Overexpression Of miR-125b Research Articles

MiR-19 3b regulated the formation of coat colors by targeting WNT10A and GNAI2 in Cashmere goats

MiRNAs as a series of small noncoding RNAs that play a crucial part in regulating coat color and hair follicle development. In the previous Solexa sequencing experiments, there were many miRNAs expressed differentially in alpacas with different coat color, including miR-193b.But the mechanism of miR-193b in mammalian pigmentation is still unknown. In this study, bioinformatics analysis showed that WNT10A and GNAI2 might be the target genes of miR-193b. qRT-PCR showed the expression of miR-193b in white Cashmere goats’ skins was obviously lower than that in browns, and the expression of WNT10A and GNAI2 were similar with miR-193b. The protein levels of WNT10A and GNAI2 indicated the same findings. Furthermore, the expression of WNT10A and GNAI2 in keratinocytes were analyzed from mRNA and protein levels, the results manifested that the group of overexpression of miR-193b in HaCaT cells increased the expressions of target genes, and miR-193b inhibition group reduced expressions. Luciferase report assays confirmed that the targeting relationship between miR-193b and target genes (WNT10A and GNAI2), the results showed that miR-193b was positively correlated with target genes. These experimental data showed that miR-193b might participate in adjustment of coat color in skin tissue of Cashmere goat by targeting WNT10A and GNAI2.

Effects of miR-133b on oxLDL-induced vascular endothelial cell injury by targeting SGTB

Objective: To investigate the effect of microRNA-133b (miR-133b) on oxidized low density lipoprotein (oxLDL) induced vascular endothelial cell injury by targeting small protein molecules rich in glutamine 34-tetrapeptide repeats (SGTB). Methods: Human umbilical vein endothelial cells (EVC-304) were induced by 100 μg/ml oxLDL for 24 h to construct a vascular endothelial cell injury model. EVC-304 cells were divided into control group, oxLDL group (oxLDL treatment), oxLDL+miR-NC group (transfectted with 20 nmol/L miR-NC+oxLDL treatment), oxLDL+miR-133b group (transfectted with 20 nmol/L miR-133b mimics +oxLDL treatment), oxLDL+si-NC group (transfectted with 20 nmol/L si-NC+oxLDL treatment), oxLDL+si-SGTB group (transfected with 20 nmol/L si-SGTB+oxLDL treatment), oxLDL+miR-133b+ pcDNA group (transfected with 20 nmol/L si-SGTB and pcDNA+oxLDL), oxLDL+miR-133b+pcDNA-SGTB group (transfected with 20 nmol/L si-SGTB and pcDNA-SGTB), 9 wells in each group. Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-133b and SGTB; flow cytometry was used to detect cell apoptosis; kits were used to detect malondialdehyde (MDA) content and the activities of superoxide disproportionation enzyme (SOD) and glutathione peroxidase (GSH-Px). The expression levels of Bcl-2 and Bax protein were detected by Western blot. The dual luciferase reporter gene assay and Western blot were used to verify the targeted and regulatory between miR-133b and SGTB. Results: Compared with the control group, the expressions of miR-133b and Bcl-2 in EVC-304 cells were decreased significantly after oxLDL induction, while the expression levels of SGTB and Bax were sincreased ignificantly (P＜0.05), the MDA content and apoptosis rate were increased significantly (P＜0.05), and the activities of SOD and GSH-Px were decreased significantly (P＜0.05). Over-expression of miR-133b or interfering with SGTB inhibited oxLDL-induced apoptosis and oxidative stress in EVC-304 cells (P＜ 0.05). miR-133b directly bound to SGTB, miR-133b overexpression significantly down-regulated SGTB expression (P＜0.05), miR-133b inhibition significantly up-regulated SGTB expression (P＜0.05) Over-expression of SGTB reversed the effect of over-expressing miR-133b on oxLDL-induced vascular endothelial cell injury (P＜0.05). Conclusion: miR-133b could attenuate oxidative stress damage and apoptosis induced by oxLDL in vascular endothelial cells by targeting and inhibiting SGTB expression.

Liraglutide suppresses obesity and promotes browning of white fat via miR-27b in vivo and in vitro.

ObjectiveTo investigate the effect of liraglutide on the browning of white fat and the suppression of obesity via regulating microRNA (miR)-27b in vivo and in vitro.MethodsSprague-Dawley rats were fed a high-fat (HF) diet and 3T3-L1 pre-adipocytes were differentiated into mature white adipocytes. Rats and mature adipocytes were then treated with different doses of liraglutide. The mRNA and protein levels of browning-associated proteins, including uncoupling protein 1 (UCP1), PR domain containing 16 (PRDM16), CCAAT enhancer binding protein β (CEBPβ), cell death-inducing DFFA-like effector A (CIDEA) and peroxisome proliferator-activated receptor-γ-coactivator 1α (PGC-1α), were detected using quantitative real-time polymerase chain reaction and Western blotting.ResultsLiraglutide decreased body weight and reduced the levels of blood glucose, triglyceride and low-density lipoprotein cholesterol in HF diet-fed rats. Liraglutide increased the levels of UCP1, PRDM16, CEBPβ, CIDEA and PGC-1α in vivo and vitro. The levels of miR-27b were upregulated in HF diet-fed rats, whereas liraglutide reduced the levels of miR-27b. In vitro, overexpression of miR-27b decreased the mRNA and protein levels of UCP1, PRDM16, CEBPβ, CIDEA and PGC-1α. Transfection with the miR-27b mimics attenuated the effect of liraglutide on the browning of white adipocytes.ConclusionLiraglutide induced browning of white adipose through regulation of miR-27b.

MiR-133b suppresses the proliferation, migration and invasion of lung adenocarcinoma cells by targeting SKA3

ABSTRACT Spindle and Kinetochore Associated Complex Subunit 3 (SKA3) is crucial for anaphase mitosis. However, the relationship between SKA3 and lung adenocarcinoma (LUAD) has not been fully clarified. Differentially expressed genes were first identified by analyzing data from TCGA. It was found that miR-133b was significantly lowly expressed in LUAD, while SKA3 was remarkably highly expressed. Cell Counting Kit-8 (CCK8), wound healing assay and Transwell assay uncovered that overexpressing miR-133b could inhibit the proliferation, invasion and migration of LUAD cells. In addition, the targeting relationship between miR-133b and SKA3 was also verified by dual-luciferase analysis. Moreover, it was proved by the rescue assay that the overexpression of miR-133b significantly downregulated SKA3 in LUAD cells. All in all, these findings revealed the role of miR-133b and SKA3 in regulating the proliferation, migration, and invasion of LUAD cells. This study could yield new information about the mechanisms of LUAD.

Potential prognostic value of miRNAs as biomarker for progression and recurrence after nephrectomy in renal cell carcinoma: a literature review.

We provide a systematic literature review on tissue miRNAs in patients with RCC to evaluate and summarize their usefulness as prognostic markers. We undertook a systematic search for articles in English using the PubMed-Medline database from January 2010 to December 2020. Studies were identified and selected according to the PRISMA criteria and the PICO methodology. The population consisted of RCC patients undergoing nephrectomy and the main outcome of interest was recurrence-free survival (RFS). Only studies providing hazard ratios (HRs) from multivariate or univariate analyzes with corresponding 95% confidence intervals (CI) and/or area under the curve (AUC) were considered. All nine included studies (1,541 patients) analyzed the relationship between tissue miRNA expression levels (up or downregulated) and RFS. Some of these found that the methylation status of miR-9-1, miR-9-3 and miR-124 was associated with a high risk of relapse. Moreover, miR-200b overexpression was associated with OS. MiR-210 overexpression indicated a shorter OS than those who were miR-210 negative. Finally, patients with high miR-125b expression had shorter cancer-specific survival (CSS) than those with low expression; similarly, patients with low miR-126 expression also had shorter CSS time. Several studies tested the usefulness of specific miRNAs to predict RCC recurrence. Some of them showed a fair accuracy and strong relationship between specific miRNA over or under-expression and survival outcomes. However, results from these studies are preliminary and miRNAs use in routine clinical practice is still far to come.

Hsa_circ_0001806 promotes glycolysis and cell progression in hepatocellular carcinoma through miR-125b/HK2.

ObjectiveHepatocellular carcinoma (HCC) is one of the most common malignant tumours and a leading cause of cancer death. Circular RNA (circRNA) has been demonstrated to play an important role in regulating tumour development. The current study aims to explore the specific role of hsa_circ_0001806 during HCC progression.MethodsThe expression of hsa_circ_0001806 in HCC tissues and cells was measured through qRT‐PCR. Cell proliferation, apoptosis and migration were measured using CCK‐8 and Annexin V/PI staining kits, and Transwell assay. Bioinformatics prediction and dual‐luciferase reporter assay were adopted to explore the mechanism underlying the cell function of hsa_circ_0001806 in HCC cells. In addition, glycolysis was assessed by measuring the glucose uptake, lactate production and ATP level using a glucose assay kit, fluorometric lactate assay kit and ATP detection assay kit.ResultsHsa_circ_0001806 was up‐regulated in HCC tissues and cells and positively associated with the advanced TNM stage, metastasis and poor overall survival. The overexpression of hsa_circ_0001806 promoted HCC cell proliferation, migration and glycolysis and inhibited cell apoptosis, while the silence of hsa_circ_0001806 showed an opposite effect. Furthermore, hsa_circ_0001806 acted as a sponge of miR‐125b to up‐regulate hexokinase II (HK2) expression. In addition, the inhibition of miR‐125b and HK2 overexpression partly reversed the inhibitory effect of hsa_circ_0001806 silencing on HCC cell proliferation, migration and glycolysis.ConclusionThe inhibition of hsa_circ_0001806 suppressed HCC cell proliferation, migration and glycolysis through mediating miR‐125b/HK2 axis.

Brown adipose tissue transplantation ameliorates diabetic nephropathy through the miR-30b pathway by targeting Runx1

ObjectiveAdipose tissue is a major source of circulating microRNAs (miRNAs) that can regulate target genes in distant organs. However, the role of brown adipose tissue (BAT) in diabetic kidney disease (DKD) is still unknown. We studied the original BAT miR-30b targeting two key fibrotic regulators, Runt-related transcription factor 1 (Runx1) and snail family zinc finger 1 (Snail1), to combat DKD. MethodsFirst, we transplanted healthy BAT from normal mouse donors into diabetic mice (induced by a high-fat diet and streptozotocin injection). In vitro, we observed extracellular vesicles (EVs) secreted from brown adipocytes. AgomiR-30b was directly administered to the BAT of diabetic mice twice weekly for 4 consecutive weeks. Next, the role of Runx1 in DKD was determined by using siRUNX1 or pCMV-RUNX1 in HK-2 cells and in diabetic mice treated with AAV9-U6-shRunx1 or AAV9-EF1a-Runx1. ResultsBAT transplantation reactivated endogenous BAT activity in diabetic mice, increased circulating miR-30b levels and significantly ameliorated DKD. In TGFβ1-treated HK-2 cells, miR-30b expression was significantly suppressed. miR-30b overexpression markedly decreased fibronectin and downregulated Runx1 and Snail1 expression, while silencing of miR-30b had the opposite effects. Next, Runx1 knockdown and overexpression mimicked the above phenotype of miR-30b mimics and inhibitors, respectively, both in vitro and in vivo. Moreover, Runx1 promoted TGFβ1-induced fibrosis by upregulating the PI3K pathway. ConclusionBAT-derived miRNAs might be a promising target for kidney protection in diabetes mellitus.

MiR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.

MicroRNA-10b promotes the apoptosis of bovine ovarian granulosa cells by targeting plasminogen activator inhibitor-1

Granulosa cells (GCs) are essential somatic cells in the ovaries, and apoptosis of GCs causes follicular atresia. microRNA-10b (miR-10b) is pivotal for cell apoptosis. However, currently, little is known about the role of miR-10b in bovine ovarian GCs (BGCs). In this study, the effect of miR-10b on the apoptosis of BGCs was investigated. Our results showed that the overexpression of miR-10b could increase the apoptosis rate of BGCs, which is associated with the increased expression of Caspase-3 and decreased expression ratio of Bcl-2/Bax (P < 0.05). Furthermore, plasminogen activator inhibitor-1 (PAI-1) was confirmed to be a validated target of miR-10b in BGCs using dual-luciferase reporter analysis, and transfection of miR-10b mimics decreased the expression of PAI-1 (P < 0.05). In addition, overexpression of PAI-1 significantly inhibited BGC apoptosis (P < 0.05), and PAI-1 could alleviate BGC apoptosis induced by miR-10b (P < 0.05). Subsequently, phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) was found to be the downstream pathway of PAI-1 by RNA-Seq analysis and verified by Western blot. Finally, a PI3K/AKT inhibitor (Miltefosine) was used to inhibit the PI3K/AKT pathway, which reversed the inhibitory effect of PAI-1 on the apoptosis of BGCs (P < 0.05), and enhanced the promotion effect of miR-10b on the apoptosis of BGCs (P < 0.05). Our results indicated that miR-10b promotes BGC apoptosis by targeting PAI-1 to regulate the PI3K/AKT pathway.

P-081: A novel circulating miRNA involved in T cell senescence of multiple myeloma patients

<h3>Background</h3> Many circulating miRNAs were reported as biomarkers of multiple myeloma (MM),but the functions of these circulating miRNAs are not clarified recently. The aim of this work is to identify novel biomarkers of circulating miRNAs in diagnosis and prognosis for MM and reveal their function in the pathogenesis of MM. <h3>Methods</h3> MiRNAs profiling in serum(n=15) and MM cells(CD138+, n=10) were analyzed by RNA-sequencing of MM patients. The common dysregulated miRNAs(P<0.05) were identified via Veen Diagram analysis in serum and MM cells between MM patients and healthy donors, then validated by RT-PCR. ROC analysis and Kaplan-Meier(K-M) analysis were performed to evaluate the differently expressed circulating miRNAs in diagnosis and prognosis in MM patients, respectively. Exosomes of serum were isolated and characterized by dynamic light scattering, transmission microscopy, and western blot analysis. <h3>Results</h3> There were stable and specific miRNA expression profiles in serum and tumor cells of MM patients. The miRNA profiling of MM serum showed that there were 74 differential circulating miRNAs(P<0.05), among which 56 miRNAs(75.7%) were down-regulated and 18 miRNAs were up-regulated(24.3%). MiRNA profiling of MM cells showed that there were 376 miRNAs differentially expressed(P<0.05), of which 87(23.1%) were down-regulated and 289(76.9%) were up-regulated. Veen analysis found 36 common dysregulated miRNAs both in serum and tumor cells of MM. There were 22.3% of differential miRNAs located on chromosome 1 and 17. Five circulating miRNAs(miR-27b-3p, miR-145-3p, miR-628-3p, miR-342-5p and miR-30e-3p) were further confirmed differentially expressed in MM patients(n=201) by RT-PCR. K-M analysis showed that MM patients with low level of miR-27b, miR-145, and miR-628 had shortened PFS and OS. Strikingly, comparing to the decreased level of miR-27b in MM patients serum, miR-27b was enriched in the exosome of serum. MM patients with low level of serum miR-27b displayed lower CD3+T cells and CD3+CD4+T cells in peripheral blood. Our further study found increased proportation of senescent T cells in MM patients with low level of serum miR-27b, especially in CD8+T cells. In order to confirm T cell senescent phenotype, PBMCs were co-cultured with MM patients serum exosomes which mimic the in vivo situation. Flow cytometry analysis showed the CD28 expression was notably decreased on the CD3+ T cells in the co-culture group. In addition, RT-PCR and flow cytometry analysis showed that overexpression of miR-27b significantly down-regulated the level of CD28 in jurkat cells, a T cell line. These findings suggested an immune suppressive microenvironment in MM patients with lower level of serum miR-27b. <h3>Conclusion</h3> Circulating miRNA works as a usful biomarker in diagnosis and prognosis of MM. Strigently, serum miR-27b is a novel circulating miRNA involved in T cell senescence of multiple myeloma patients which indicated the poor outcome of patients with MM.

MiR-125b enhances autophagic flux to improve septic cardiomyopathy via targeting STAT3/HMGB1

We explore the role of miR-125b in septic cardiomyopathy, focusing on miR-125b/STAT3/HMGB1 axis. CLP mouse model and LPS-stimulated primary rat cardiomyocytes (CMs) and H9C2 cell were used as in vivo and in vitro models of septic cardiomyopathy, respectively. qRT-PCR and western blot were performed to measure expression levels of miR-125b, STAT3, HMGB1, and autophagy-related proteins. MTT assay was employed to examine LPS toxicity. Dual luciferase activity assay and CHIP were performed to validate interactions of miR-125b/STAT3 and STAT3/HMGB1 promoter. Immunostaining was used to assess the level of autophagic flux. ROS level was measured by fluorescence assay. Heart functions were examined via intracoronary Doppler ultrasound. miR-125b was diminished while STAT3 and HMGB1 were elevated in the heart tissue following CLP surgery and in LPS-treated H9C2 cells. LPS treatment up-regulated ROS generation and suppressed autophagic flux. Overexpression of miR-125b mimics or knockdown of STAT3 or HMGB1 alleviated LPS-induced hindrance of autophagic flux and ROS production. miR-125b directly targeted STAT3 mRNA and STAT3 bound with HMGB1 promoter. Overexpression of miR-125b mitigated myocardial dysfunction induced by CLP in vivo. Hyperactivation of STAT3/HMGB1 caused by reduced miR-125b contributes to ROS generation and the hindrance of autophagic flux during septic cardiomyopathy, leading to myocardial dysfunction.

MiRNA-26b suppresses the TGF-β2-induced progression of HLE-B3 cells via the PI3K/Akt pathway

To study the effect of miR-26b on lens epithelial cells induced by transforming growth factor beta (TGF-β) 2 and the underlying signaling pathways. Human lens epithelial cell line B-3 (HLE-B3) was incubated with TGF-β2 (5 ng/mL) and then transfected with miR-26b mimics. The expression of miR-26b was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), while 5'-bromodeoxyuridine (BrdU) and wound-healing assays were used to measure the growth and migration of HLE-B3 cells, respectively. The expression of epithelial-mesenchymal transition (EMT) markers and the activity of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway were measured by Western blotting assay and immunofluorescence staining. Electron microscopy was also used to observe cellular morphology. The expression levels of miR-26b were significantly reduced in human posterior capsular opacification-attached lens tissue and TGF-β2-stimulated HLE-B3 cells. In the presence of TGF-β2, the growth, migration, and EMT of HLE-B3 cells were distinctly enhanced; these effects were attenuated by the administration of miR-26b mimics. Furthermore, the overexpression of miR-26b significantly reduced upregulation of the PI3K/Akt pathway when stimulated by TGF-β2 in HLE-B3 cells. Moreover, the addition of an activator (740 Y-P) led to the upregulation of the PI3K/Akt pathway and abolished the protective effect of miR-26b on the HLE-B3 cells that was mediated by TGF-β2. The miR-26b suppresses TGF-β2-induced growth, migration, and EMT in HLE-B3 cells by regulating the PI3K/Akt signaling pathway.

MiR-125b regulates inflammation in bovine mammary epithelial cells by targeting the NKIRAS2 gene

Mastitis is a complex inflammatory disease caused by pathogenic infection of mammary tissue in dairy cows. The molecular mechanism behind its occurrence, development, and regulation consists of a multi-gene network including microRNA (miRNA). Until now, there is no report on the role of miR-125b in regulating mastitis in dairy cows. This study found that miR-125b expression is significantly decreased in lipopolysaccharide (LPS)-induced MAC-T bovine mammary epithelial cells. Also, its expression is negatively correlated with the expression of NF-κB inhibitor interacting Ras-like 2 (NKIRAS2) gene. MiR-125b target genes were identified using a double luciferase reporter gene assay, which showed that miR-125b can bind to the 3′ untranslated region (3′ UTR) of the NKIRAS2, but not the 3′UTR of the TNF-α induced protein 3 (TNFAIP3). In addition, miR-125b overexpression and silencing were used to investigate the role of miR-125b on inflammation in LPS-induced MAC-T. The results demonstrate that a reduction in miR-125b expression in LPS-induced MAC-T cells increases NKIRAS2 expression, which then reduces NF-κB activity, leading to low expression of the inflammatory factors IL-6 and TNF-α. Ultimately, this reduces the inflammatory response in MAC-T cells. These results indicate that miR-125b is a pro-inflammatory regulator and that its silencing can alleviate bovine mastitis. These findings lay a foundation for elucidating the molecular regulation mechanism of cow mastitis.

CircRNA MFACR Is Upregulated in Myocardial Infarction and Downregulates miR-125b to Promote Cardiomyocyte Apoptosis Induced by Hypoxia.

:Circular RNA (circRNA) MFACR promotes cardiomyocyte death that leads to myocardial infarction (MI). This study aimed to explore the role of MFACR in MI. T-qPCRs were performed to measure the expression levels of MFACR and miR-125b in plasma samples from both MI patients (n = 61) and healthy controls (n = 61). MFACR or miR-125b was overexpressed in AC16 cells (cardiomyocytes) to explore the interaction between them. Methylation of miR-125b gene in cells with the overexpression of MFACR was detected by methylation-specific PCR. Cell apoptosis after transfections was detected by cell apoptosis assay. MI model was constructed to further demonstrate the effect of MFACR in vivo. We found that MFACR was upregulated in MI and inversely correlated with miR-125b. In AC16 cells, hypoxia treatment increased the expression levels of MFACR and decreased the expression levels of miR-125b. In AC16 cells, overexpression of MFACR decreased the expression levels of miR-125b and increased the methylation of miR-125b gene. Under hypoxia treatment, overexpression of MFACR increased AC16 cell apoptosis, and overexpression of miR-125b decreased cell apoptosis. In addition, overexpression of miR-125b reversed the effects of overexpression of MFACR on cell apoptosis both in vivo and in vitro.

Abstract MP13: Small RNA Profiling Identifies Mir-200b As A Repressor Of Vascular Smooth Muscle Cell Migration And Proliferation

Background and Objectives: A large body of research has identified miRNA as pivotal regulators of smooth muscle cell (SMC) fate in various vascular diseases. However, global changes of miRNA expression profile during SMC phenotypic modulation have not been well characterized. As the targetome of a given miRNA is highly dependent on the cell type and context, comparative analysis of mRNA and miRNA expression profiles can provide decisive information for assessing the versatility in miRNA functions and discovering novel miRNA modulating SMC phenotype. Our goal is to define the miRNA signatures in differentiated and de-differentiated SMC, the miRNA overall contribution to the transcriptome, and novel functionally relevant miRNA. Methods: By performing RNAseq and small RNAseq, we defined the mRNA/miRNA expression profiles in aortic SMC cultured in differentiation or de-differentiation media with Platelet Derived Growth Factor-BB (PDGF-BB) and performed functional studies on differentially expressed miRNAs (DEmiR). Results: We found that PDGF-BB-induced SMC de-differentiation generated 48 significant DEmiRs, including 15 downregulated and 33 upregulated DEmiRs. Integrative analysis predicted that nearly 40% (1335 of 3523) of differentially expressed transcripts were direct targets of DEmiRs. The miR-200 cluster (miR-200a, miR-200b and miR-429) was found potently downregulated after PDGF-BB treatment, as well as Tet2 and Myocardin knockdown, while its expression increased in rapamycin treated SMC. We found that miR-200b overexpression prevented PDGF-BB-induced SMC migration and proliferation, while miR-200b inhibition had an opposite effect. Finally, we identified Quaking, a potent inducer of SMC phenotypic switching, as a primary target of miR-200b. Conclusion: Our study predicted a marked contribution of miRNA in regulating SMC transcriptomic profile. miR-200b was identified as a novel pro-differentiation miRNA in SMC through inhibition of phenotypic switching.

Baicalein Mediates Mitochondrial Autophagy via miR-30b and the NIX/BNIP3 Signaling Pathway in Parkinson's Disease.

Parkinson's disease (PD) is regarded as a severe neurodegenerative disorder. Baicalein is involved in the treatment of PD. This study explored the mechanism of baicalein in PD. The PD rat model was established using 6-hydroxydopamine. The neurologic score, dopamine (DA) content, apoptotic cells, and neuronal damage were evaluated after rats were treated with baicalein. Autophagy in PD rats was inhibited using 3-methyladenine (3-MA). The mitochondrial membrane potential (MMP) and autophagy-related proteins (LC3, P62) were detected. Next, agomiR-30b was transfected into PD rats. The targeting relation between miR-30b and NIX was predicted and verified. Then, sh-NIX was transfected into PD rats, and the effects of miR-30b and NIX on MMP, LC3, and P62 were assessed. When miR-30b was overexpressed using agomiR-30b, the NIX and BNIP3 levels were detected. Baicalein increased the neurological score and restored DA content, neurons, MMP, and mitochondrial autophagy protein levels. Baicalein inhibited miR-30b expression and miR-30b targeted NIX. miR-30b upregulation or NIX silencing reversed the effect of baicalein on MMP and mitochondrial autophagy. Baicalein upregulated NIX and BNIP3 expressions, while miR-30b overexpression inhibited NIX and BNIP3 expressions. In summary, baicalein mediated mitochondrial autophagy and restored neuronal activity by downregulating miR-30b and activating the NIX/BNIP3 pathway, thus protecting against PD.

MicroRNA-29b reduces myocardial ischemia-reperfusion injury in rats via down-regulating PTEN and activating the Akt/eNOS signaling pathway.

Reperfusion may cause injuries to the myocardium in ischemia situation, which is called ischemia/reperfusion (I/R) injury. The study aimed to explore the roles of microRNA-29b (miR-29b) in myocardial I/R injury. Myocardial I/R injury rat model was established. Differentially expressed miRNAs between the model rats and the sham-operated rats were analyzed. miR-29b expression in myocardial tissues was measured. Gain-of-function of miR-29b was performed, and then the morphological changes, infarct size, myocardial function, oxidative stress, and the cell apoptosis in myocardial tissues were detected. The target relation between miR-29b and PTEN was detected through bio-information prediction and dual luciferase reporter gene assay. Activation of Akt/eNOS signaling was detected. H9C2 cells were subjected to hypoxia/reoxygenation treatment to perform in vitro experiments. I/R rats presented severe inflammatory infiltration, increased infarct size and cell apoptosis, increased oxidative stress and decreased myocardial function. miR-29b was downregulated in I/R rats, and up-regulation of miR-29b reversed the above changes. miR-29b directly bound to PTEN, and overexpression of miR-29b reduced PTEN expression level and increased the protein levels of p-Akt/Akt and p-eNOS/eNOS. In vivo results were confirmed in in vitro experiments. This study provided evidence that miR-29b could alleviate the myocardial I/R injury in vivo and in vitro by inhibiting PTEN expression and activating the Akt/eNOS signaling pathway.

Long Non-Coding RNA CCAT2 Promotes the Development of Esophageal Squamous Cell Carcinoma by Inhibiting miR-200b to Upregulate the IGF2BP2/TK1 Axis.

Long non-coding RNAs (lncRNAs) have been shown to play important roles in human cancers, including esophageal squamous cell carcinoma (ESCC). In the current study, we identified CCAT2 as a relevant lncRNA and investigated its role in the progression of ESCC. RT-qPCR was adopted to detect CCAT2 expression in collected clinical samples, ESCC cell lines, and a normal cell line. We tested the correlation between CCAT2 expression and the prognosis of ESCC. RT-qPCR or immunoblotting was adopted to detect the expression of relevant factors in ESCC tissues or cells. Cell proliferation, apoptosis, migration, and invasion were examined by colony formation assay, flow cytometry, scratch assay, and Transwell assay, respectively, while subcutaneous tumorigenesis in nude mice was adopted to examine the role of CCAT2 in tumorigenesis of ESCC cells in vivo. Bioinformatics analysis, dual luciferase reporter assay, and RIP were conducted for the target relationship profiling. Me-RIP was adopted to detect m6A modification level of TK1 in ESCC tissues or cells. Upregulated CCAT2, IGF2BP2, and TK1 expression and inhibited miR-200b expression were observed in ESCC cells and tissues. CCAT2 bound to miR-200b and reduced its expression, leading to upregulated IGF2BP2 expression. IGF2BP2 improved TK1 mRNA stability to enhance its expression by recognizing its m6A modification. CCAT2 promoted the migration and invasion of ESCC cells in vitro, and tumorigenesis in vivo by upregulating TK1 expression, while overexpression of miR-200b reversed these effects of CCAT2. Overall, this study suggests that CCAT2 competitively binds to miR-200b to alleviate its inhibitory effects on IGF2BP2 expression, resulting in elevated TK1 expression, and an ensuing promotion of the development of ESCC.

CircFBXL5 promotes the 5-FU resistance of breast cancer via modulating miR-216b/HMGA2 axis

BackgroundCircular RNAs (circRNAs) have been confirmed to be relevant to the 5-fluorouracil (5-FU) resistance of breast cancer. Nevertheless, how and whether circRNA F-box and leucine-rich repeat protein 5 (circFBXL5) regulates the 5-FU resistance of breast cancer is uncertain. This study aims to explore the function and mechanism of circFBXL5 in the 5-FU resistance of breast cancer.MethodsThirty nine paired breast cancer and normal tissues were harvested. circFBXL5, microRNA-216b (miR-216b) and high-mobility group AT-hook 2 (HMGA2) abundances were examined via quantitative reverse transcription polymerase chain reaction or western blot. Cell viability, 5-FU resistance, migration, invasion, and apoptosis were tested via cell counting kit-8 assay, wound healing analysis, transwell analysis, and flow cytometry. The relationship of miR-216b and circFBXL5 or HMGA2 was tested via dual-luciferase reporter analysis and RNA pull-down assay. The impact of circFBXL5 on breast cancer tumor growth in vivo was analyzed via xenograft model.ResultscircFBXL5 was highly expressed in breast cancer tissues and cells, and was more upregulated in 5-FU-resistant breast cancer cells. Function experiments showed that circFBXL5 knockdown inhibited the 5-FU resistance of breast cancer by inhibiting cell migration, invasion and promoting apoptosis. In the terms of mechanism, miR-216b could be sponged by circFBXL5, and its inhibitor could also reverse the influence of circFBXL5 silencing on the 5-FU resistance of breast cancer cells. In addition, HMGA2 was a target of miR-216b, and its overexpression also reversed the regulation of miR-216b overexpression on the 5-FU resistance of breast cancer. Furthermore, circFBXL5 interference declined breast cancer tumor growth in xenograft model.ConclusionOur data showed that circFBXL5 could promote the 5-FU resistance of breast cancer by regulating miR-216b/HMGA2 axis.

MiR-125b inhibits cardiomyocyte apoptosis by targeting BAK1 in heart failure

BackgroundAlthough miR-125b plays a crucial role in many human cancers. However, its function in heart failure (HF) remains unclear. Our study aimed to investigate its involvement in heart failure.MethodsIn this study, the mouse HF model was successfully constructed through transverse aortic constriction (TAC) operation. Changes in mRNA and protein levels in isolated myocytes and heart tissues were examined using qRT-PCR, Western blot and Immunohistochemical staining and immunofluorescent staining. Changes in cardiac functions were examined using ultrasound. Interactions between miR-125b and BAK1 was analyzed using the luciferase reporter assay. Cardiomyocyte apoptosis was evaluated using the TUNEL staining.ResultsWe found that miR-125b expression was significantly downregulated in myocardial tissues of HF mice. Moreover, miR-125b upregulation in HF mice injected with agomir-125b efficiently ameliorated cardiac function. Further, miR-125b upregulation significantly decreased the protein levels of apoptosis-related makers c-caspase 3 and Bax, while increased Bcl-2 expression. In addition, BAK1 was identified as a direct target of miR-125b. As expected, BAK1 overexpression observably reversed the effect of agomir-125b on cardiac function and on the expression of apoptosis-related makers in the heart tissues of HF mice.ConclusionsTaken together, miR-125b overexpression efficiently attenuated cardiac function injury of HF mice by targeting BAK1 through inhibiting cardiomyocyte apoptosis, suggesting that miR-125b/BAK1 axis might be a potential target for the diagnosis or treatment of HF.