MiR-34b-3p Impaired HUVECs Viability and Migration via Targeting PDK1 in an In Vitro Model of Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) leads to poor pregnancy outcomes. The methods for GDM early diagnosis and treatment are still unknown. This study aimed to investigate the expression and diagnostic potential of miR-34b-3p in GDM patients and further analyzed the effects of miR-34b-3p on HUVECs viability and migration. The expression of miR-34b-3p was detected in HUVECs of GDM and normal pregnant women by qRT-PCR. Then the HUVECs were isolated from normal pregnant women. High glucose (HG) was used to treat the HUVECs to mimic the GDM in vitro. The cell viability and migration were determined by MTT, wound healing assay, and transwell assay. The interaction between miR-34b-3p and PDK1 was evaluated by luciferase activity assay. Our results showed that miR-34b-3p was up-regulated in HUVECs of GDM patients. Then the HUVECs were isolated from normal pregnant women and they were treated with HG to mimic the GDM in vitro. Interestingly, knockdown of miR-34b-3p restored the impairment of HG treatment-induced effects in HUVECs. More importantly, PDK1 was proved to be a potential target of miR-34b-3p. Finally, the rescue experiments confirmed that miR-34b-3p impaired cell viability and migration ability in HUVECs by targeting PDK1. These findings concluded that miR-34b-3p impaired HUVECs viability and migration in GDM by targeting PDK1, which might provide a novel perspective for the pathogenesis and underlying therapeutic target for GDM.

     

MicroRNA-135b-5p Downregulation Causes Antidepressant Effects by Regulating SIRT1 Expression

Depression is a serious and potentially life-threatening mental illness. Recently, the role of sirtuin 1 (SIRT1) in chronic unpredictable mild stress (CUMS) management has been examined. The present study explored and clarified whether microRNA (miR)-135b-5p could play a role in depression by regulating the expression of SIRT1. SIRT1 was identified as the target gene of miR-135b-5p using TargetScan and the dual luciferase reporter assay. In addition, the expression levels of SIRT1 were significantly reduced in mouse peripheral blood and hippocampal tissue samples, while the expression of miR-135b-5p exhibited the opposite effects. Subsequently, the effects of miR-135b-5p inhibition were investigated in mice with depression. The results indicated that the miR-135b-5p inhibitor significantly increased the weight loss induced by CUMS compared with the model group, while reducing the expression levels of miR-135b-5p and further alleviating the depression-like behavior induced by CUMS. Concomitantly, the results indicated that the miR-135b-5p inhibitor inhibited CUMS-induced hippocampal cell apoptosis and significantly reduced the expression levels of cleaved caspase-3 and the ratio of cleaved caspase-3/caspase-3. Moreover, the miR-135b-5p inhibitor significantly reduced the CUMS-induced increase of the inflammatory factors IL-1β, IL-6 and TNF-α in the hippocampal mouse samples, while significantly increasing the expression levels of SIRT1. Finally, the results demonstrated that all the effects of the miR-135b-5p inhibitor on CUMS-induced mice were significantly reversed by SIRT1 silencing. In conclusion, the present study indicated that the miR-135b-5p/SIRT1 pathway was a key mediator of antidepressant effects induced in depressed mice. Therefore, it could be considered a potential therapeutic target for the treatment of CUMS-induced depression.

     

MicroRNA-146b-5p promotes atrial fibrosis in atrial fibrillation by repressing TIMP4

Alteration of tissue inhibitors of matrix metalloproteinases (TIMP)/matrix metalloproteinases (MMP) associated with collagen upregulation has an important role in sustained atrial fibrillation (AF). The expression of miR-146b-5p, whose the targeted gene is TIMPs, is upregulated in atrial cardiomyocytes during AF. This study was to determine whether miR-146b-5p could regulate the gene expression of TIMP4 and the contribution of miRNA to atrial fibrosis in AF. Collagen synthesis was observed after miR-146b-5p transfection in human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs)-fibroblast co-culture cellular model in vitro. Furthermore, a myocardial infarction (MI) mouse model was used to confirm the protective effect of miR-146b-5p downregulation on atrial fibrosis. The expression level of miR-146b-5p was upregulated, while the expression level of TIMP4 was downregulated in the fibrotic atrium of canine with AF. miR-146b-5p transfection in hiPSC-aCMs-fibroblast co-culture cellular model increased collagen synthesis by regulating TIMP4/MMP9 mediated extracellular matrix proteins synthesis. The inhibition of miR-146b-5p expression reduced the phenotypes of cardiac fibrosis in the MI mouse model. Fibrotic marker MMP9, TGFB1 and COL1A1 were significantly downregulated, while TIMP4 was significantly upregulated (at both mRNA and protein levels) by miR-146b-5p inhibition in cardiomyocytes of MI heart. We concluded that collagen fibres were accumulated in extracellular space on miR-146b-5p overexpressed co-culture cellular model. Moreover, the cardiac fibrosis induced by MI was attenuated in antagomiR-146 treated mice by increasing the expression of TIMP4, which indicated that the inhibition of miR-146b-5p might become an effective therapeutic approach for preventing atrial fibrosis.     

Decreased miR-30b-5p expression by DNMT1 methylation regulation involved in gastric cancer metastasis

miRNAs have emerged as crucial regulators in the regulation of development as well as human diseases, especially tumorigenesis. The aims of this study are to evaluate miR-30b-5p expression pattern and mechanism in gastric carcinogenesis due to which remains to be determined. Expression of miR-30b-5p was analyzed in 51 gastric cancer cases and 4 cell lines by qRT-PCR. The effect of DNA methylation on miR-30b-5p expression was assessed by MSP and BGS. In order to know whether DNMT1 increased miR-30b-5p promoter methylation, DNMT1 was depleted in cell lines AGS and BGC-823. The role of miR-30b-5p on cell migration was evaluated by wound healing assays. Decreased expression of miR-30b-5p was found in gastric cancer samples. In tumor, the expression level of miR-30b-5p was profound correlated with lymph node metastasis (P = 0.019). The level of miR-30b-5p may be restored by DNA demethylation and DNMT1 induced miR-30b-5p promoter methylation. In vitro functional assays implied that enforced miR-30b-5p expression affected cell migration, consistent with tissues analysis. Our findings uncovered that miR-30b-5p is significantly diminished in gastric cancer tissues, providing the first insight into the epigenetic mechanism of miR-30b-5p down-regulation, induced by DNMT1, and the role of miR-30b-5p in gastric cancer carcinogenesis. Overexpression of miR-30b-5p inhibited cell migration. Thus, miR-30b-5p may represent a potential therapeutic target for gastric cancer therapy.     

Long noncoding RNA ANRIL contributes to the development of ulcerative colitis by miR-323b-5p/TLR4/MyD88/NF-κB pathway

The aim of this study was to investigate the role and possible mechanism of long noncoding RNA ANRIL in the development of ulcerative colitis (UC). The expression of ANRIL in colonic mucosa tissues collected from the sigmoid colon of UC patients and healthy control was determined. Subsequently, fetal human cells (FHCs) were treated with lipopolysaccharide (LPS) to stimulate UC-caused inflammatory injury, followed by detection of the effects of suppression of ANRIL on cell viability, apoptosis and cytokines production in LPS-stimulated FHCs. Moreover, the regulatory relationship between ANRIL and miR-323b-5p as well as the target relationship between miR-323b-5p and TLR4 were investigated. Furthermore, the effects of ANRIL/miR-323b-5p axis on the activation of TLR4/MyD88/NF-κB pathway in LPS-stimulated FHCs were investigated. LncRNA ANRIL was highly expressed in colonic mucosa tissues of UC patients. In addition, LPS markedly induced cell injury in FHC cells (inhibited cell viability and promoted cell apoptosis and cytokine production). Suppression of ANRIL alleviated LPS-induced injury in FHC cells, which was achieved by negatively regulating miR-323b-5p. Moreover, miR-323b-5p negatively regulated TLR4 expression and TLR4 was a target of miR-323b-5p. Knockdown of TLR4 reversed the effects of miR-323b-5p suppression on LPS-induced injury in LPS-stimulated FHCs. Furthermore, the effects of ANRIL on LPS-induced cell injury were achieved by TLR4/MyD88/NF-κB pathway. Our data indicate that suppression of ANRIL may inhibit the development of UC by regulating miR-323b-5p/TLR4/MyD88/NF-κB pathway. ANRIL/miR-323b-5p/TLR4/MyD88/NF-κB pathway may provide a new strategy for UC therapy.     

长链非编码RNAIFNG-AS1靶向miR-19b-1-5p调控oxLDL诱导的血管内皮细胞增殖、凋亡的机制研究

为了探讨长链非编码RNA干扰素活化基因的反义核糖核酸(lncRNA IFNG-AS1)对氧化型低密度脂蛋白(oxLDL)诱导的人脐静脉血管内皮细胞EVC-304增殖、凋亡的影响和调控机制,该研究采用100 μg/mL的oxLDL分别处理转染si-IFNG-AS1、miR-19b-1-5p mimics或共转染si-IF...     

Inheritable changes in miRNAs expression in HeLa cells after X-ray and mitomycin C treatment

We identified 40 miRNAs with inherited aberrant expression by multiple parallel sequencing of human HeLa cells irradiated with X rays and mitomycin C. Twenty-two miRNAs were repressed and 15 miRNAs were induced after radiation and mytomycin C treatment. The expression of three miRNAs (miR-10b-5p, miR-148a-3p, and miR-340-5p) decreased after X-ray exposure and increased after mitomycin C treatment. The spectrum of aberrantly expressed miRNAs after X-ray and mitomycin C treatment is different, except for three miRNAs (mir-100-5p, miR-99b-5p, miR-501-3p), which showed the inherited decreased expression after both mutagens. It has been ascertained that for five miRNAs (miR-21-3p, miR-182-5p, miR-19b-3p, miR-30a-3p, and miR-30e-3p) with increased inherited expression, the targets are well-described tumor suppressor genes. For 9 miRNAs (miR-99b-5p, miR-148a-3p, miR-365a-3p, miR-193a-3p, miR-100-5p, miR-99a-5p, miR-29b-3p, miR-340-5p, and miR-23b-3p) with reduced inherited expression, the targets are oncogenes. The obtained results provide further support of the idea that induced epigenetic changes in the genome should be considered when assessing the long-term genetic effects of ionizing radiation and chemical compounds.     

下调lncRNA MCF2L-AS1靶向miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡

摘要 目的：探讨lncRNA MCF2L-AS1对胃癌细胞恶性生物学行为的影响及分子机制。方法：选取45例胃癌患者的癌组织及癌旁正常组织,或培养胃黏膜上皮细胞GES-1、胃癌细胞HGC-27,采用RT-qPCR检测MCF2L-AS1和miR-33b-5p的表达水平。采用双荧光素酶报告实验检测MCF2L-AS1和miR-33b-5p的靶向关系。将HGC-27细胞分为si-NC组、si-MCF2L-AS1组、mimic NC组、miR-33b-5p mimic组、si-MCF2L-AS1+inhibitor NC组、si-MCF2L-AS1+miR-33b-5p inhibitor组,分别转染si-NC、si-MCF2L-AS1、mimic NC、miR-33b-5p mimic或共转染si-MCF2L-AS1+inhibitor NC、si-MCF2L-AS1+miR-33b-5p inhibitor。采用MTT实验检测细胞增殖情况,流式细胞术检测细胞凋亡率,克隆形成实验检测细胞克隆形成数,Transwell实验检测迁移和侵袭细胞数。结果：与癌旁正常组织或GES-1细胞相比,胃癌组织或HGC-27细胞中MCF2L-AS1表达水平升高、miR-33b-5p表达水平降低,差异均有统计学意义（P<0.05）。MCF2L-AS1可靶向调控miR-33b-5p。下调MCF2L-AS1或过表达miR-33b-5p,miR-33b-5p表达水平升高,HGC-27细胞凋亡率升高,但细胞增殖、克隆形成数、迁移和侵袭数均减少,差异均有统计学意义（P<0.05）。抑制miR-33b-5p可减弱下调MCF2L-AS1对HGC-27细胞的生物学作用。结论：下调MCF2L-AS1通过上调miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡;MCF2L-AS1通过靶向调控miR-33b-5p表达进而参与胃癌细胞的恶性生物学行为。     

miR-193b-5p regulates chondrocytes metabolism by directly targeting histone deacetylase 7 in interleukin-1β-induced osteoarthritis

There is increasing evidence regarding the pivotal roles of microRNAs (miRNAs) and histone deacetylases (HDACs) in the development of osteoarthritis (OA). This study aimed to determine whether miR-193b-5p regulates HDAC7 expression directly to affect cartilage degeneration. Expression levels of miR-193b-5p, HDAC7, matrix metalloproteinase 3 (MMP3), and MMP13 were determined in normal and OA cartilage and primary human chondrocytes (PHCs) stimulated with interleukin-1β (IL-1β). PHCs were transfected with a miR-193b-5p mimic or inhibitor to verify whether miR-193b-5p influences the expression of HDAC7 and MMPs. A luciferase reporter assay was performed to demonstrate the binding between miR-193b-5p and the 3′-untranslated region (UTR) of HDAC7. Expression of miR-193b-5p was reduced in IL-1β-stimulated PHCs and in OA cartilage compared to that in normal cartilage. Luciferase reporter assay exhibited the repressed activity of the reporter construct containing the 3′UTR of HDAC7. Both miR-193b-5p overexpression and HDAC7 inhibition decreased the expression of MMP3 and MMP13, whereas the inhibition of miR-193b-5p enhanced HDAC7, MMP3, and MMP13 expression. miR-193b-5p downregulates HDAC7 directly and, as a result, inhibits MMP3 and MMP13 expression, which suggests that miR-193b-5p has a protective role in OA.     

microRNA-141 is associated with hepatic steatosis by downregulating the sirtuin1/AMP-activated protein kinase pathway in hepatocytes

Sirtuin1 (SIRT1) is a crucial regulator of metabolism and it is implicated in the metabolic pathophysiology of several disorders inclusive of Type 2 diabetes and fatty liver disease (NAFLD). The aim of this study was to investigate the role of miR-141 in hepatic steatosis via regulation of SIRT1/AMP-activated protein kinase (AMPK) pathway in hepatocytes. Liver hepatocellular cells (HepG2) were treated with high concentration of glucose to be subsequently used for the assessment of miR-141 and SIRT1 levels in a model of hepatic steatosis. On the other hand, cells were transfected with miR-141 to investigate its effect on hepatocyte steatosis and viability as well as SIRT1 expression and activity along with AMPK phosphorylation. Targeting of SIRT1 by miR-141 was evaluated by bioinformatics tools and confirmed by luciferase reporter assay. Following the intracellular accumulation of lipids in HepG2 cells, the level of miR-141 was increased while SIRT1 mRNA and protein levels, as well as AMPK phosphorylation, was decreased. Transfection with miR-141 mimic significantly downregulated SIRT1 expression and activity while miR-141 inhibitor had the opposite effects. Additionally, modulation of miR-141 levels significantly influenced AMPK phosphorylation status. The results of luciferase reporter assay verified SIRT1 to be directly targeted by miR-141. miR-141 could effectively suppress SIRT1 and lead to decreased AMPK phosphorylation in HepG2 cells. Thus, miR-141/SIRT1/AMPK signaling pathway may be considered a potential target for the therapeutic management of NAFLD.     

Long noncoding RNA LINC00629 restrains the progression of gastric cancer by upregulating AQP4 through competitively binding to miR-196b-5p

Gastric cancer continues to be a common cancer in the world with high incidence and mortality. Accumulating evidence has implicated long noncoding RNAs (lncRNAs) in gastric cancer progression. Here, this study identified the potential role of a novel lncRNA, LINC00629 in gastric cancer and to elucidate the underlying mechanism. Initially, microarray-based gene expression profiling of gastric cancer was employed to identify differentially expressed genes. Next, the expression of LINC00629, microRNA-196b-5p (miR-196b-5p) and aquaporin 4 (AQP4) in clinical gastric cancer tissues was determined and the cell line presenting with the lowest LINC00629 expression was selected. The interaction among LINC00629, miR-196b-5p, and AQP4 was identified. Expression of LINC00629, miR-196b-5p, and AQP4 in gastric cancer cells were altered and then biological behaviors of gastric cancer cells were assessed by 5-ethynyl-2′-deoxyuridine and Transwell assays. Tumor formation in vivo was evaluated in nude mice. In gastric cancer, expression of LINC00629 and AQP4 was downregulated, and expression of miR-196b-5p was upregulated. Proliferation, invasion, and migration of gastric cancer cells were reduced after overexpression of LINC00629. LINC00629 competitively bound to miR-196b-5p, while AQP4 was a target of miR-196b-5p. Either downregulating miR-196b-5p or upregulating AQP4 could restrain the development of gastric cancer in vitro. LINC00629 overexpression repressed the growth of transplanted tumors in vivo. Taken together, LINC00629 competitively bound to miR-196b-5p to upregulate AQP4 expression, thereby inhibiting gastric cancer progression. Therefore, understanding of this mechanism may help to improve gastric cancer treatment.     

miRNA-130b-5p promotes hepatic stellate cell activation and the development of liver fibrosis by suppressing SIRT4 expression

Liver fibrosis is a progressive disease accompanied by the deposition of extracellular matrix (ECM). Numerous reports have demonstrated that alterations in the expression of microRNAs (miRNAs) are related to liver disease. However, the effect of individual miRNAs on liver fibrosis has not been studied. Hepatic stellate cells (HSCs), being responsible for producing ECM, exert an important influence on liver fibrosis. Then, microarray analysis of non-activated and activated HSCs induced by transforming growth factor β1 (TGF-β1) showed that miR-130b-5p expression was strongly up-regulated during HSC activation. Moreover, the progression of liver fibrosis had a close connection with the expression of miR-130b-5p in different liver fibrosis mouse models. Then, we identified that there were specific binding sites between miR-130b-5p and the 3′ UTR of Sirtuin 4 (SIRT4) via a luciferase reporter assay. Knockdown of miR-130b-5p increased SIRT4 expression and ameliorated liver fibrosis in mice transfected with antagomiR-130b-5p oligos. In general, our results suggested that miR-130b-5p promoted HSC activation by targeting SIRT4, which participates in the AMPK/TGF-β/Smad2/3 signalling pathway. Hence, regulating miR-130b-5p maybe serve as a crucial therapeutic treatment for hepatic fibrosis.     

miR-125b-5p促进分枝杆菌在宿主细胞和小鼠体内存活的研究

通过观察miR-125b-5p对分枝杆菌在宿主细胞和小鼠体内存活情况的影响,探究其在抗结核免疫过程中的作用。采用不同培养基对分枝杆菌进行培养并计数;以1640培养基加10%胎牛血清培养所有实验用细胞。将终浓度50 nmol/L的miR-125b-5p 模拟物、miR-125b-5p 抑制剂及磷酸盐缓冲液(PBS)对照加入细胞后,在不同时间点收集细胞。用分枝杆菌分别感染宿主细胞(A549、THP-1和RAW264.7)以及C57BL/6小鼠。采用定量聚合酶链反应检测miR-125b-5p的表达量。结果miR-125b-5p在分枝杆菌感染的多种宿主细胞及小鼠中都显著上调表达,其中小鼠肺部的表达量提高了约15倍。分别转染模拟物和抑制剂后,再用分枝杆菌感染细胞,结果发现miR-125b-5p可促进分枝杆菌在宿主细胞内的生长。当miR-125b-5p抑制剂注射到卡介苗(BCG)感染的小鼠体内时,小鼠体内的细菌载量显著降低(P<0.05)。本研究证明miR-125b-5p可调控分枝杆菌在宿主细胞及小鼠体内的生长,在抗结核免疫过程中发挥了重要作用。进一步对其作用机制的深入研究将为临床结核病的治疗提供理论指导。     

MiR-181b-5p Downregulates NOVA1 to Suppress Proliferation,Migration and Invasion and Promote Apoptosis in Astrocytoma

MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3''-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma.     

miR-125b-5p对人血管瘤内皮细胞HemES增殖和凋亡的影响及其机制

目的:研究miR-125b-5p对人血管瘤内皮细胞HemECs增殖、凋亡的影响.方法:RT-qPCR检测人血管瘤内皮细胞HemECs及其旁系组织细胞中miR-125b-5p与MCL-1 mRNA的表达;选取HemECs细胞分为对照组、miR-NC 组、miR-125b-5p mimic 组、miR-125b-5p in...     

miR-216b-5p regulates proliferation and apoptosis of ox-LDL-stimulated VSMCs and HUVECs via IGF2

Atherosclerosis (AS) is one of the principal causes of cardiovascular disorder. Reportedly, vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) play key roles in AS development, and microRNAs (miRNAs) regulate their functions. The function of miR-216b-5p in AS remains unknown. Human VSMCs and human HUVECs were treated with ox-LDL to establish the in vitro model of AS. MiR-216b-5p and IGF2 expressions in VSMCs and HUVECs were probed by qRT-PCR and western blot. The viability, cell cycle progression, and apoptosis of VSMCs and HUVECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine, and flow cytometry assays, respectively. The binding sites between IGF2 3′UTR and miR-216b-5p were validated by dual-luciferase reporter assay. miR-216b-5p expression was declined in ox-LDL-induced VSMCs and HUVECs. In VSMCs, miR-216b-5p overexpression inhibited excessive proliferation and induced apoptosis. MiR-216b-5p could markedly restrain the viabiblity of VSMCs induced by ox-LDL and enhanced the viability of HUVECs. Additionally, IGF2 was confirmed as the direct target of miR-216b-5p and transfection of IGF2 overexpression plasmids rescued the effects of miR-216b-5p on VSMCs and HUVECs. miR-216b-5p alleviates the dysfunction of VSMCs and HUVECs caused by ox-LDL via repressing IGF2, and exerts protective functions to block the development of AS.