miR-425-5p靶向FOXJ3对乳腺癌细胞的作用研究

目的 探讨微小RNA-425-5p(miR-425-5p)靶向FOXJ3对乳腺癌细胞的作用.方法 用Lipofiectamine2000分别将miR-425-5p inhibitor及NC转染至MDA-MB-231乳腺癌细胞作为miR-425-5p inhibitor组和NC组,不做任何处理的为对照组.通过RT-qPCR测乳腺上皮细胞MCF10A及miR-425-5p inhibitor组、NC组、对照组中miR-425-5p及FOXJ3mRNA表达水平.通过MTT法测miR-425-5p inhibitor组、NC组、对照组细胞增殖情况;通过流式细胞仪测3组细胞凋亡情况;Transwell小室实验测细胞侵袭情况;Western-blot测细胞中FOXJ3、CyclinD1、Bcl-2、MMP-2蛋白表达情况;双荧光素酶实验验证miR-425-5p与FOXJ3的靶向关系.结果 与乳腺上皮细胞MCF10A比,乳腺癌细胞MDA-MB-231中miR-425-5p表达水平显著升高(P<0.05),FOXJ3mRNA表达水平显著下降(P<0.05);与对照组和NC组比,miR-425-5p inhibitor组细胞增殖、迁移、侵袭能力受到抑制,细胞凋亡率升高(P<0.05);双荧光素酶实验结果显示抑制miR-425-5p表达显著增加野生型FOXJ33'UTR萤光素酶活性,对突变型FOXJ33'UTR的萤光素酶活性无影响.结论 下调miR-425-5p可能通过靶向FOXJ3抑制乳腺癌细胞增殖、迁移、侵袭,并促进细胞凋亡.     

用双萤光素酶报告基因技术验证小鼠lncRNA-H19与miR-199a-5p的靶向关系

目的:构建长链非编码RNA-H19(lncRNA-H19)萤光素酶报告质粒,利用双萤光素酶报告基因技术验证小鼠lncRNA-H19与微小RNA-199a-5p(miR-199a-5p)的靶向关系。方法:通过生物信息学网站RegRNA2.0预测获取小鼠lncRNA-H19与miR-199a-5p潜在的互补结合位点。将H19及其突变体克隆到萤光素酶载体psi CHECK-2中,构建H19野生型和突变型质粒,并采用酶切和测序方法鉴定psi CHECK-2-H19载体是否构建成功。将H19野生型和突变型质粒分别与miR-199a-5p模拟物、miR-199a-5p抑制剂、miR-199a-5p模拟物阴性对照或miR-199a-5p抑制剂阴性对照在293T细胞中共转染。收集细胞后通过双萤光素酶报告系统检测不同组别的萤光素酶活性,从而对lncRNA-H19与miR-199a-5p的靶向调节关系进行验证。结果:构建的重组萤光素酶报告质粒经酶切及测序鉴定正确,双萤光素酶报告基因检测显示,与miR-199a-5p模拟物阴性对照组相比,miR-199a-5p模拟物组H19野生型报告基因的萤光素酶活性显著降低,下降约49%左右(P0.01),而miR-199a-5p抑制剂组H19野生型报告基因的萤光素酶活性较miR-199a-5p模拟物组明显增高(P0.01)。miR-199a-5p模拟物、miR-199a-5p抑制剂、miR-199a-5p模拟物阴性对照以及miR-199a-5p抑制剂阴性对照对H19突变型的萤光素酶活性均无明显影响。结论:lncRNA-H19能够靶向结合miR-199a-5p,并在转录后水平对其有直接抑制作用。     

miR-142-3p通过调控Hmgb1抑制雨蛙素诱导的大鼠胰腺外分泌细胞系AR42J凋亡

目的 探讨miR-142-3p调控Hmgb1对大鼠胰腺外分泌细胞系AR42J凋亡的影响。方法 将AR42J细胞分为空白组(blank)、急性胰腺炎模型组(AP,100 nmol/L雨蛙素作用24 h),再分别用miR-142-3p mimics、 mimics NC、miR-142-3p inhibitor和inhibitor NC转染模型组细胞,记为miR-142-3p mimics组、 mimics NC组、miR-142-3p inhibitor组和inhibitor NC组。用RT-qPCR检测细胞中miR-142-3p表达;Western blot检测HMGB1、caspase-3、Bax、Bcl-2蛋白表达;Hoechst染色测定细胞凋亡;流式细胞测量术检测细胞凋亡率;双荧光素酶报告基因实验明确miR-142-3p和Hmgb1的靶向关系。结果 与空白组相比,AP组中miR-142-3p表达水平显著下调(P<0.01),HMGB1、caspase-3蛋白表达量上调(P<0.05),Bax蛋白表达量显著上调(P<0.01),Bcl-2蛋白表达量显著降低(P&l...     

miR-99a-5p通过靶向mTOR抑制前列腺癌细胞增殖及肿瘤生长

目的 探讨miR-99a-5p靶向mTOR对前列腺癌细胞增殖及肿瘤生长的影响。方法 CCK-8法检测miR-99a-5p对DU-145细胞增殖的影响;miR-99a-5p mimics转染前列腺癌DU-145细胞,通过实时定量PCR检测miR-99a-5p和mTOR mRNA表达,Western blot检测miR-99a-5p过表达后DU-145细胞mTOR蛋白的表达;通过生物信息学网站预测mTOR是否为miR-99a-5p潜在的靶基因,并通过双荧光素酶报告基因实验进行验证,CCK-8和肿瘤异种移植裸鼠模型验证miR-99a-5p通过靶向mTOR对前列腺癌细胞增殖及肿瘤生长的影响。结果 转染miR-99a-5p mimics抑制DU-145细胞体外增殖活性,降低mTOR mRNA和蛋白的表达,miR-99a-5p结合mTOR mRNA 3’UTR区域,且miR-99a-5p通过靶向mTOR抑制前列腺癌细胞增殖及肿瘤生长。结论 miR-99a-5p通过靶向调控mTOR抑制前列腺癌细胞的增殖及肿瘤生长。     

甘草己烷-乙醇提取物通过miR-151a-5p/GABARAPL1轴调控前列腺癌细胞增殖、凋亡北大核心CSCD

目的:探讨甘草己烷-乙醇提取物(HEGU)对前列腺癌细胞增殖、凋亡的影响及分子机制。方法:人前列腺癌细胞PC-3分为对照(NC)组、不同剂量HEGU组、anti-con组、anti-miR-151a-5p组、HEGU+miR-con组、HEGU+miR-151a-5p组。MTT检测细胞增殖;流式细胞术检测细胞凋亡;Western blot检测细胞周期蛋白D1(CyclinD1)、细胞周期蛋白依赖性激酶抑制剂1A(p21)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X(Bax)蛋白表达;RT-qPCR检测miR-151a-5p和GABA A型受体相关蛋白样1(GABARAPL1)mRNA表达;双荧光素酶报告实验检测miR-151a-5p与GABARAPL1的靶向关系。结果:与NC组相比,不同剂量HEGU组前列腺癌细胞活性降低,凋亡率升高,CyclinD1、Bcl-2表达降低,p21、Bax表达升高,miR-151a-5p表达降低,GABARAPL1 mRNA表达升高,且呈剂量依赖性(P<0.05)。与anti-con组相比,anti-miR-151a-5p组细胞活性降低,凋亡率升高,CyclinD1、Bcl-2表达降低,p21、Bax表达升高(P<0.05)。miR-151a-5p靶向负调控GABARAPL1表达。过表达miR-151a-5p逆转了HEGU对前列腺癌细胞增殖、凋亡的影响。结论:HEGU可能通过调控miR-151a-5p/GABARAPL1抑制前列腺癌细胞增殖,促进细胞凋亡。     

miR-301a-3p对大鼠星形胶质细胞Cx43转录后的靶向调控作用

目的:研究大鼠星形胶质细胞中微小RNA-301a-3p(miR-301a-3p)对缝隙连接蛋白43(Cx43)表达的靶向调控作用及其作用位点。方法:合成miR-301a-3p agomir和miR-301a-3p antagomir,转染至星形胶质细胞,Western blot检测各组细胞中Cx43蛋白的表达情况;构建重组载体wt-pEZX-MT05-Cx43和mut-pEZX-MT05-Cx43,采用双萤光素酶报告基因实验验证miR-301a-3p的靶基因;构建表达载体pcDNA3.1-Cx43,通过回复实验分析miR-301a-3p对细胞凋亡的影响。结果:将miR-301a-3p agomir转染到星形胶质细胞后,Western blot检测显示,与对照组相比,Cx43蛋白表达显著降低(P<0.05)。双萤光素酶报告基因实验结果表明,miR-301a-3p能够与Cx43的3′-UTR结合,对其表达产生负调控;将不含Cx43 3′-UTR的重组载体pcDNA3.1-Cx43转染星形胶质细胞后,能够回复miR-301a-3p对Cx43蛋白表达的负调控作用,引起细胞凋亡。结论:Cx...     

circAGFG1对非小细胞肺癌生物学行为的影响机制研究

目的 探讨环状RNA(circRNA)AGFG1通过靶向微小RNA(miR)-374a-5p/血管内皮生长因子C(VEGFC)轴对非小细胞肺癌（NSCLC）生物学行为的影响机制。方法 qRT-PCR检测55例NSCLC患者的癌组织和癌旁组织以及人支气管上皮细胞系（BEAS-2B）、人NSCLC细胞系（HCC827、H1975、A549、H1299）中circAGFG1、miR-374a-5p、VEGFC mRNA表达水平。以A549细胞为研究对象,设置对照组、circAGFG1小干扰RNA(si circAGFG1)组及阴性对照（si NC）组、si circAGFG1+miR-374a-5p抑制物（miR-374a-5p inhibitor）组以及si circAGFG1+抑制物对照组（inhibitor NC）组。qRT-PCR检测各组A549细胞中circAGFG1、miR-374a-5p、VEGFC mRNA表达水平;CCK-8及Transwell实验分别检测细胞活力及迁移、侵袭能力;Western blot检测增殖蛋白Ki-67、基质金属蛋白酶-9(MMP-9)以及VEGFC...     

黄芩素调控miR-378a-5p对脂多糖诱导的心肌细胞损伤和凋亡的影响北大核心CSCD

目的:探讨黄芩素对脂多糖(LPS)诱导的心肌细胞(H9C2)损伤和凋亡的影响,并分析其机制是否与调控miR-378a-5p表达有关。方法:将H9C2细胞分为对照组、LPS组、LPS+10μmol/L黄芩素组、LPS+20μmol/L黄芩素组、LPS+40μmol/L黄芩素组、LPS+20μmol/L黄芩素+miR-con组、LPS+20μmol/L黄芩素+miR-378a-5p组。细胞计数法、流式细胞术分析细胞活力和凋亡。试剂盒检测丙二醛(MDA)水平、乳酸盐脱氢酶(LDH)释放量、TNF-α和IL-6分泌量以及超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性。实时定量PCR分析miR-378a-5p表达量。结果:LPS处理显著降低H9C2细胞存活率、促进细胞凋亡,增加MDA水平、LDH释放量、TNF-α和IL-6分泌量、miR-378a-5p表达量,降低SOD和GSH-Px活性(P<0.05)。黄芩素显著提高LPS处理的H9C2细胞存活率,抑制细胞凋亡,降低MDA水平、LDH释放量、TNF-α和IL-6分泌量、miR-378a-5p表达量,并增加SOD和GSH-Px活性(P<0.05)。过表达miR-378a-5p显著减弱黄芩素对LPS处理的H9C2细胞存活率、凋亡、MDA水平、LDH释放量、TNF-α和IL-6分泌量以及SOD和GSH-Px活性的影响(P<0.05)。结论:黄芩素可减轻LPS诱导的心肌细胞损伤和凋亡,其机制可能与下调受损心肌细胞miR-378a-5p表达有关。     

下调miR-199a-5p对阿霉素诱导的心肌细胞凋亡的影响和机制研究

目的:探讨下调miR-199a-5p对阿霉素诱导的心肌细胞凋亡的影响和机制。方法:将心肌细胞H9C2分成Control组(常规培养的对照细胞)、DOX组(经含1μM阿霉素的细胞培养液培养)、DOX+Anti-miR-NC组(转染inhibitor control,经含1μM阿霉素的细胞培养液培养)和DOX+Anti-miR-199a-5p组(转染miR-199a-5p inhibitor,经含1μM阿霉素的细胞培养液培养)、DOX+Anti-miR-199a-5p+DKK1组(转染miR-199a-5p inhibitor,经含1μM阿霉素和20ng/ml的Wnt/β-catenin信号抑制剂DKK1的细胞培养液培养)。各组细胞处理24h以后,Realtime PCR测定miR-199a-5p表达,CCK-8测定细胞增殖,流式细胞术测定细胞凋亡,Western blot测定C-Caspase-3、β-catenin、c-Myc蛋白表达。结果:与Control组比较,DOX组细胞中miR-199a-5p水平升高,细胞增殖活性下降,细胞凋亡和C-Caspase-3蛋白表达水平升高,β-catenin、c-Myc蛋白表达水平降低(P均<0.01)。与DOX+Anti-miR-NC组比较,DOX+Anti-miR-199a-5p组细胞中miR-199a-5p水平降低,细胞增殖活性升高,细胞凋亡水平和C-Caspase-3蛋白表达水平降低,β-catenin、c-Myc蛋白表达水平升高(P均<0.01)。与DOX+Anti-miR-199a-5p组相比,DOX+Anti-miR-199a-5p+DKK1组心肌细胞存活率降低,细胞凋亡率升高,细胞中C-Caspase-3蛋白表达水平升高,β-catenin、c-Myc蛋白表达水平降低(P均<0.01)。结论:下调miR-199a-5p通过激活Wnt/β-catenin信号抑制阿霉素诱导的心肌细胞凋亡。     

miR-20a-3p、miR-449b-5p在妊娠糖尿病中的表达及临床意义

目的 通过基于基因表达数据库（GEO）的基因表达数据，描述miR-20a-3p、miR-449b-5p与妊娠糖尿病之间的关系。方法 本研究选择了GEO数据库中的GSE182737及GSE98403两个数据集，将妊娠糖尿病组（GDM）和正常组（normal）的外周血标本分别作为实验组和对照组，分别筛选表达差异cicrRNA及miRNA，通过cicrRNA数据库筛选靶向miRNA，取交集miRNA。利用mirPATH软件对交集的差异miRNA行京都基因与基因组百科全书（KEGG）、基因本体（GO）功能分析。收集2019年1月至2020年12月在常熟市第二人民医院产科就诊的GDM的患者60例，同期正常妊娠的产妇60例，对采集的血液标本进行qPCR验证miR-20a-3p、miR-449b-5p的表达。结果 GSE182737的差异cicrRNA为10个，GSE98403的差异miRNA为73个，两个数据集表达差异miRNA交集为7个。通过miPATH软件对交集的差异miRNA行KEGG富集，结果显示在脂肪酸代谢及合成等通路上显著富集。GO富集分析显示在应激反应及细胞死亡等通路上显著富集。通过...     

Combination of tumor suppressor miR-20b-5p,miR-30a-5p,and miR-196a-5p as a serum diagnostic panel for renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) accounts for 3 % of cancer patients. Early detection influences the therapeutic strategy and significantly improves patients’ survival rates. Stable existing circulating miRNAs could be a promising diagnostic biomarker.MethodsPreviously our team demonstrated the anti-tumor effect of miR-20b-5p, miR-30a-5p and miR-196a-5p in RCC tissue and cell lines. Here, based on 110 RCC patients and 110 health control, we investigated serum expression of these three miRNAs in the testing set and the validation set separately by using quantitative real-time PCR. A three-miRNA panel with high diagnostic efficiency was constructed. Correlations between these miRNAs and clinical parameters were investigated. Additionally, the TCGA dataset and bioinformatic analysis are used for the functional exploration of these miRNAs.ResultsSerum expression levels of miR-20b-5p, miR-30a-5p were significantly reduced in RCC patients, while miR-196a-5p expression level was up-regulated (p < 0.001). miR-20b-5p, miR-30a-5p and miR-196a-5p had moderate diagnostic ability for RCC (AUC = 0.807, 0.766 and 0.719 in the testing set, respectively). The AUC of the three-miRNA panel was 0.949 in the testing set and 0.938 in the validation set. Specifically, the serum expression level of miR-196a-5p was significantly down-regulated in RCC patients with higher Fuhrman grade (p = 0.051). TCGA dataset analysis showed that the three-miRNA panel probably participated in RCC by targeting ITGA4 and NRP2.ConclusionThe three-miRNA panel could serve as a promising non-invasive biomarker for RCC detection.     

miR-30a-3p靶向NOD1对心肌细胞缺氧复氧损伤的影响

目的　探究miR-30a-3p与NOD1的靶向关系，及其对心肌细胞缺氧复氧损伤的影响和机制。 方法　将细胞分为Ctrl组、H/R组、miR-30a-3p mimic组和miR-30a-3p inhibitor组，经过缺氧复氧处理后，转染相应的miRNA处理细胞，RT-PCR检测miR-30a-3p和NOD1基因表达水平，荧光素酶报告检测miR-30a-3p和NOD1靶向关系，Western blot检测NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平，CCK8法检测细胞活性，Hoechst检测细胞凋亡，试剂盒检测LDH、CK、MDA、T-SOD水平。 结果　miR-30a-3p表达水平随缺氧复氧处理时间增长而下降，NOD1基因表达水平随缺氧复氧处理时间增长而升高。在荧光素酶报告实验中，NOD1 WT+miR-30a-3p mimic荧光素酶活性显著低于NOD1 WT+NC组。与Ctrl组比较，H/R组miR-30a-3p基因表达水平、细胞活性、T-SOD水平降低，NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率升高；与H/R组比较，miR-30a-3p mimic组miR-30a-3p基因表达水平、细胞活性、T-SOD水平升高，NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率降低，miR-30a-3p inhibitor组miR-30a-3p基因表达水平、细胞活性、T-SOD水平降低，NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率升高。 结论　miR-30a-3p可靶向作用于NOD1，缓解心肌细胞缺氧复氧造成的损伤，其作用机制可能与调控NF-κB信号通路有关。     

miR-30a-3p靶向NOD1对心肌细胞缺氧复氧损伤的影响

目的　探究miR-30a-3p与NOD1的靶向关系,及其对心肌细胞缺氧复氧损伤的影响和机制。 方法　将细胞分为Ctrl组、H/R组、miR-30a-3p mimic组和miR-30a-3p inhibitor组,经过缺氧复氧处理后,转染相应的miRNA处理细胞,RT-PCR检测miR-30a-3p和NOD1基因表达水平,荧光素酶报告检测miR-30a-3p和NOD1靶向关系,Western blot检测NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平,CCK8法检测细胞活性,Hoechst检测细胞凋亡,试剂盒检测LDH、CK、MDA、T-SOD水平。 结果　miR-30a-3p表达水平随缺氧复氧处理时间增长而下降,NOD1基因表达水平随缺氧复氧处理时间增长而升高。在荧光素酶报告实验中,NOD1 WT+miR-30a-3p mimic荧光素酶活性显著低于NOD1 WT+NC组。与Ctrl组比较,H/R组miR-30a-3p基因表达水平、细胞活性、T-SOD水平降低,NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率升高;与H/R组比较,miR-30a-3p mimic组miR-30a-3p基因表达水平、细胞活性、T-SOD水平升高,NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率降低,miR-30a-3p inhibitor组miR-30a-3p基因表达水平、细胞活性、T-SOD水平降低,NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率升高。 结论　miR-30a-3p可靶向作用于NOD1,缓解心肌细胞缺氧复氧造成的损伤,其作用机制可能与调控NF-κB信号通路有关。     

Circulating miR-29c-3p is downregulated in patients with acromegaly

Background/aimmiRNAs control various biological functions, such as cell proliferation, differentiation, signaling pathways, apoptosis and metabolism. Recently, it has been shown that there is a relationship between changes in miRNA expression and the development of acromegaly. Studies are needed to identify new disease-specific miRNAs. The aim of the current study is to evaluate plasma miR-29c-3p, miR-31-5p and miR-18a-5p steady-state levels in acromegaly. Another aim is to investigate whether there is a difference in the levels of these miRNAs in patients with inadequate control and controlled acromegaly with somatostatin analog (SSA) therapy. These miRNAs targeting the IGF-1 gene were determined by in silico estimation.Materials and methodsThe study included 30 healthy controls (HC) and 20 patients with acromegaly. Anterior pituitary functions and disease activities of patients with acromegaly were evaluated at the time of study. The miR-29c-3p, miR-31-5p and miR-18a-5p levels were measured using quantitative real-time PCR (RT-qPCR).ResultsThe expression level of miR-29c-3p was significantly lower in patients with acromegaly compared to the HC group (p < 0.001). This downregulation was more pronounced in patients with inadequately controlled acromegaly than in patients with acromegaly controlled with somatostatin analogues (SSA) therapy (p = 0.016). Univariate logistic regression analysis results showed that down regulation of miR-29c-3p expression increases the risk of developing acromegaly [OR (95% Cl) = 1.605 (1.142-2.257), p = 0.006]. There was no significant difference between the groups in terms of miR-31-5p and miR-18a-5p expression levels (p = 0.375 and p = 0.649, respectively). ConclusionPlasma miR-29c-3p expression level is downregulated in patients with acromegaly, and this is more pronounced in patients with inadequate control.     

miR-203a-3p promotes loureirin A-induced hair follicle stem cells differentiation by targeting Smad1

MicroRNAs (miRNAs) participate in the repair of skin trauma. Our previous study indicated that loureirin A promoted hair follicle stem cells (HFSCs) to repair skin epidermis. However, the mechanism of miRNA-mediated regulation of loureirin A-induced HFSC differentiation remained to be explored. In the present study, HFSCs from rat vibrissa were identified by immunofluorescence in vitro. Microarray and quantitative real time polymerase chain reaction analyses demonstrated that miR-203a-3p was upregulated in differentiated HFSCs induced by loureirin A. The expression of cytoskeletal keratin (CK) 5 and involucrin was promoted by miR-203a-3p mimics while repressed by a miR-203a-3p inhibitor. Smad1 was identified as a key target of miR-203a-3p using target prediction tools. Luciferase reporter gene test confirmed a special target association between miR-203a-3p and Smad1. Short interfering Smad1 was transfected into HFSCs, and the expression levels of CK5 and involucrin were upregulated. Thus, it can be inferred that miR-203a-3p negatively regulated the expression of Smad1 and promoted the differentiation of loureirin A-induced HFSCs. Bone morphogenetic protein (BMP) signal inhibition and Wnt activation coregulate skin injury repair. BMP/Smad1 signaling is involved in maintaining the characteristics of HFSCs and inhibiting their differentiation. Our results showed that miR-203a-3p reduces Smad1 to release BMP inhibition. Taken together, miR-203a-3p/Smad1 is a potential therapeutic molecular target in skin wound healing, and may play an active role in wound repair and regenerative medicine.     

微小RNA-199a-5p通过靶向SIRT1促进心肌纤维化相关基因表达

目的:研究微小RNA-199a-5p(miR-199a-5p)对心肌成纤维细胞中纤维化相关基因表达的调控作用及其可能作用的靶基因。方法:原代分离并体外培养成体C57BL/6小鼠心肌成纤维细胞;双萤光素酶报告基因实验检测miR-199a-5p与潜在靶基因沉默信息调节因子1(SIRT1)3’端非翻译区(3’-UTR)的结合作用;实时荧光定量PCR(RT-q PCR)和Western blot法分别检测SIRT1以及纤维化标志物胶原蛋白(Col)1a1、Col3a1和α-平滑肌肌动蛋白(α-SMA)的mRNA和蛋白表达。结果:在血管紧张素Ⅱ(AngⅡ)诱导的小鼠心肌成纤维细胞中,Col1a1、Col3a1和α-SMA的表达增强,miR-199a-5p表达上调。在心肌成纤维细胞中过表达miR-199a-5p可以增强Col1a1、Col3a1和α-SMA的表达。双萤光素酶报告基因实验显示miR-199a-5p与SIRT1 3’-UTR有结合作用。RT-q PCR和Western blot结果证实miR-199a-5p可在转录水平抑制SIRT1表达。过表达miR-199a-5p和沉默SIRT1均能一致性促进心肌成纤维细胞中Col1a1、Col3a1和α-SMA的表达。抑制AngⅡ诱导的小鼠心肌成纤维细胞中NF-κB激活,可显著降低miR-199a-5p表达。结论:SIRT1是miR-199a-5p的作用靶基因,并介导miR-199a-5p促进纤维化标志物Col1a1、Col3a1和α-SMA的表达。     

miR-199a-3p 通过靶向 CABLES-1 调控流体剪切力介导的成骨细胞增殖

目的 探讨miR-199a-3p在流体剪切力(fluid shear stress, FSS)诱导成骨细胞增殖中的作用及其可能的分子机制。方法 对成骨细胞MC3T3-E1加载1.2 Pa FSS,时间分别为0、15、30、45、60、75、90 min。使用miR-199a-3p模拟物或miR-199a-3p抑制物转染MC3T3-E1细胞。使用将过表达的miR-199a-3p以及其阴性对照分别转染MC3T3-E1细胞,并以1.2 Pa FSS处理45 min。将pcDNA NC、pcDNA-CABLES-1、si RNA NC、si RNA CABLES-1转染至MC3T3-E1细胞中。分别共转染pc DNA-CABLES-1与miR-199a-3p mimic以及si RNA-CABLES-1与miR-199a-3p inhibitor。CCK-8实验检测细胞活性;RT-qPCR检测CABLES-1、 miR-199a-3p、CDK 6、Cyclin D1、PCNA表达水平;荧光素酶报告实验检测CABLES-1和miR-199a-3p的靶向关系。免疫荧光检测CABLES-1蛋白表达。...     

过表达miR-130a-3p对心肌细胞肥大的缓解作用研究

本研究旨在探索miR-130a-3p对心肌细胞肥大的作用及其可能机制。通过胸主动脉缩窄法(TAC)构建压力超负荷所致心肌肥厚小鼠模型。使用去甲肾上腺素(NE)刺激SD乳鼠原代心肌细胞(NRCMs)及H9c2大鼠心肌细胞系,诱导这两种心肌细胞发生肥大表型转变。检测miR-130a-3p的表达变化,并进一步探索其对心肌细胞肥大是否有调控作用。结果表明,miR-130a-3p在肥厚心肌组织、肥大NRCMs及H9c2细胞中的表达均明显降低。给予miR-130a-3p mimics使其过表达后,H9c2细胞中肥大标志基因心房利钠肽(ANP)、脑利钠肽(BNP)和肌球蛋白重链β(β-MHC)的表达较对照组(mimics N.C.+NE组)明显下调,且细胞面积明显减小。而给予miR-130a-3p inhibitor抑制其表达后,肥大心肌细胞中ANP、BNP、β-MHC的表达进一步上升,且细胞面积进一步增加。Western blot检测发现,过表达miR-130a-3p后心肌细胞中磷酸化Akt和磷酸化mTOR的表达水平下调。以上结果提示,miR-130a-3p mimics可缓解心肌细胞肥大的程度;其inhibitor则可使心肌细胞肥大进一步加剧。过表达miR-130a-3p可能通过影响Akt通路来缓解H9c2心肌细胞肥大的程度。