下调miR-199a-5p抑制脂多糖诱导的大鼠Ⅱ型肺泡上皮细胞系AECⅡ凋亡

目的 探讨miR-199a-5p在慢性阻塞性肺疾病(COPD)患者血清中的表达及其对脂多糖(LPS)诱导的大鼠Ⅱ型肺泡上皮细胞系(AECⅡ)凋亡的影响和可能机制.方法 选取2017年4月至2018年12月于湖南中医药大学第一附属医院就诊的45例COPD患者和同时期45名健康体检者.体外培养AECⅡ,分为对照组(cont...     

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。     

miR-181a-5p对坐骨神经损伤后神经细胞凋亡的影响

目的 探究miR-181a-5p对坐骨神经损伤后神经细胞再生及凋亡的影响。方法 建立坐骨神经损伤大鼠模型,通过FISH和qRT-PCR检测坐骨神经miR-181a-5p表达。鞘内注射antagomiR-181a-5p,通过qRT-PCR检测坐骨神经miR-181a-5p表达,通过HE染色检测坐骨神经病理学变化,通过TUNEL检测坐骨神经细胞凋亡。结果 miR-181a-5p在坐骨神经损伤大鼠坐骨神经近侧断端表达升高。鞘内注射antagomiR-181a-5p降低坐骨神经损伤大鼠坐骨神经miR-181a-5p表达,改善大鼠坐骨神经损伤,降低坐骨神经细胞凋亡水平。结论 miR-181a-5p在损伤的坐骨神经中高表达,抑制miR-181a-5p表达降低坐骨神经细胞凋亡水平。     

miR-106a-5p靶向GAB1影响人心肌细胞增殖和凋亡的机制研究

目的 探究心力衰竭患者血浆中miR-106a-5p的表达水平及其影响人心肌细胞增殖和凋亡的潜在机制.方法 利用RT-qPCR检测45例心力衰竭患者和健康志愿者血浆中miR-106a-5p的表达水平;利用Lipofectamine 2000转染miR-106a-5p模拟物及模拟物对照至人心肌细胞系,RT-qPCR验证转染...     

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。     

miR-148a-5p参与了高蛋氨酸饮食诱导ApoE-/-小鼠的肝细胞凋亡

文题释义：凋亡：是一种程序性细胞死亡,其特征是进行性的核和细胞质收缩、染色质浓缩和凋亡小体脱落。凋亡由称为半胱天冬酶的特定类型的半胱氨酸蛋白酶执行,一旦激活,就可以裂解细胞底物并导致细胞死亡。最常见的凋亡指标为抑制凋亡基因Bcl-2和促进凋亡基因Bax,Bcl-2增高,细胞凋亡减少;Bax增高,细胞凋亡增加。微小RNA(miRNA)：是一组内源性非编码小RNA,在动物、植物、病毒和其他生物中普遍存在,可调节将近60%的基因表达,这些基因负责细胞的增殖、分化、凋亡和其他生理过程。miRNA可以在多种疾病中异常表达,并对这些疾病的诊断和预后评估起重要作用。背景：高蛋氨酸饮食可以导致ApoE^-/-小鼠发生肝损伤,微小RNA(miRNA)参与细胞存活、分化和细胞凋亡等各种细胞过程,具有重要意义。 目的：探讨miR-148a-5p在高蛋氨酸饮食诱导ApoE^-/-小鼠肝细胞凋亡中的作用。方法：12只ApoE^-/-小鼠随机分为2组,每组6只,ApoE^-/-对照组为普通饮食,ApoE^-/-高蛋氨酸组为高蛋氨酸饮食。苏木精-伊红染色观察2组肝组织形态学变化;TUNEL染色观察肝细胞的凋亡情况;Western blot测定Bax、Bcl-2的表达改变;荧光定量PCR检测miR-148a-5p的表达;运用Target Scan靶基因预测软件预测miR-148a-5p的靶基因;双荧光素酶活性实验明确其靶向关系。结果与结论：①与对照组相比,ApoE-/-高蛋氨酸组肝脏组织肝小叶结构发生明显紊乱,部分细胞呈胞浆疏松化变性,肝细胞凋亡增加,Bax的表达明显上升且Bcl-2的表达显著下降(P < 0.01);②荧光定量PCR结果显示,ApoE-/-高蛋氨酸组miR-148a-5p的表达增加(P < 0.05);③靶基因预测软件提示,Bcl-2为miR-148a-5p的潜在靶基因;④双荧光素酶活性实验确定了miR-148a-5p与Bcl-2的靶向关系(P < 0.05);⑤肝细胞中过表达miR-148a-5p后Bax的表达显著上升且Bcl-2的表达明显下降(P < 0.01);⑥提示在高蛋氨酸饮食诱导的ApoE-/-小鼠肝细胞中,miR-148a-5p通过负调控Bcl-2促进肝细胞凋亡,这可能是造成肝损伤的机制之一。 ORCID: 0000-0003-1524-1117(马芳) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

circFADS2靶向miR-125a-5p调控MPP+诱导的SK-N-SH细胞损伤的机制

目的：探究circFADS2靶向miR-125a-5p调控帕金森病（PD）细胞炎症反应和凋亡的机制。方法：100μmol/L MPP+诱导SK-N-SH细胞建立PD模型,分为Con组、PD组、PD+pcDNA组、PD+pcDNA-circFADS2组、PD+anti-miR-NC组、PD+anti-miR-125a-5p组、PD+pcDNA-circFADS2+miR-NC组、PD+pcDNA-circFADS2+miR-125a-5p组。qRT-PCR检测circFADS2和miR-125a-5p表达;ELISA检测IL-1β、TNF-α含量;流式细胞术检测细胞凋亡;Western blot检测cleaved-caspase3、cleaved-caspase9蛋白表达;双荧光素酶报告实验检测circFADS2与miR-125a-5p的靶向关系。结果：与Con组相比,PD组circFADS2表达降低,miR-125a-5p表达、凋亡率、IL-1β、TNF-α含量、cleaved-caspase3、cleaved-caspase9蛋白表达升高（P<0.05）。circFADS2过表...     

miR-125a-5p靶向 Scarb1基因对缺氧/复氧心肌细胞损伤的影响及其机制研究

目的探讨miR-125a-5p靶向调控清道夫受体B1(Scarb1)基因对缺氧/复氧大鼠心肌细胞损伤的影响及作用机制。方法将体外培养的H9c2大鼠心肌细胞随机分为空白对照组、缺氧/复氧组、转染对照组和miR-125a-5p转染组。检测各组miR-125a-5p的表达量、心肌细胞的活力、凋亡率、ATP含量以及Scarb1、Cyt C、Bax、Bcl-2及NF-κB信号通路相关蛋白的表达情况。利用Targetscan软件预测miR-125a-5p的靶基因, 用双荧光素酶报告基因实验验证miR-125a-5p和Scarb1的靶向关系。结果与空白对照组相比, 缺氧/复氧组心肌细胞miR-125a-5p、Bax、Cyt C的表达和凋亡率明显升高(P<0.05), 细胞活力、Scarb1、Bcl-2的表达及ATP含量明显降低(P<0.05)。与转染对照组相比, miR-125a-5p转染组的情况与上述变化相反。双荧光素酶报告基因实验证实Scarb1为miR-125a-5p的靶基因。缺氧/复氧能够促进心肌细胞中NF-κB p65、c-Myc和Cyclin D1蛋白的表达, 而下调miR-...     

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.     

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。     

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。     

LncRNA TSIX promotes osteoblast apoptosis in particle-induced osteolysis by down-regulating miR-30a-5p

Objective: This study aims to investigate the role of TSIX/miR-30a-5p axis in particle-induced osteolysis (PIO).

Method: PIO mouse model was established by the implantation of Co-Cr-Mo metal particles (CoPs). MC3T3-E1 cells received CoPs stimulation. Bone mineral density (BMD) in the skull was detected to evaluate PIO development. The expression of TSIX and miR-30a-5p was detected by using qRT-PCR. Osteoblast apoptosis was measured using flow cytometry. RNA pull-down was used to verify the regulatory relationship between TSIX and miR-30a-5p.

Result: The results showed that BMD of the skull in PIO mice was significantly decreased compared with control mice, which indicated that the PIO model was established successfully. Moreover, CoPs could up-regulate TSIX level, down-regulate miR-30a-5p expression, and promote osteoblast apoptosis in vivo and in vitro. The results also found that TSIX negatively regulated miR-30a-5p expression, and knockdown of TSIX inhibited Runx2 expression. As expected, miR-30a-5p inhibitor could reverse the inhibition of si-TSIX on osteoblast apoptosis.

Conclusion: TSIX played a pivotal role in PIO development by negatively regulating miR-30a-5p.     

微小RNA-181a-5p靶向PTEN对人白血病HL-60细胞增殖和侵袭的影响

目的 探讨微小RNA-181a-5p对白血病HL-60细胞增殖和侵袭的影响,并初步分析其机制。方法体外培养人白血病HL-60细胞,将miR-181a-5p抑制物转染白血病HL-60细胞,MTT法检测各组HL-60细胞的增殖能力,Transwell实验检测各组HL-60细胞的侵袭能力。实时定量PCR和Western blot检测各组细胞miR-181a-5p和PTEN的表达,双荧光素酶报告基因实验分析miR-181a-5p和PTEN的靶向关系。结果 MTT和Transwell实验结果显示,转染miR-181a-5p抑制物后,HL-60细胞的增殖和侵袭能力下降（P<0.05）,miR-181a-5p的表达下降,PTEN的表达上升（P<0.05）。双荧光素酶实验表明PTEN是miR-181a-5p的靶基因。结论 抑制miR-181a-5p使白血病细胞HL-60的增殖和侵袭能力下降,其机制可能与上调PTEN表达有关。     

miR-15a-5p抑制人肝癌SMMC-7721细胞增殖与迁移

目的:观察高表达的miR-15a-5p对人肝细胞癌SMMC-7721细胞增殖和迁移能力的影响。方法:化学合成加入EcoRⅠ和HindⅢ酶切位点的miR-15a-5p寡聚核苷酸,并进行测序确认;利用pcDNA6.2-GW/EmGFP质粒构建miR-15a-5p真核表达载体,瞬时转染SMMC-7721细胞,实时荧光定量PCR检测miR-15a-5p的表达;CCK-8法和台盼蓝染色活细胞计数检测SMMC-7721细胞的增殖能力,划痕实验检测细胞迁移能力的变化。结果:设计的miR-15a-5p序列与寡核苷酸测序结果匹配达100%;真核表达质粒瞬转后人肝癌SMMC-7721细胞miR-15a-5p的表达量与对照组相比显著增加(P0.05);miR-15a-5p高表达SMMC-7721细胞的增殖能力与对照组相比均显著下降(P0.05);miR-15a-5p高表达组细胞迁移速度低于对照组。结论:高表达的miR-15a-5p可抑制人肝癌SMMC-7721细胞的增殖和迁移能力。     

Circular RNA circEGFR regulates tumor progression via the miR-106a-5p/DDX5 axis in colorectal cancer

Recently, an increasing number of studies have reported that dysregulation of circular RNA (circRNA) expression plays critical roles in the progression of several cancers, including colorectal cancer (CRC). However, the detailed molecular mechanisms of circRNAs involvement in CRC remain largely unknown. Here, we confirmed that the level of circEGFR was significantly increased in CRC tissues compared to matched adjacent non-tumor tissues, and a high level of circEGFR was correlated with poor clinicopathological characteristics and poor prognosis in patients with CRC. Moreover, increased circEGFR expression promoted CRC cell proliferation, migration, and invasion in vitro. Mechanistically, circEGFR acted as a ceRNA for miR-106a-5p to relieve the repressive effect of miR-106a-5p on DDX5 mRNA. Moreover, circEGFR enhanced DDX5 expression, thereby upregulating p-AKT levels. Together, these findings showed that circEGFR promoted CRC cell proliferation, migration, and invasion through the miR-106a-5p/DDX5/AKT axis, and may serve as a promising diagnostic marker and therapeutic target for CRC patients.     

miR-30a-5p启动子区DNA甲基化在同型半胱氨酸致肝损伤中的作用

目的:探讨微小RNA-30a-5p(miR-30a-5p)启动子区DNA甲基化在肝损伤中的作用。方法:随机选取4周龄胱硫醚β-合成酶(CBS)基因正常(CBS^+/+)小鼠(n=12)及单基因敲除(CBS^+/-)小鼠(n=12),均给予高蛋氨酸饮食8周。HL-7702细胞体外常规培养,分为对照(control)组、同型半胱氨酸(Hcy)组和Hcy+5-氮杂胞苷(AZC)组。全自动生化分析仪检测小鼠血清Hcy、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平;微板法测定肝细胞ALT和AST水平;小鼠肝脏石蜡切片行HE染色观察肝脏损伤情况;细胞活力染色检测肝细胞活力;RT-qPCR法检测小鼠肝脏组织和肝细胞中miR-30a-5p的表达;Pearson相关性分析肝脏miR-30a-5p表达与血清ALT和AST水平的相关性;巢式降落式甲基化特异性PCR (nMS-PCR)检测小鼠肝脏组织以及肝细胞中miR-30a-5p启动子区DNA甲基化水平的变化。结果:与CBS^+/+对照组相比,CBS^+/-组...     

CircRNA has_circ_0001806 promotes hepatocellular carcinoma progression via the miR-193a-5p/MMP16 pathway

Reportedly, circular RNAs (circRNAs) are crucial regulators in cancer progression. Nonetheless, the molecular mechanism of circRNAs in hepatocellular carcinoma (HCC) has not been fully clarified. Gene expression omnibus (GEO) database was employed to screen out the differentially expressed circRNAs in HCC. qRT-PCR and western blot were executed to detect circ_0001806 expression, miR-193a-5p expression, and MMP16 mRNA and protein expressions in HCC. The effect of circ_0001806 on HCC was analyzed by the CCK-8 method and Transwell experiment. RIP assay, pull-down experiment, and dual-luciferase reporter gene experiment were applied to validate the targeting relationships among circ_0001806, miR-193a-5p, and MMP16. Circ_0001806 was up-modulated in HCC tissues and cell lines. Knockdown of circ_0001806 impeded the multiplication, migration, and invasion of HCC cells. Circ_0001806 could up-regulate MMP16 expression through repressing miR-193a-5p, thereby facilitating the malignant biological behaviors of HCC. Circ_0001806 promoted HCC progression by regulating miR-193a-5p/MMP16 axis.