Target Gene Of miR-21 Research Articles

Inhibition of miR-182 represses growth of hepatocellular carcinoma cells by targeting SOX11

Purpose: To investigate the effect of microRNA-182 (miR-182) on hepatocellular carcinoma (HCC) and its mechanisms of action.Methods: Sixty-five HCC tissues and matched adjacent tissues were obtained, and miR-182 and sexdetermining region on the Y Chromosome (SRY)-related HMG box 11 (SOX11) expression were quantified using reverse transcription-polymerase chain reaction (RT-PCR). SOX11-positive cells were identified by immunohistochemistry and the correlation between SOX11 and miR-182 expression was analyzed by Spearman correlation analysis. Huh-7 cells were transfected with the miR-182 mimic, the miR-182 inhibitor, and/or si-SOX11, and cell proliferation was measured by cell counting kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry. Luciferase reporter assay was used to investigate the putative target gene of miR-182. A xenograft nude mouse model was established by transfection with miR-182 antagomir, while tumor volume and weight were calculated. SOX11, cleaved caspase-3, cleaved poly (ADP ribose) polymerase (PARP), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) protein levels were analyzed by western blot.Results: MiR-182 expression increased and SOX11 expression was decreased in HCC tissues (p < 0.05). Luciferase reporter assay data confirmed that miR-182 directly targets SOX11. Inhibition of miR-182 repressed proliferation and Bcl-2 expression, but increased protein expression of cleaved caspase-3, cleaved RAPP, and Bax in Huh-7 cells (p < 0.05). In addition, suppression of SOX11 reversed the effects of miR-182 on cell proliferation and apoptosis. The miR-182 antagomir decreased tumor growth and miR-182 expression but increased SOX11 expression in vivo (p < 0.05).Conclusion: MiR-182 and SOX11 may be novel therapeutic targets for HCC patients.

Dual regulation of miR-375 and CREM genes in pancreatic beta cells

ABSTRACT MicroRNA-375 (miR-375) is upregulated in the islets of some diabetics and is correlated with poor outcome. Previous work in our laboratory showed that cyclic adenosine monophosphate (cAMP) reduces miR-375 expression and could provide a way to restore normal miR-375 levels, however the transcription repression mechanism is unknown. Using a chromatin immunoprecipitation assay we show that cAMP response element modulator (CREM) binds to the miR-375 promoter 3-fold above background and we find that CREM represses transcription from the miR-375 promoter 1.8-fold. While investigating miR-375 target genes we discovered that several microRNA:mRNA target prediction algorithms listed human CREM as a target gene of miR-375. The predicted binding site is conserved in primates but not in other species. We found that indeed miR-375 binds to the predicted site on human CREM and represses translation of a green fluorescent protein reporter gene by 30%. These findings suggest a primate-specific double-negative feedback loop, a mechanism that would keep these important β-cell regulators in check.

MicroRNA-122 inhibits hepatic stellate cell proliferation and activation in vitro and represses carbon tetrachloride-induced liver cirrhosis in mice

ObjectiveThis study aimed to determine the roles of microRNA (miR)-122 in the activation of hepatic stellate cells (HSCs) and liver cirrhosis. MethodsRat primary HSCs were incubated with transforming growth factor-beta (TGF-β), during which miR-122 and EphB2 expression was measured. miR-122 mimic and/or pcDNA3.1 EphB2 was transfected into TGF-β-induced HSCs. A mouse model of liver cirrhosis was established via an intraperitoneal injection of carbon tetrachloride (CCl4), followed by the injection of miR-122 agomir. Levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured. Fibronectin (FN), alpha smooth muscle actin (α-SMA), Collagen I, miR-122, and EphB2 expression was evaluated in liver tissues and HSCs. Cell proliferation was measured using CCK-8 assay. Interactions between miR-122 and EphB2 were assessed using dual luciferase reporter assay. ResultsmiR-122 (0.15-fold) was downregulated and EphB2 (mRNA: 5.06-fold; protein: 2.35-fold) was upregulated after TGF-β induction of HSCs. Overexpressed miR-122 decreased proliferation and EphB2 (mRNA: 0.46-fold; protein: 0.62-fold), FN (mRNA: 0.45-fold; protein: 0.64-fold), α-SMA (mRNA: 0.48-fold; protein: 0.51-fold), and Collagen I (mRNA: 0.44-fold; protein: 0.51-fold) expression in HSCs, which was abrogated by EphB2 upregulation. miR-122 expression was reduced by 0.21-fold and serum ALT and AST levels were enhanced in mice following 8-week CCl4 induction along with increased expression of FN, α-SMA, and Collagen I in liver tissues, which was blocked by miR-122 overexpression. Moreover, EphB2 was a target gene of miR-122. ConclusionmiR-122 curtails HSC proliferation and activation by targeting EphB2 and suppresses liver cirrhosis in mice.

Pseudogene AK4P1 promotes pancreatic ductal adenocarcinoma progression through relieving miR-375-mediated YAP1 degradation.

Pseudogenes have been reported to play oncogenic or tumor-suppressive roles in cancer progression. However, the molecular mechanism of most pseudogenes in pancreatic ductal adenocarcinoma (PDAC) remains unknown. Herein, we characterized a novel pseudogene-miRNA-mRNA network associated with PDAC progression using bioinformatics analysis. After screening by dreamBase and GEPIA, 12 up-regulated and 7 down-regulated differentially expressed pseudogenes (DEPs) were identified. According to survival analysis, only elevated AK4P1 indicated a poor prognosis for PDAC patients. Moreover, we found that AK4 acts as a cognate gene of AK4P1 and also predicts worse survival for PDAC patients. Furthermore, 32 miRNAs were predicted to bind to AK4P1 by starBase, among which miR-375 was identified as the most potential binding miRNA of AK4P1. A total of 477 potential target genes of miR-375 were obtained by miRNet, in which 49 hub genes with node degree ≥ 20 were identified by STRING. Subsequent analysis for hub genes demonstrated that YAP1 may be a functional downstream target of AK4P1. To confirmed the above findings, microarray, and qRT-PCR assay revealed that YAP1 was dramatically upregulated in both PDAC cells and tissues. Functional experiments showed that knockdown of YAP1 significantly suppressed PDAC cells growth, increased apoptosis, and decreased the ability of invasion. In conclusion, amplification of AK4P1 may fuel the onset and development of PDAC by targeting YAP1 through competitively binding to miR-375, and serve as a promising biomarker and therapeutic target for PDAC.

ELF3-induced miR-182 inhibits adipogenic differentiation in Graves’ orbitopathy by targeting thyrotropin receptor

Graves' orbitopathy (GO) is a common autoimmune disease, but its specific pathogenesis has not been fully elucidated. MicroRNAs (miRNAs) possess an important regulatory function in the occurrence and development of autoimmune diseases. In the present study, we explored the potential role of miR-182 in the diagnosis of GO. The expression of miR-182, thyrotropin receptor (TSHR) and adipocytokines was ascertained by qRT-PCR assay. The triglyceride (TG) content was ascertained by ELISA assay. The lipid droplet content was identified by Oil Red O staining. The relationship between E74-like factor 3 (ELF3), miR-182 and TSHR was confirmed by ChIP, dual-luciferase reporter assay and RIP. The expression of miR-182 decreased, while TSHR increased, and adipocytokine (adiponectin, leptin, PPAR-γ, and AP2) levels were upregulated in preorbital adipose tissue of patients with GO and differential medium induced (DM-induced) 3T3-L1 cells. MiR-182 mimics inhibited adipocytokine expression, TG content and lipid droplets; however, miR-182 inhibitor had the opposite results. TSHR was a target gene of miR-182, and TSHR overexpression (oe-TSHR) reversed the effect of miR-182 mimics on adipogenic differentiation of 3T3-L1 by DM treatment. ELF3 transcription promoted miR-182 expression. Oe-ELF3 inhibited adipocytokine expression and reduced TG content and lipid droplets in DM-induced 3T3-L1 cells. MiR-182 inhibitor reversed the effect of oe-ELF3 on adipogenic differentiation in 3T3-L1. ELF3/miR-182/TSHR axis alleviated Graves' orbitopathy by inhibiting adipogenic differentiation.

MiR-375 and miR-5691 exert anti-fibroproliferative effects on hypertrophic scar fibroblasts by suppressing thrombospondin 1 expression

Background: Hypertrophic scar (HS) is characterized by the hyperproliferation of fibroblasts and the excessive deposition of extracellular matrix (ECM). Thrombospondin 1 (THBS1) is a component of the ECM, which has been implicated in HS formation. Objectives: This study aimed to explore whether miR-375/miR-5691 could modulate HS formation by targeting THBS1. Methods: The expression levels of miR-375/miR-5691/THBS1 in HS and normal skin tissues were measured by quantitative reverse transcription-polymerase chain reaction. 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide and Western blot assays were performed on fibroblasts isolated from HS tissues (HSFBs) to determine cell proliferation and the expression levels of proliferating cell nuclear antigen (PCNA), apoptosis-related proteins (caspase3/9, cleaved caspase3/9, Bax, and Bcl-2), and ECM-related proteins. The binding sites between THBS1 and miR-375/miR-5691 were predicted by the TargetScan. Dual-luciferase reporter and anti-Ago2 immunoprecipitation assays were applied to confirm the interactions between THBS1 and miR-375/miR-5691. Results: The expression levels of both miR-375 and miR-5691 were downregulated in HS tissues and HSFBs, which were negatively correlated with THBS1 expression levels. The overexpression of miR-375/miR-5691 inhibited cell proliferation and ECM production, and promoted apoptosis of HSFBs, while silencing of miR-375/miR-5691 led to an opposite result. In the mechanism analysis, THBS1 was confirmed as the direct target gene of miR-375/miR-5691. Furthermore, rescue experiments showed that the suppressed growth of HSFBs and ECM production induced by silencing of THBS1 was reversed by miR-375/miR-5691 inhibitors. Conclusion: MiR-375/miR-5691 was downregulated in HS tissues, and it could suppress the hyperproliferation and ECM production of HSFBs by targeting THBS1.

MicroRNA-126 and VEGF enhance the function of endothelial progenitor cells in acute myocardial infarction.

Previous studies have found that microRNA-126 (miR-126) overexpression can exert beneficial effects on endothelial function and angiogenesis. The role of miR-126 was previously reported to be by directly limiting the activities of negative regulators of the vascular endothelial growth factor (VEGF) pathway, such as PI3K regulation subunit 2 (PIK3R2). The aim of the present study was to investigate the role of the miR-126/PIK3R2/VEGF axis in endothelial progenitor cells (EPCs) under hypoxic conditions. An in vitro hypoxia model in EPCs was established by exposing EPCs to hypoxia (O2/N2/CO2, 1/94/5) for 72 h, before reverse transcription-quantitative PCR (RT-qPCR) and western blot analyzes were used to measure miR-126 and PIK3R2 expression in EPCs. The proliferation, migration and tube-forming ability of the transfected cells were measured using MTT, Transwell and tube formation assays, respectively. miR-126 expression was found to be lower in EPCs in the hypoxia group compared with that in the control group (P<0.01). The expression of PIK3R2, a direct target gene of miR-126, was found to be higher in the hypoxia group compared with that in the control group (P<0.01). miR-126 mimic and VEGF-plasmid co-transfection improved the proliferation, migration, tube-forming ability and restored the phosphorylation of AKT in EPCs under hypoxic conditions (all P<0.01). In addition, the effects of miR-126 mimic on hypoxia-induced EPCs were reversed by PIK3R2-plasmid co-transfection, whilst the effects of VEGF-plasmid were enhanced further by co-transfection with the miR-126 mimic. In conclusion, miR-126 promoted the functions of EPCs under hypoxic conditions by negatively targeting PIK3R2, whilst the combined overexpression of miR-126 and VEGF enhanced these aforementioned effects.

Tumor suppressor functions of miRNA-375 in nasopharyngeal carcinoma through inhibition of ubiquitin-specific protease 1 expression.

Nasopharyngeal carcinoma (NPC) development involves many genetic alterations. This study profiled differentially expressed microRNAs (DE-miRNAs) and selected miR-375 for further study. DE-miRNAs were screened using online databases and subjected to various analyzes. miR-375 mimics with negative control (NC) cDNA, and a ubiquitin-specific protease 1 (USP1) as well as a NC group were transfected into NPC cells for analysis by quantitative PCR, western blotting, wound healing, Transwell, flow cytometry, cell counting kit-8 (CCK-8), and luciferase gene reporter assays. Among these DE-miRNAs, miR-375 was downregulated and miR-21 was upregulated in NPC cells. Bioinformatical analysis identified USP1 as a potential target gene of miR-375. Increased USP1 expression was associated with poor survival of head and neck cancer patients. The luciferase assay confirmed miR-375 binding to the USP1 3'-untranslated region (UTR), while the transfection experiment confirmed miR-375 expression reduced USP1 expression. USP1 overexpression reversed the anti-tumor activity of miR-375 in NPC cells as determined by tumor cell migration, invasion, apoptosis, and viability assays. In addition, USP1 overexpression activated phosphoinositide 3-kinase (PI3K) signaling, whereas a selective PI3K inhibitor (S2739) could reverse the effects of USP1 on NPC cells in vitro. miR-375 and miR-21 are both related to NPC and miR-375 can target USP1. Further experiments revealed that up-regulated miR-375 expression led to USP1 down-regulation, and miR-375 overexpression suppressed PI3K/Akt signaling and inhibited NPC cell migration and invasion, but promoted NPC cell apoptosis.

Global identification of circular RNAs in imatinib (IM) resistance of chronic myeloid leukemia (CML) by modulating signaling pathways of circ_0080145/miR-203/ABL1 and circ 0051886/miR-637/ABL1

Imatinib (IM), targeting of BCR-ABL1 tyrosine kinase, is currently one of the first-line choices in the treatment of chronic myeloid leukemia (CML). This study aims to explore the molecular mechanisms underlying IM resistance in CML treatment. 108 CML patients were recruited and grouped according to their sensitivity to IM as the responder group (N = 66) and the non-responder group (N = 42). Real-time quantitative PCR (RT-qPCR) was performed to evaluate the expression of candidate circular RNAs (circRNAs), microRNA (miRNAs) and messenger RNA (mRNAs). No significant difference was noted regarding demographic and clinicopathological characteristics between the responder group and the non-responder group. The expression of circ_0080145, circ_0051886 and ABL1 mRNA was significantly increased, while the expression of miR-203 and miR-637 was decreased in the non-responder group as compared with the responders. By using in-silicon analysis, it was predicted that circ_0080145 and circ_0051886 targeted miR-203 and miR-637 respectively, and ABL1 was found to be shared direct target gene of miR-203 and miR-637. Ectopic over-expression of circ_0080145 and circ_0051886 respectively reduced the expression of miR-203 and miR-637. The expression of ABL1 mRNA/protein was most upregulated in culture cells co-transfected with circ_0080145 and circ_0051886 as compared with those cells individually transfected. This study established the signaling pathways of circ_0080145/miR-203/ABL1 and circ 0051886/miR-637/ABL1. The deregulation of circ_0080145 and circ_0051886 is, at least partially, responsible for the development of IM chemoresistance in CML by regulating expression of ABL1 via modulating expression of miR-203 and miR-637.

Circ_0043532 regulates miR-182/SGK3 axis to promote granulosa cell progression in polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women at childbearing age. Several circular RNAs (circRNAs) have been demonstrated to be involved in PCOS. In this study, we aimed to explore the function and mechanism of circ_0043532 in PCOS.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the expression of circ_0043532, miR-182 and serum/glucocorticoid regulated kinase family member 3 (SGK3). Cell proliferation was assessed by 5-ethynyl-2′-deoxyuridine (EdU) assay and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Flow cytometry analysis was employed to evaluate cell cycle and cell apoptosis. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the association between miR-182 and SGK3. Western blot assay was carried out to determine the protein level of SGK3.ResultsCirc_0043532 was markedly elevated in PCOS granulosa cells (GCs) and KGN cells. Silencing of circ_0043532 suppressed cell proliferation and cell cycle process and promoted cell apoptosis in PCOS GCs and KGN cells. For mechanistic analysis, circ_0043532 was identified as a sponge of miR-182 and SGK3 was confirmed to be a target gene of miR-182. Inhibition of miR-182 rescued the impacts of circ_0043532 interference on PCOS GCs and KGN cell progression. Moreover, miR-182 overexpression suppressed cell proliferation and cell cycle process and promoted cell apoptosis in PCOS GCs and KGN cells by targeting SGK3.ConclusionDeficiency of circ_0043532 suppressed cell proliferation and induced cell cycle arrest and cell apoptosis in PCOS by modulation of miR-182/SGK3 axis.

Long Non-Coding RNA GRIK1-AS1 Inhibits the Proliferation and Invasion of Gastric Cancer Cells by Regulating the miR-375/IFIT2 Axis.

Long non-coding RNAs (lncRNAs) play important roles in various biological processes and human diseases, including cancer. In this study, we demonstrated a regulatory relationship between lncRNA GRIK1-AS1 and miR-375/IFIT2 axis in gastric cancer. Our results show a decreased expression of GRIK1-AS1 in gastric cancer tissues compared to adjacent normal gastric tissues. Gastric cell lines also have reduced levels of GRIK1-AS1 compared to gastric epithelial cell line GES-1. Ectopic expression of GRIK1-AS1 in gastric cancer cell lines significantly inhibits cellular viability, migration, and invasion. RNA-pull down and the luciferase activity assays show that GRIK1-AS1 mainly interacts specifically with miR-375. We further demonstrate a negatively regulatory relationship between lncRNA GRIK1-AS1 and miR-375. We discovered that IFIT2 was one of the direct key downstream target genes of miR-375, and established the important role of the GRIK1-AS1/miR-375/IFIT2 axis in the progression of gastric cancer. Taken together, our results revealed a novel mechanism of GRIK1-AS1 as a sponge to miR-375 that impacts gastric cancer progression via modulating target mRNA IFIT2 translation, and as a result, opens a new strategy to future GRIK1-AS1 based therapeutic development.

MiR-122 Inhibits the Cervical Cancer Development by Targeting the Oncogene RAD21.

Cervical cancer (CC) is one of the most frequentlydiagnosed tumors in female. miR-122 has been proved to be dominant in CC. The particular role of miR-122 in CC is unclear. Thus, we attempted to investigate the prognostic role of miR-122 in CC. We used the database of Kaplan-Meier curve plot. Growth and apoptosis of C33A cells were detected by CCK-8, colony formation assay, transwell assays and flow cytometry analysis. The target gene of miR-122 was identified using bioinformatics, q-PCR, western blot and luciferase assay. It showed that CC patients with overexpression of miR-122 have a better prognosis in the Kaplan-Meier plot database analysis. Overexpressed miR-122 inhibited the malignant growth and induced apoptosis of CC. miR-122 targeting of RAD21 cohesin complex component (RAD21) was identified using bioinformatics, Q-PCR, western blot and luciferase assay analyses. Moreover, we found miR-122 conduct its functions via RAD21 via the PI3K/AKT signaling pathway. Importantly, overexpression of RAD21 restored the roles of miR-122 in CC. Our data suggested that miR-122 could block malignant growth and promoted apoptosis by targeting RAD21 in CC. Our finding indicates miR-122 could potentially participate in the pathogenesis and be a biomarker or the potential therapeutic target of CC.

Blood Orange Juice Intake Modulates the Expression of miR-126–3p and let-7f-5p in PBMC of Overweight and Insulin Resistance Women

ObjectivesTo investigate the effect of chronic blood orange juice intake on the microRNA profile in peripheral blood mononuclear cells (PBMC) of overweight and insulin resistance women. MethodsInterventional and chronic study with women (n = 8) aged 18 to 40 years, diagnosed with overweight [body mass index (BMI) 25–29.9 kg/m2] and insulin resistance [homeostatic model assessment insulin resistance (HOMA-IR) index >2,71]. For four weeks, the volunteers ingested 500 mL/day of blood orange juice (Moro variety), with blood samples collected at baseline and four weeks after the beginning of drink ingestion. Evaluation of the expression of 137 microRNAs in PBMC was performed by real-time polymerase chain reaction (PCR). ResultsBlood orange juice intake decreased the expression of miR-126-3p (p = 0.004) and let-7f-5p (p = 0.005) in PBMC. These microRNAs are involved in suppressing the synthesis of several proteins of the insulin signaling pathway. Insulin receptor substrates (IRS) 1 and 2 were identified as target genes of mir-126. Insulin-like growth factor 1 receptor (IGF1R), insulin receptor (INSR), IRS2, phosphatidylinositol-3-kinase interacting protein 1 (PIK3IP1), and protein kinase B/Akt 2 (AKT2) were identified as target genes of let-7f. ConclusionsBlood orange juice, rich in vitamin C, flavonoids, and anthocyanins, downregulates the expression of microRNA involved in impairing the insulin signaling pathway. Funding SourcesFood Research Center (FoRC), São Paulo Research Foundation (FAPESP)

Sevoflurane alleviates hepatic ischaemia/reperfusion injury by up-regulating miR-96 and down-regulating FOXO4.

Hepatic ischaemia/reperfusion (I/R) injury represents an event characterized by anoxic cell death and an inflammatory response, that can limit the treatment efficacy of liver surgery. Ischaemic preconditioning agents such as sevoflurane (Sevo) have been highlighted to play protective roles in hepatic I/R injury. The current study aimed to investigate the molecular mechanism underlying the effects associated with Sevo in hepatic I/R injury. Initially, mouse hepatic I/R injury models were established via occlusion of the hepatic portal vein and subsequent reperfusion. The expression of forkhead box protein O4 (FOXO4) was detected using reverse transcription quantitative polymerase chain reaction and Western blot analysis from clinical liver tissue samples obtained from patients who had previously undergone liver transplantation, mouse I/R models and oxygen‐deprived hepatocytes. The morphology of the liver tissues was analysed using haematoxylin‐eosin (HE) staining, with apoptosis detected via TUNEL staining. Immunohistochemistry methods were employed to identify the FOXO4‐positive cells. Mice with knocked out FOXO4 (FOXO4‐KO mice) were subjected to I/R. In this study, we found FOXO4 was highly expressed following hepatic I/R injury. After treatment with Sevo, I/R modelled mice exhibited an alleviated degree of liver injury, fewer apoptotic cells and FOXO4‐positive cells. FOXO4 was a target gene of miR‐96. Knockdown of FOXO4 could alleviate hepatic I/R injury and decrease cell apoptosis. Taken together, the key observations of our study suggest that Sevo alleviates hepatic I/R injury by means of promoting the expression of miR‐96 while inhibiting FOXO4 expression. This study highlights the molecular mechanism mediated by Sevo in hepatic I/R injury.

MicroRNA-126 engineered muscle-derived stem cells attenuates cavernosa injury-induced erectile dysfunction in rats.

Background: Cavernosa injury is a common cause of organic erectile dysfunction (ED), which requires safe and effective treatments. In the present study, the therapeutic efficiency of muscle-derived stem cells (MDSCs) modified with microRNA-126 (miR-126) was determined in rats with cavernosa injury.Methods: MDSCs were transfected with miR-126 and then were transplanted into rats with cavernosa injury. Erectile function, vascular function (western blot and immunofluorescence), extraction, and detection of exosomes were then undertaken.Results: On the 28th day after transplantation, the highest value of intra-cavernous pressure (ICP)/mean arterial pressure (MAP) in rats of miRNA-126 group (0.84 ± 0.14) was observed (Control: 0.38 ± 0.07; MDSC: 0.54 ± 0.11, Vector: 0.60 ± 0.02; respectively). Treatment of miRNA-126-modified-MDSCs remarkably strengthened vascular structure, supported by hematoxylin-eosin staining. The expression of CD31, von Willebrand Factor and vascular endothelial factors were higher than those in other groups, indicating improved vascular function. In vitro mechanism studies showed that exosomes containing miR-126 isolated from MDSCs promoted angiogenesis and attenuated apoptosis of human umbilical venous endothelial cells. Finally, insulin receptor substrate 1 and Krüppel-like factor 10 were determined as the direct target genes of miR-126.Conclusions: MiR-126 engineered MDSCs notably repaired cavernosa injury in rats via vascular reconstruction by directly targeting IRS1 and KLF10, in which the exosomes secreted by MDSCs played a critical role.

EZH2 is associated with cartilage degeneration in osteoarthritis by promoting SDC1 expression via histone methylation of the microRNA-138 promoter

Cartilage degeneration has been reported to deteriorate osteoarthritis (OA), a prevalent joint disease caused by intrinsic and epigenetic factors. This study aimed to examine the molecular mechanism of enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)/microRNA-138 (miR-138)/syndecan 1 (SDC1) and its epigenetic regulation in cartilage degeneration in OA. An OA cell model was induced by stimulating chondrocytes with interleukin (IL)-1β at a final concentration of 10 ng/mL, followed by alterations in EZH2 and miR-138 expression. Afterwards, cell apoptosis was analyzed using flow cytometry. The expression patterns of cartilage catabolism-related factors (MMP-13, ADAMTS-4, and ADAMTS-5) were determined using RT-qPCR and western blot analyses. The EZH2 and H3K27me3 enrichment at the miR-138 promoter region were determined using ChIP-qPCR. Finally, an OA mouse model was constructed to verify the function of EZH2 in vivo. EZH2 was expressed at high levels in OA models. EZH2 depletion ameliorated OA, as evidenced by reduced cell apoptosis in IL-1β-treated chondrocytes and decreased levels of cartilage catabolism-related factors. Moreover, EZH2 promoted histone methylation at the miR-138 promoter to suppress miR-138 expression, thereby upregulating the expression of SDC1, a target gene of miR-138. Changes in this pathway increased the expression of cartilage catabolism-related factors in vitro while promoting cartilage degeneration in vivo. Our data provided evidence that EZH2 inhibits miR-138 expression by promoting the histone methylation of its promoter, which induces cartilage degeneration in OA models by upregulating SDC1 expression, suggesting a novel mechanistic strategy for OA treatment.

MiR-375 Promotes Pancreatic Differentiation In Vitro by Affecting Different Target Genes at Different Stages.

Human embryonic stem cells (hESCs) possess the ability to differentiate into insulin-producing cells (IPCs), which can be used to treat type I diabetes. miR-375 is an essential transcriptional regulator in the development and maturation of the pancreas. In this study, we optimized a protocol to differentiate hESCs into IPCs and successfully obtained IPCs. Then, we performed overexpression and inhibition experiments of miR-375 on cells at different stages of differentiation and performed RNA-seq. The results showed that the expression of miR-375 fluctuated during hESC differentiation and was affected by miR-375 mimics and inhibitors. miR-375 influences global gene expression and the target genes of miR-375. The overexpression of miR-375 can cause changes in multiple signaling pathways during pancreatic development. miR-375 is a major participant in the differentiation of pancreatic β-cells through different target genes at different stages. This study provides new ideas for further investigation of how microRNAs affect cell fate and gene transcription.

MiR-218-2 regulates cognitive functions in the hippocampus through complement component 3–dependent modulation of synaptic vesicle release

microRNA-218 (miR-218) has been linked to several cognition related neurodegenerative and neuropsychiatric disorders. However, whether miR-218 plays a direct role in cognitive functions remains unknown. Here, using the miR-218 knockout (KO) mouse model and the sponge/overexpression approaches, we showed that miR-218-2 but not miR-218-1 could bidirectionally regulate the contextual and spatial memory in the mice. Furthermore, miR-218-2 deficiency induced deficits in the morphology and presynaptic neurotransmitter release in the hippocampus to impair the long term potentiation. Combining the RNA sequencing analysis and luciferase reporter assay, we identified complement component 3 (C3) as a main target gene of miR-218 in the hippocampus to regulate the presynaptic functions. Finally, we showed that restoring the C3 activity in the miR-218-2 KO mice could rescue the synaptic and learning deficits. Therefore, miR-218-2 played an important role in the cognitive functions of mice through C3, which can be a mechanism for the defective cognition of miR-218 related neuronal disorders.

LncRNA MALAT1 Functions as a Competing Endogenous RNA to Regulate BMI1 Expression by Sponging miR-200c/miR-203 in the Control of the Differentiation of Pulp Cells.

Long non-coding RNAs (lncRNAs) and miRNAs (microRNAs) are considered as key regulators of several biological processes, including dental development. In this study, we explored the lncRNAs and miRNAs which are involved in dental development. Real-time PCR was performed to identify the candidate lncRNAs and miRNAs involved in dental development. Bioinformatics analysis and luciferase assay were carried out to establish the regulatory relationships between MALAT1, miR-203 and miR-200c in dental development. Among all candidate lncRNAs, only MALAT1 was highly expressed in differentiated human dental pulp cells (hDPCs), and among all candidate miRNAs which are down-regulated in differentiated hDPCs, miR-203, and miR-200c are most decreased. Furthermore, MALAT1 was up-regulated while miR-203 and miR-200c were down-regulated in differentiated hDPCs in a time-dependent manner. MiR-203 and miR-200c were proved to bind to MALAT1. Moreover, BMI1 was identified as a target gene of miR-203 or miR-200c, and BMI1 was time-dependently decreased in hDPCs cultured with odontogenic medium. On the contrary, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), osteocalcin (OCN), and alkaline phosphatase (ALP), were time-dependently increased in hDPCs cultured with odontogenic medium. Finally, the overexpression of MALAT1 and the knockdown of miR-203/miR-200c both significantly increased the levels of BMI1, DSPP, DMP-1, OCN, and ALP, while the effect of knockdown of miR-203/miR-200c was much stronger than that of the overexpression of MALAT1. Our results demonstrated that MALAT1 functions as a competing endogenous RNA of miR-203 and miR-200c and accordingly promotes BMI1 expression. Therefore, MALAT1 may serve as a biomarker for dental development.

The microRNA-429/DUSP4 axis regulates thesensitivity of colorectal cancer cells to nintedanib.

Colorectal cancer (CRC) is recognized as one of the most common malignancies, which ranks third among all cancer-related deaths worldwide. Nintedanib is an orally available tyrosine kinase inhibitor that can treat CRC; however, drug resistance to nintedanib leads to unsatisfactory treatments for patients with CRC. The aim of the present study was to explore whether overexpression of miR-429 elevated the sensitivity of CRC cells to nintedanib by downregulating dual specificity protein phosphatase 4 (DUSP4). The nintedanib-resistant CRC cell model was established via the treatment of cells with nintedanib in a dose-dependent manner. Reverse transcription-quantitative PCR was used to detect the expression levels of miR-429 and DUSP4, and to confirm the transfection efficiency of miR-429 mimic and DUSP4 overexpression plasmid. Cell Counting Kit-8 assay was utilized to measure the inhibition rate of cells. Western blotting was conducted to observe the expression levels of DUSP4 protein, apoptosis-related proteins and proteins related to the JNK signaling pathway. Dual-luciferase reporter assay was performed to evaluate luciferase activity and TUNEL assay was conducted to detect the apoptosis of cells. The results revealed that miR-429 mimic elevated the sensitivity of CRC cells to nintedanib. Moreover, by ENCORI prediction, DUSP4 was identified as a target gene of miR-429, and overexpression of DUSP4 reversed the inducing effect of miR-429 overexpression on the sensitivity of CRC cells to nintedanib. In conclusion, overexpression of miR-429 may elevate the sensitivity of CRC cells to nintedanib through inhibition of the JNK signaling pathway by targeting DUSP4. These findings may aid in the prevention of drug resistance of CRC cells to nintedanib.