miR-135a-5p靶向抑制Kif3B调控小鼠腭胚突间充质细胞的自噬

背景：研究表明，在地塞米松诱导腭裂的小鼠胚胎腭突间充质细胞中miR-135a-5p呈高表达，初级纤毛及其介导的Shh信号通路参与小鼠胚胎腭突间充质细胞的自噬。由此猜测miR-135a-5p可能通过初级纤毛及其介导的Shh信号途径调控小鼠胚胎腭突间充质细胞的自噬。目的：探讨miR-135a-5p对小鼠胚胎腭突间充质细胞自噬的调控作用。方法：体外提取并培养C57BL/6J小鼠胚胎腭突间充质细胞。细胞转染分别设置为：(1)对照组、miR-135a-5p阴性对照组、miR-135a-5p模拟物组。(2)NC+miR-NC组、KIF3B过表达组、miR-135a-5p+KIF3B组。qRT-PCR验证miR-135a-5p、KIF3B的转染效率；透射电镜观察各组细胞中自噬小体/自噬溶酶体的数目；免疫荧光技术测定自噬标记物LC3B的荧光表达程度；Western blot检测KIF3B、LC3和P62的蛋白表达。(3)miR-135a-5p阴性对照组、SAG处理组、SAG+miR-135a-5p组。qRT-PCR检测Shh信号下游关键转录因子Gli3的mRNA表达水平；Western blot检测自...     

过表达miR-151a-3p抑制PC-3前列腺癌细胞增殖和迁移

目的研究上调微小RNA-151a-3p(miR-151a-3p)对前列腺癌细胞增殖和迁移的影响和机制。方法采用实时荧光定量PCR检测PC-3M、C4-2B、22RV1、DU-145、PC-3、LNCaP人前列腺癌细胞和RWPE-1人正常前列腺上皮细胞中miR-151a-3p的表达量。以miR-151a-3p表达量最低的PC-3细胞为研究对象,生物信息学预测并采用双荧光素酶报告基因实验检验miR-151a-3p的潜在靶基因。转染miR-151a-3p模拟物或阴性对照微小RNA(miR-NC)至PC-3细胞。实时荧光定量PCR检测miR-151a-3p和潜在靶基因mRNA的表达量,Western blot法检测靶基因及下游信号通路蛋白表达量。采用MTT法检测PC-3细胞的增殖,Transwell~(TM)实验检测PC-3细胞的迁移能力。结果 miR-151a-3p在前列腺癌细胞的表达量明显低于RWPE-1人正常前列腺上皮细胞,其中PC-3细胞中的表达量最低。生物信息学预测及双荧光素酶报告基因实验表明NIMA相关激酶2(NEK2)是miR-151a-3p的潜在靶基因。转染miR-151a-3p模拟物后,PC-3细胞中miR-151a-3p的表达量明显上升,NEK2基因的表达量明显降低,NEK2基因下游磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)信号通路蛋白水平降低。上调miR-151a-3p明显抑制前列腺癌PC-3细胞的增殖和迁移。结论miR-151a-3p在前列腺癌细胞中表达降低,上调miR-151a-3p的表达可通过降低NEK2基因及下游PI3K-AKT-mTOR信号通路蛋白的表达抑制前列腺癌PC-3细胞的增殖和迁移。     

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。     

mi R-34a-3p抑制胰腺癌细胞增殖和侵袭转移并下调Smad2/TGF-β信号通路

目的 探讨miR-34a-3p对胰腺癌细胞的作用及可能机制。方法 RT-qPCR方法检测miR-34a-3p在人胰腺导管腺癌细胞SW1990和胰腺导管上皮细胞HPDE中的表达情况;转染miR-34a-3p mimics后,采用CCK-8法检测细胞增殖;Transwell小室实验检测细胞侵袭情况;Western blot检测细胞上皮-间质转化情况;进一步用生物信息学预测miR-34a-3p的下游靶基因,并用荧光素酶报告基因实验验证;然后采用Western blot检测靶蛋白以及靶蛋白下游调控蛋白的表达。结果 RT-qPCR检测发现miR-34a-3p在SW1990中的表达低于HPDE细胞。miR-34a-3p在SW1990细胞中过表达后,SW1990细胞增殖受到抑制、侵袭能力降低、E-cadherin表达上调、N-cadherin表达下调。生物信息学网站预测、双荧光素酶报告基因实验和Western blot证实Smad2是miR-34a-3p的靶蛋白,同时发现其下游蛋白TGF-β表达也显著下调。结论 miR-34-3p在胰腺癌细胞中的表达下调,miR-34-3p能够抑制胰腺癌细胞增殖、侵...     

lnc AL928768.3通过miR-92a-3p/ADAM10轴对类风湿关节炎滑膜成纤维细胞增殖和凋亡的影响北大核心CSCD

目的:探讨lnc AL928768.3如何通过miR-92a-3p/解整合素金属蛋白酶10(ADAM10)轴调控类风湿关节炎(RA)滑膜成纤维细胞(SFs)增殖和凋亡。方法:qPCR检测RA患者滑膜组织lnc AL928768.3和miR-92a-3p表达。生物信息学手段和双荧光素酶报告基因实验分析lnc AL928768.3、miR-92a-3p与ADAM10的靶向关系,Western blot分析ADAM10表达。MTT检测MH7A细胞增殖,流式细胞术检测细胞凋亡。Western blot检测MH7A细胞增殖和凋亡相关蛋白增殖细胞核抗原(PCNA)和活化的半胱氨酸蛋白酶-3(Cleaved Caspase-3)表达。结果:lnc AL928768.3在RA患者滑膜组织中呈高表达,而miR-92a-3p呈低表达。生物信息学和双荧光素酶报告基因实验证实,lnc AL928768.3可靶向负调控miR-92a-3p表达,且ADAM10是miR-92a-3p的直接靶基因。抑制lnc AL928768.3表达或敲低ADAM10表达均可抑制细胞增殖并诱导细胞凋亡;干扰miR-92a-3p能够逆转抑制lnc AL928768.3对MH7A细胞增殖抑制和凋亡的促进作用;过表达lnc AL928768.3或干扰miR-92a-3p表达能够逆转敲低ADAM10对MH7A细胞增殖抑制和凋亡的促进作用。结论:lnc AL928768.3在RA患者滑膜组织中表达上调,抑制lnc AL928768.3通过miR-92a-3p/ADAM10轴抑制MH7A细胞增殖并促进细胞凋亡。     

miR-196a-5p抑制小鼠胚胎干细胞的自我更新

目的 研究miR-196a-5p在小鼠胚胎干细胞(mESC)自我更新中的功能.方法 定量PCR法确定miR-196a-5p在mESC早期分化过程中的表达.在胚胎干细胞中过表达miR-196a-5p类似物,通过集落形成实验、细胞周期分析、碱性磷酸酶染色和Oct4染色确定miR-196a-5p过表达对mESC自我更新及增殖的影响.结果 miR-196a-5p在mESC早期分化过程中表达上升(P＜0.05),在mESC中过表达miR-196a-5p类似物后,ESC的集落形成及增殖能力被明显抑制(P＜0.01),碱性磷酸酶活性下降,Oct4蛋白水平也出现明显下降.结论 miR-196a-5p具有抑制mESC自我更新的能力,在mESC早期分化过程中可能发挥重要作用.     

miR-130a-3p 通过调控 PDK1 影响肝癌细胞糖代谢的研究

目的 探讨 miR-130a-3p 通过靶向糖代谢关键酶 PDK1 对肝癌细胞糖代谢以及增殖迁移的影响, 为肝癌的诊断及治疗提供新的方向。 方法 qRT-PCR 技术检测肝癌的临床组织和细胞中 miR-130a-3p 的表 达, 克隆形成、 CCK-8、 Transwell 和细胞划痕实验检测细胞的增殖和迁移; Western 印迹检测相关蛋白的表 达; 乳酸检测试剂盒, ATP 检测试剂盒和 PDH 活性检测试剂盒分别检测细胞中乳酸的生成、 ATP 的生成和 PDH 的活性; 双荧光素酶报告基因实验验证 miR-130a-3p 与 PDK1 3′UTR 靶向结合。 结果 在肝癌组织和 细胞中 miR-130a-3p 呈低表达, 并与患者肿瘤组织的大小和 TNM 分期有关; 过表达 miR-130a-3p 能够增强 肝癌细胞中 PDH 活性, 抑制乳酸和 ATP 的生成, 从而抑制肝癌细胞的增殖和迁移; miR-130a-3p 可以靶向 调控 PDK1; 抑制 PDK1 的表达, 能够逆转 miR-130a-3p 过表达对细胞增殖和迁移能力的增强, 此外对 PDH 活 性、 乳酸和 ATP 的影响也被逆转。 结论 miR-130a-3p 通过靶向调控 PDK1 影响肝癌细胞糖酵解及恶性进展。     

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。     

HSF1基因缺失通过上调microRNA-195a-3p加速压力超负荷下心脏重构

目的:探讨热休克转录因子1(HSF1)调控微小RNA-195a-3p (miR-195a-3p)对心肌微血管内皮细胞血管新生功能的影响,旨在阐明HSF1基因缺失加重压力超负荷下心脏重构的病理分子机制。方法:压力超负荷动物模型采用小鼠主动脉弓缩窄(TAC),辅以心脏超声功能评价。在体实验分组为HSF1基因敲除(HSF1~(-/-))小鼠假手术组、C57BL/6野生型(WT)小鼠假手术组、HSF1~(-/-)小鼠TAC模型组和C57BL/6 WT小鼠TAC组;细胞实验分组为对照组、miR-195a-3p模拟物干预组和阴性对照microRNA(miR-NC)干预组。TAC术后4周,通过病理组织切片检测各组小鼠心肌肥厚(HE染色)和血管新生(CD31染色),小鼠心超检查心脏主要功能指标变化;通过生物信息软件TargetScan 6.2结合萤光素酶(luciferase)报告基因检测,以及Western blot验证,测定miR-195a-3p调控血管新生的下游分子靶点;并用miR-195a-3p诱导微血管内皮细胞,观察其对细胞成管能力的影响。结果:HSF1缺失会导致TAC诱导的小鼠左心室重构加重。芯片筛查结果表明HSF1缺失可促进心脏12种microRNAs表达上调,5种microRNAs在心肌微血管内皮细胞中表达显著升高,其中miR-195a-3p的增高具有统计学显著性。miR-195a-3p过表达可以有效抑制微血管内皮细胞CD31和血管内皮生长因子(VEGF)的表达,阻滞细胞形成管腔样结构。TargetScan 6.2预测miR-195a-3p的靶点为AMP活化蛋白激酶α2(AMPKα2),在微血管内皮细胞用miR-195a-3p模拟物干预可以有效抑制AMPKα2的表达,同时miR-195a-3p模拟物也抑制了CD31和VEGF的表达。结论:HSF1基因缺失导致的miR-195a-3p表达上调是加速压力超负荷下心脏重构的重要诱因,其机制可能是通过抑制AMPKα2介导的血管新生信号通路的激活。     

血清外泌体中mi R-642a-3p下调HK1对胰岛素分泌和儿童1型糖尿病进展的影响北大核心CSCD

目的:探讨血清外泌体中miR-642a-3p靶向HK1对1型糖尿病(T1DM)患儿胰岛β细胞增殖、凋亡及胰岛素分泌的影响。方法:GEO数据库筛选T1DM患儿血清中的差异表达miRNAs,Targetscan预测miRNAs的靶基因,双荧光素酶报告实验验证靶向关系。收集68例T1DM患儿外周血标本,分离并鉴定血清外泌体。高糖培养基处理胰岛β细胞。将转染后的外泌体与胰岛β细胞共培养并分组。采用qRT-PCR分别检测miR-642a-3p在外周血、血清外泌体中的表达水平。流式细胞术、CCK-8试验检测胰岛β细胞的凋亡和增殖情况。ELISA检测胰高血糖素样肽-1(GLP-1)和胰岛素分泌浓度。结果:相对于健康志愿者,T1DM患儿血清及血清外泌体中miR-642a-3p表达明显升高(均P<0.05)。HK1被证实为miR-642a-3p的靶点。相对于NC组,抑制血清外泌体中miR-642a-3p的表达后胰岛β细胞凋亡率降低,增殖活性增加,胰岛素分泌增多,GLP-1浓度上调(均P<0.05)。过表达miR-642a-3p能够提高胰岛β细胞凋亡率,减弱其增殖活性,使胰岛素分泌减少,抑制GLP-1表达(均P<0.05),从而进一步促进T1DM,但该作用被HK1部分挽救(均P<0.05)。结论:血清外泌体源性miR-642a-3p能够通过调控HK1抑制胰岛β细胞增殖,并促进其凋亡,从而促进儿童T1DM进展。血清外泌体源性miR-642a-3p有望在儿童T1DM的靶向治疗中发挥作用。     

下调lncRNA DNM3OS通过靶向miR-193a-3p增强人直肠癌细胞系SW-480的放疗敏感性

目的 探讨lncRNA DNM3OS对直肠癌SW-480细胞的增殖、凋亡以及放射敏感性的影响及分子机制.方法 选取30例直肠癌患者癌组织及癌旁组织,RT-qPCR检测DNM3OS和miR-193a-3p的表达水平;将抑制DNM3OS的表达载体、过表达miR-193a-3p载体转染至SW-480细胞,将DNM3OS与抑制...     

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-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.     

Lidocaine inhibits proliferation and induces apoptosis in colorectal cancer cells by upregulating mir-520a-3p and targeting EGFR

Lidocaine is a conventional local anesthetic which is shown antiproliferative of colorectal cancer (CRC) in patients. MicroRNAs (miRNAs) have been consistently demonstrated to be involved in CRC, and miR-520a-3p could suppress CRC migration, promote apoptosis by targeting epidermal growth factor receptor (EGFR). However, the mechanism by which lidocaine regulated CRC proliferation and apoptosis remains unknown. In this study, quantitative RT-PCR were used to measure miR-520a-3p and EGFR expression levels, and western blotting assays ware performed to measure EGFR expression in CRC cells. Luciferase reporter assay was employed to validate the direct targeting of EGFR by miR-520a-3p. Cell proliferation and apoptosis assays ware utilized to analyze the role of lidocaine in CRC cells. The results indicated that 500 and 1000 μM lidocaine over 24?h inhibited proliferation and induced apoptosis of CRC cells. Compared with the control group, the expression of EGFR was suppressed by lidocaine (500 μM) in CRC cells. Furthermore, miR-520a-3p could directly targets EGFR in CRC cells. Lidocaine (500 μM) increased the expression of miR-520a-3p and rescued the reduction of miR-520a-3p caused by miR-520a-3p inhibitor. The results suggested that lidocaine could suppress the expression of EGFR by upregulating miR-520a-3p, and it could induce apoptosis and inhibit proliferation in CRC cells. Lidocaine may serve as potential therapeutic regimen for colorectal cancer.     

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。     

甲基莲心碱通过调控miR-29a-3p/AQP4表达抑制Aβ1-42致PC12细胞的损伤

目的探讨甲基莲心碱(Nef)在β淀粉样蛋白(Aβ1-42)损伤大鼠肾上腺髓质嗜铬细胞瘤细胞PC12的作用及其分子机制。方法将PC12细胞分为对照组、模型组(4μg/mL Aβ1-42培养24 h)和干预组(低、中、高剂量甲基莲心碱)。MTT法检测PC12细胞存活;流式细胞计量术检测细胞凋亡;Western blot检测水通道蛋白4(AQP4)、Bcl-2和Bax表达量;qPCR检测细胞中miR-29a-3p和AQP4 mRNA表达。生物学信息预测和双荧光素酶基因报告分析miR-29a-3p和AQP4的靶向关系。在模型组转染miR-29a-3p、si-AQP4,或转染anti-miR-29a-3p并进行高剂量Nef干预,考察其对Aβ1-42损伤PC12细胞存活和凋亡的影响。结果与模型组相比,甲基莲心碱组细胞存活率和Bcl-2、miR-29a-3p表达明显升高,细胞凋亡率和Bax、AQP4 mRNA和蛋白表达明显降低(P<0.05)。AQP4是miR-29a-3p的靶基因。miR-29a-3p过表达和抑制AQP4表达均显著提高PC12细胞存活率和Bcl-2表达量(P<0.05),降低细胞凋亡率和Bax蛋白水平(P<0.05)。抑制miR-29a-3p表达逆转甲基莲心碱对Aβ1-42损伤PC12细胞存活、Bcl-2蛋白水平的促进作用,以及逆转甲基莲心碱对细胞凋亡率、Bax蛋白表达的抑制作用。结论甲基莲心碱通过miR-29a-3p/AQP4抑制Aβ1-42所致PC12细胞损伤,促进细胞存活,并抑制细胞凋亡。     

Geminin is an indispensable inhibitor of Cdt1 in mouse embryonic stem cells

Geminin is implicated in regulation of the cell cycle and differentiation. Although loss of Geminin triggers unscheduled DNA rereplication as a result of interruption of its interaction with Cdt1 in some somatic cancer cells, whether such cell cycle regulation also operates in embryonic stem cells (ESCs) has remained unclear. To characterize the Geminin‐Cdt1 axis in ESCs and compare it with that in somatic cells, we established conditional knockout (KO) of Geminin in mouse ESCs and mouse embryonic fibroblasts (MEFs). Geminin KO ESCs manifest a large flattened morphology, develop polyploidy accompanied by DNA damage and G₂‐M checkpoint activation, and subsequently undergo apoptosis. Rereplication in Geminin KO ESCs was attenuated by inhibition of G₂‐M checkpoint signaling or by expression of wild‐type Geminin, but not by expression of a Geminin mutant that does not bind to Cdt1, indicating the importance of sequestration of Cdt1 by Geminin in G₂ phase. In contrast, Geminin KO MEFs did not manifest disturbance of the cell cycle unless they were treated to force abnormal accumulation of Cdt1. Together, our results indicate that Geminin is a key inhibitor of Cdt1 in mouse ESCs, but that it plays a backup role in MEFs to compensate for accidental up‐regulation of Cdt1.     

MiR-103a-3p Contributes to the Progression of Colorectal Cancer by Regulating GREM2 Expression

PurposeOur research aimed to investigate the influence of miR-103a-3p on the growth and apoptosis of colorectal cancer (CRC) cells.Materials and MethodsBioinformatics was employed to analyze differentially expressed microRNAs and predict target genes. qRT-PCR was applied to detect the expression of miR-103a-3p in CRC and normal cells. HCT116 and Caco-2 were chosen, and miR-103a-3p mimics, miR-103a-3p inhibitor, as well as specific siRNAs targeting GREM2, were constructed. We subsequently evaluated alternations in cell proliferation, cell cycle and cell cycle regulators, apoptosis, and related proteins (Bcl-2 and Bax) by CCK-8 testing, Western blotting, luciferase reporter, colony formation, and Annexin V-FITC/PI. Possible binding sites for miR-103a-3p on the 3''UTR of GREM2 were checked with luciferase assay, and the impact of GREM2 on miR-103a-3p activity was also validated with above biological function testing. Additionally, the effect of miR-103a-3p knockdown in CRC cells and the molecular mechanism of miR-103a-3p targeting GREM2 were also studied.ResultsBioinformatics analysis revealed that miR-103a-3p expression increased remarkably in CRC, and targeted regulatory correlation existed between miR-103a-3p and GREM2. MiR-103a-3p inhibitor significantly impeded proliferative capacity and caused cell cycle arrest, as well as apoptosis, in HCT116 and Caco-2 cells. Consistent with this finding, overexpression of GREM2 showed similar effects to miR-103a-3p inhibition. Moreover, we demonstrated that miR-103a-3p connected target GREM2 and GREM2 knockdown reversed the effects of miR-103a-3p inhibitor on HCT116 and Caco-2 cell proliferation, cell cycle, and apoptosis. Further study showed that miR-103a-3p targeting GREM2 appeared to affect CRC progression via the transforming growth factor-β pathway.ConclusionMiR-103a-3p could augment CRC progression by targeting GREM2 and that miR-103a-3p/GREM2 could be potential novel targets for CRC therapy.