024 and 0.001, respectively). Furthermore, multivariate analysis confirmed that low miR-34c expression was an independent risk factor not only in whole-cohort AML (P = 0.040) but also in non-M3 AML (P = 0.015) and CN-AML patients (P = 0.021)., Conclusions: Our findings indicate that low miR-34c level is a novel promising biomarker in predicting prognosis in patients with de novo AML., (© 2016 John Wiley & Sons Ltd.)

To date, diagnosis of schizophrenia is still based on clinical interviews and careful observations, which is subjective and variable, and can lead to misdiagnosis and/or delay in diagnosis. As early intervention in schizophrenia is important in improving outcomes, objective tests that can be used for schizophrenia diagnosis or treatment monitoring are thus in great need. MicroRNAs (miRNAs) negatively regulate target gene expression and their biogenesis is tightly controlled by various factors including transcription factors (TFs). Dysregulation of miRNAs in brain tissue and peripheral blood mononuclear cells (PBMNCs) from patients with schizophrenia has been well documented, but analysis of the sensitivity and specificity for potential diagnostic utility of these alternations is limited. In this study, we explored the TF-miRNA-30-target gene axis as a novel biomarker for schizophrenia diagnosis and treatment monitoring. Using bioinformatics analysis, we retrieved all TFs that control the biogenesis of miRNA 30 members as well as all target genes that are regulated by miRNA-30 members. Further, reverse transcription-quantitative PCR analysis revealed that the early growth response protein 1 (EGR1) and miR-30a-5p were remarkably downregulated, whereas neurogenic differentiation factor 1 (NEUROD1) was significantly upregulated in PBMNCs from patients in acute psychotic state. Antipsychotics treatment resulted in the elevation of EGR1 and miR-30a-5p but the reduction of NEUROD1. Receiver operating characteristic analysis showed that the EGR1-miR-30a-5p-NEUROD1 axis possessed significantly greater diagnostic value than miR-30a-5p alone. Our data suggest the EGR1-miR-30a-5p-NEUROD1 axis might serve as a promising biomarker for diagnosis and treatment monitoring for those patients in acute psychotic state.

Previous research has showed that miR-99a-5p was a tumor suppressor. The aim of our study was to explore the effect of miR-99a-5p on the vitality and proliferation, migration together with the invasion of oral tumor cells via inhibiting the expression of NOX4. QRT-PCR and Western blot were applied to examine the expression level of miR-99a-5p and NOX4 in human oral tumorous and adjacent tissues. Dual luciferase reporter gene assay was applied to confirm that miR-99a-5p negatively regulated directly on NOX4 in TSCC1 cells. Cell transfection and lentiviral vectors were used to up-regulate expression of miR-99a-5p and NOX4, respectively. Cell proliferation, cell cycle, apoptosis and invasion along with the migration in different groups were assessed using MTT assay, colony formation assay, the flow cytometry, transwell assay and the wound healing assay, respectively. MiR-99a-5p was under-expressed in human oral tumor, while NOX4 was over-expressed. There was a negative relationship between miR-99a-5p and NOX4. Up-regulating miR-99a-5p or down-regulating NOX4 suppressed the vitality, proliferation, migration together with invasion of TSCC1 cells. MiR-99a-5p affected the vitality and proliferation, migration together with the invasion of oral tumor cells through targeting NOX4.

Objective: To investigate the K562 cells biological function and related molecular changes in PTEN-PI3K/AKT signaling pathway of leukemia K562 cells by inhibiting the miRNA-21 expression to explore its pathogenesis of leukemia. Methods: The chemical synthetic miRNA-21 inhibitor was transfered into K562 cells by electrotransfection. RT-PCR was used to detect the miRNA-21 expression changes. Cell proliferation and apoptosis were determined by using MTT and flow cytometry. Western-blot were used to detect the protein expression changes of PTEN, PI3K and p-AKT respectively. Results: The relative expression of miRNA-21 in experimental group was (8.070 ± 5.138)% at 24 hours, which was lower than control groups ( P <0.05). The apoptotic rate of (13.370±0.250)% at 24 hours in experimental group was obviously higher than control groups. The cellular proliferation were significantly different at 24 hours. The proliferation inhibition rate was (8.1±0.9)% at 24 hours, which was up to (43.1±2.1)% at 60 hours, but the control groups showed no difference. K562 cell proliferation significantly decreased, while cell apoptosis markedly increased by inhibiting miRNA-21 expression ( P <0.01). Western-blot analysis revealed up-regulation of PTEN and down-regulation of PI3K and p-AKT protein expressions after successfully suppressed miRNA-21 expression ( P <0.01). Conclusion: Inhibiting miRNA-21 expression in K562 cell could suppress the PI3K/AKT pathway by up-regulation of PTEN expression and promote cell antiproliferative and pro-apoptosis effects.

Mesenchymal stem cells (MSC) are known to have the ability to differentiate into various lineages of mesenchymal tissue. They are widely distributed in a variety of tissues in the body and are also present in the foetal environment. MicroRNAs (miRNAs) are a fundamental class of biological molecules that play a crucial role in development. In this study, microRNA-378 and its predicted target (Sufu, suppressor of fused homolog) were used to study proliferation of rabbit umbilical cord mesenymal stem cells, luciferase reporter assay was used to assess and confirm the binding sequence of 3'untranslated region between microRNA-378 and Sufu. The results showed that miRNA-378 overexpression reduced Sufu gene, and promoted cell proliferation of rabbit umbilical cord mesenymal stem cells via cell viability and BrdU testing, which molecular mechanisms were down-regulation of apoptosis genes after miRNA-378 overexpression. In conclusion, our research shows that the microRNA-378 promoted the cell proliferation of rabbit umbilical cord mesenymal stem cells, and this mechanism was miRNA-378 reduced the expression of Sufu, increased cell proliferation.

Background: Several recent studies have revealed that microRNAs (miRNAs) are stably detectable in the circulation and can be used as biomarkers for diagnosis and prognosis of malignancy. The aim of this manuscript is to investigate serum miR-218 expression in patients with hepatocellular carcinoma (HCC) and to analyze its potential diagnostic and prognostic value in HCC., Methods: Quantitative real-time quantitative PCR (qPCR) was conducted to detect serum miR-218 expression from 156 HCC and 98 benign liver diseases (BLD) as well as 64 healthy controls. The relevance of serum miR-218 expression to the clinicopathological factors was assessed. In addition, the prediction of cutoff values of the markers was performed by the receiver operating characteristic (ROC) curve. Moreover, the Kaplan-Meier method was used to plot survival curves and univariable and multivariable Cox regression analyses were used to evaluate independent prognostic factors., Results: Consequently, our findings revealed that serum miR-218 levels were remarkably underexpressed in HCC patients as compared to BLD patients and healthy controls. And its low level was obviously related to tumor size (p = 0.048), tumor number (p = 0.018), vascular invasion (p = 0.039), Edmondson grade (p = 0.042), and higher TNM stage (III-IV). ROC curve analysis showed that miR-218 had a significant diagnostic accuracy, yielded an AUC (the areas under the ROC curve) of 0.734 (95 % confidence interval (CI) 0.68-0.789, p < 0.01), thus providing a sensitivity of 66.7 % and a specificity of 69.1 % in discriminating HCC from BLD and healthy controls. Meanwhile, miR-218 can act as a useful biomarker in distinguishing the patients with large tumors (>5 cm) from patients with small tumors (<5 cm) (p < 0.01). In addition, the combination of miR-218 and AFP had greater diagnosis capacity with an AUC of 0.908 (95 % CI 0.876-0.940; p < 0.01). Both log-rank test and Cox regression analysis demonstrated that the decreased serum expression of miR-218 had a significant impact on overall survival of the patients with HCC (HR = 3.049, 95 % CI 2.028-4.585, p < 0.01)., Conclusion: Taken together, this study suggested that serum expression of miR-218 might be a potential noninvasive tumor biomarker in the diagnosis and assessment of prognosis of HCC.

Objective: To explore the effect of let-7 miRNA silencing on Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and the underling mechanism., Methods: The pEGFP-C2-let-7 sponge vector was transfected into BCBL-1 and 293T cells with Lipofectamine 2000 to silence the expression of let-7 miRNAs. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of let-7 miRNAs, the transcriptional levels of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), cyclooxygenase-2 (COX-2) and matrix metalloproteinase 13 (MMP-13), and the DNA copy numbers of KSHV open reading frame 50 (ORF50) and open reading frame 72 (ORF72). Western blot was used to detect the total and phosphorylated protein levels of MAP4K4, COX-2, extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK., Results: The expression of let-7 miRNAs was dramatically decreased in the let-7 sponge transfected BCBL-1 and 293T cells compared with that in the vector-transfected cells (P<0.05 for all). The gene copy number and mRNA transcriptional level of KSHV ORF50 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with that in the vector-transfected cells (1.00±0.10 vs. 2.33±0.18 and 1.08±0.48 vs 3.22±0.27, respectively,P<0.001 for both). The gene copy number and mRNA transcriptional level of KSHV ORF72 were also significantly increased in let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells(1.07±0.49 vs 1.67±0.45 and 1.01±0.19 vs 1.54±0.11, respectively,P<0.05 for both). Furthermore, the mRNA transcriptional levels of MAP4K4, COX-2 and MMP-13 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells (1.00±0.05 vs 5.73±0.96, 1.00±0.05 vs 2.68±0.19, 1.00±0.02 vs 2.69±0.25, respectively,P<0.001 for all). Let-7 miRNAs silencing also increased the protein expression levels of MAP4K4, COX-2 and phospho-ERK1/2, while the phospho-JNK and phospho-p38 were not changed in the BCBL-1 and 293T cells., Conclusions: Let-7 silencing may activate the replication of KSHV, possibly through up-regulating MAP4K4 and its downstream molecules COX-2, MMP-13, and phosphorylation of ERK1/2, finally results in the progression of Kaposi sarcoma.

Aim: Several factors have been implicated in the pathogenesis of colorectal cancer (CRC) associated with ulcerative colitis (UC). We investigated markers of cancer cell pluripotency, including CD44 and CD166, microRNA-21 (miR-21) and microRNA-215 (miR-215), and APC, K-ras and DCC mutations in biopsy specimens from patients with UC to evaluate any correlations with clinical risk factors., Method: We observed 18 patients with UC and collected two biopsy specimens from each patient at diagnosis and at a follow-up end-point. We examined the expression of CD44, CD166, miR-21 and miR-215, and APC, K-ras and DCC mutations. We compared these markers at the two time points and assessed their associations with clinical characteristics, including the duration of colitis, histological alterations and the age of the patient at the onset of UC., Results: Most (16/18) patients had alleviation of mucosal inflammation or remained stable during follow-up; one patient developed dysplasia and one had severe aggravation of the lesion during follow-up. Enhanced expression of CD44, CD166 and miR-21 with miR-215 was found in the specimens obtained at follow-up, despite alleviation of mucosal lesions. Coherence of cancer stem cell markers and miRNAs was seen in patients who had significant worsening of inflammation, dysplasia and a long duration of colitis. APC mutation occurred in only one patient; this patient had the longest duration of UC (23 years)., Conclusion: Enhanced markers of CRC in follow-up colonic mucosal samples support the conclusion that the duration of UC plays the most important role in UC-related carcinogenesis., (Colorectal Disease © 2016 The Association of Coloproctology of Great Britain and Ireland.)

Objective: Elevated miR-181a is associated with the transition of ovarian tissues from normal into a cancerous state. However, its regulative effect on multidrug resistance (MDR) of ovarian cancer is not quite clear. Therefore, this study aimed to investigate its regulative effects on epithelial-to-mesenchymal transition (EMT) and MDR in ovarian cancer., Materials and Methods: The expression profile of miR-181a in normal and ovarian cancer tissues was firstly quantified using qRT-PCR analysis. Then, human ovarian cancer cell line SKOV3 were transfected for miR-181a overexpression and the paclitaxel-resistant variant SKOV3/PTX cells were transfected for miR-181a knockdown. The effect of miR-181a overexpression/knockdown on EMT and PTX sensitivity were studied., Results: MiR-181a level in chemoresistant (CR) cancer tissues were significantly higher than in chemosensitive (CS) cancer tissues and in normal tissue. SKOV3/PTX cells had significantly higher expression of miR-181a and N-cadherin than SKOV3 cells. SKOV3 cells had decreased E-cadherin expression and increased N-cadherin expression after enforced miR-181a expression, while SKOV3/PTX cells had increased E-cadherin expression and decreased N-cadherin expression after miR-181a knockdown. SKOV3 cells had increased P-gp expression after enforced miR-181a expression. Following MTT assay and flow cytometry analysis both confirmed that miR-181a overexpression decreased the PTX sensitivity of SKOV3 cells and while miR-181a inhibition increased the sensitivity of SKOV3/PTX cells., Conclusions: MiR-181a is an important oncomiR significantly increased in chemoresistant ovarian cancer. Its upregulation is associated with increased level of EMT and decreased cell apoptosis induced by PTX treatment.

Background: Recent studies have shown that microRNA-21 (miR-21) is overexpressed in solid tumors and implicated in the modulation of drug-induced resistance., Methods: In this study, we investigated the anti-tumor effects of miR-21 on the sensitivity of osteosarcoma cells to CDDP., Results: Changes in the sensitivity of osteosarcoma cells to CDDP were examined after transfection with miR-21 mimics or anti-miR-21 or bcl-2 siRNA in combination with CDDP. Osteosarcoma cells transfected with miR-21 mimics were significantly resistant to CDDP, while suppression of miR-21 in osteosarcoma cells led to enhanced CDDP cytotoxicity. Moreover, the miR-21-induced changes in chemoresponse were ameliorated by down-regulation of bcl-2 by its siRNA., Conclusion: The miR-21 in osteosarcoma cells is a significant modulator of the anti-tumor effect of CDDP by regulating the expression of bcl-2, and the study reveals a novel mechanism of osteosarcoma drug resistance.

The activation of cancer-associated fibroblasts (CAFs) is a key event in tumor progression, and alternative extracellular matrix (ECM) proteins derived from CAFs induce ECM remodeling and cancer cell invasion. Here we found that miR-200 s, which are generally downregulated in activated CAFs in breast cancer tissues and in normal fibroblasts (NFs) activated by breast cancer cells, are direct mediators of NF reprogramming into CAFs and of ECM remodeling. NFs with downregulated miR-200 s displayed the traits of activated CAFs, including accelerated migration and invasion. Ectopic expression of miR-200 s in CAFs at least partially restored the phenotypes of NFs. CAF activation may be governed by the targets of miR-200 s, Fli-1 and TCF12, which are responsible for cell development and differentiation; Fli-1 and TCF12 were obviously elevated in CAFs. Furthermore, miR-200 s and their targets influenced collagen contraction by CAFs. The upregulation of fibronectin and lysyl oxidase directly by miR-200 or indirectly through Fli-1 or TCF12 contributed to ECM remodeling, triggering the invasion and metastasis of breast cancer cells both in vitro and vivo. Thus, these data provide important and novel insights into breast CAF activation and ECM remodeling, which trigger tumor cell invasion.

Background: MicroRNAs (miRNAs) have emerged as epigenetic regulators of metabolism and energy homeostasis. There is a growing body of evidence pointing to miRNAs that have important regulatory roles in insulin sensitivity., Objective: The aim of this work was to explore the expression and mechanism of action of miR-26b in obesity-related insulin resistance (IR) in adipocytes., Methods: Quantitative real-time PCR was performed to determine miR-26b expression in obese rodent models, human obesity subjects and insulin-resistant adipocytes. We analysed the roles of miR-26b overexpression and inhibition on glucose uptake in adipocytes. Western blotting was used to detect the levels of protein molecules involved in the phosphoinositide-3-kinase (PI3K) pathway. Bioinformatics and the Dual Luciferase Assay were used to identify the target gene of miR-26b. We assessed the regulatory roles of miR-26b on the phosphatase and tensin homologue (PTEN)/PI3K/AKT pathway and the relationship between miR-26b and the metabolism of human obese subjects., Results: Levels of miR-26b are reduced in visceral adipose tissue (VAT) in obese rodent models, human obesity and insulin-resistant adipocytes. MiR-26b promotes insulin-stimulated glucose uptake and increases insulin-stimulated glucose transporter type 4 translocation to the plasma membrane in human mature adipocytes. MiR-26b modulates insulin-stimulated AKT activation via inhibition of its target gene, PTEN, and significantly increases insulin sensitivity via the PTEN/PI3K/AKT pathway. The expression level of miR-26b negatively correlates with increasing body mass index and homeostasis model assessment for IR in human obese subjects., Conclusion: Decreased miR-26b expression in VAT may be involved in obesity-related IR by interrupting the PTEN/PI3K/AKT pathway.

Emerging evidence has shown that cancer stem cells (CSCs) are the cellular determinants to promote cancer invasion and metastasis. However, the mechanism underlying CSC invasion remains unknown. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression, and their expressions are often dysregulated in cancers. In the present study, we demonstrated that two functionally related microRNAs, miR-20a and -106a (miR-20a/106a), were capable of enhancing the invasiveness of CD133(+) glioma stem cells (GSCs) isolated from both glioblastoma cell line U87 and primary human glioma specimens. We found that the level of miR-20a/106a in GSCs was significantly higher than that in the committed CD133(-) glioma cells, and correlated with the invasive capability of GSCs. By bioinformatic analysis, we identified tissue inhibitor of metalloproteinases-2 (TIMP-2) as one of the miR-20a/106a-targeted genes. TIMP-2 level correlated inversely with miR-20/106 expression. Directly targeting by miR-20a/106a on 3'-untranslation region (3'-UTR) of TIMP-2 mRNA was confirmed by 3'-UTR dual-luciferase reporter assay. Knockdown of miR-20a/106a in GSCs increased endogenous TIMP-2 protein abundance, thereby inhibiting GSC invasion. We also found that Nordy, a synthetic lipoxygenase inhibitor, inhibited GSC invasiveness by elevating the expression of TIMP-2 via downregulation of miR-20a/106a. Our results indicate that miR-20a/106a has a key role in GSC invasion and may serve as targets for treatment of glioblastoma.

Recently, the role of miR-29b in colorectal carcinoma (CRC) development appears to be controversial. Until now, the expression and function of miR-29b in CRC have not been clarified clearly. We showed that decreased expression of miR-29b usually occurred in CRC cell lines and tissue samples. Loss- and gain-of-function assays in vitro revealed suppressive effects of miR-29b on cell proliferation and migration. Endogenous overexpression of miR-29b was sufficient to suppress aggressive behavioral phenotypes in mice. Proteomic analysis showed that miR-29b involved in integrate several key biological processes. In addition, miR-29b mediated the inhibition of epithelial-mesenchymal transition (EMT) and the inactivation of mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signal transduction pathway. Further studies found that T lymphoma invasion and metastasis 1 (Tiam1) was identified as a direct target of miR-29b. In contrast to the phenotypes induced by miR-29b restoration, Tiam1-induced cell proliferation and migration partly rescued miR-29b-mediated biological behaviors. Our results illustrated that miR-29b as a suppressor has a critical role in CRC progression, which suggests its potential role in the molecular therapy of patients with advanced CRC.

Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis.

Objectives: Evidence has indicated that ovarian epithelial cancer-type cells under serum-free culture conditions can form spheroid cells and exhibit characteristics expected of cancer stem-like cells (CSCs). However, the mechanism by which differentiated ovarian cancer cells acquire stem-cell properties during CSC enrichment has needed to be elucidated. Recent studies have demonstrated that induction of epithelial to mesenchymal transition (EMT) can generate CSCs and be associated with tumour aggressiveness and metastasis., Materials and Methods: Ovarian epithelial cancer cell lines, SKOV3 and HO8920, were cultured for spheroid cells and adherent cells. CSC enrichment was investigated using MTT assay, flow cytometery and qRT-PCR and expression level of PI3K/AKT pathway components was analysed by western blotting., Results: Compared to adherent cells, the spheroid cells expressed mesenchymal markers highly and exhibited significantly more motility; we also observed increases in phosphate AKT1 levels in the spheroid cells. Moreover, transfection of miR-20a or miR-200c led to corresponding reduction in endogenous PTEN protein, while AKT1 and phosphate AKT1 levels were upregulated in miRNAs-transfected cells. Finally, PI3K/AKT pathway inhibitor LY294002 reduced expressions of mesenchymal markers and stem-cell gene activity in spheroid cells, enhancing sensitivity of spheroid cells to paclitaxel treatment., Conclusions: Our findings demonstrate that EMT contributed to enrichment of ovarian CSCs in vitro, making EMT targeting in epithelial ovarian cancer a novel therapeutic option., (© 2013 John Wiley & Sons Ltd.)

A major advancement in the treatment of chronic myeloid leukemia (CML) has been the development of imatinib and other BCR-ABL tyrosine kinase inhibitors. MicroRNAs (miRNAs) are small RNA molecules that influence gene expression by post-transcriptional regulation of messenger RNA. It is not yet clear how miRNAs are able to regulate the effectiveness of imatinib in CML. Here, we show that imatinib markedly inhibits expression of miR-30a in human CML cells. miR-30a is a potent inhibitor of autophagy by downregulating Beclin 1 and ATG5 expression. miR-30a mimic or knockdown of autophagy genes (ATGs) such as Beclin 1 and ATG5 by short hairpin RNA enhances imatinib-induced cytotoxicity and promotes mitochondria-dependent intrinsic apoptosis. In contrast, knockdown of miR-30a by antagomir-30a increases the expression of Beclin 1 and ATG5, and inhibits imatinib-induced cytotoxicity. These findings indicate that dysregulation of miR-30a may interfere with the effectiveness of imatinib-mediated apoptosis by an autophagy-dependent pathway, thus representing a novel potential therapeutic target in CML.