SNHG6靶向miR-101/TGFBR1促进AMI小鼠左心室心肌纤维化

目的探讨SNHG6对急性心肌梗死(AMI)小鼠左心室心肌的影响。 方法将30只雄性C57/BL6小鼠构建成AMI小鼠后随机均分为AMI组、AMI+SNHG6组、AMI+miR-101-3p组、AMI+SNHG6+miR-101-3p组、AMI+miR-101-3p+TGFBR1组,另设正常小鼠6只为假手术组。qRT-PCR检测AMI小鼠SNHG6、miR-101-3p表达水平。心脏彩超检测各组小鼠左室射血分数(LVEF);马松和天狼星红染色法以及免疫组化分析各组小鼠左心室心肌纤维化变化。将H9C2细胞株分为阴性对照组(转染空质粒)、SNHG6组(转染质粒SNHG6)、miR-101-3p组(转染质粒miR-101-3p)。Western blotting检测各组TGFBR1蛋白表达;采用双荧光素酶报告基因法预测并验证SNHG6/miR-101-3p/TGFBR1荧光素酶活性及调控机制。 结果AMI小鼠较假手术组SNHG6表达显著增加,miR-101-3p降低(P＜0.05)。与AMI组比较,AMI+SNHG6组小鼠LVEF降低,心肌纤维化程度加重(P＜0.05);AMI+miR-101-3p组LVEF升高,心肌纤维化程度减轻(P＜0.05)。AMI+SNHG6+miR-101-3p组较AMI+SNHG6组LVEF升高、心肌纤维化程度减轻(P＜0.05),而AMI+miR-101-3p+TGFBR1组较AMI+miR-101-3p组LVEF降低、心肌纤维化程度加重(P＜0.05)。双荧光素酶报告基因法验证显示,miR-101-3p组SNHG6、TGFBR1野生型质粒的荧光素酶活性较阴性对照组明显降低(P＜0.05)。 结论SNHG6抑制miR-101-3p上调TGFBR1加重AMI小鼠左心室心肌纤维化。     

沉默circROBO1通过下调KRAS抑制鼻咽癌CNE2细胞侵袭与肺转移

目的探讨沉默环状RNA hsa_circ_0124696(circROBO1)对鼻咽癌CNE2细胞侵袭与肺转移的影响机制。 方法qRT-PCR检测鼻咽癌及癌旁组织中circROBO1表达。采用干扰小RNA(siRNA)沉默鼻咽癌CNE2细胞中circROBO1的表达,Transwell及HE染色检测circROBO1对CNE2细胞迁移能力和体内肺转移的影响。TargetScan在线软件预测circROBO1下游miR-217与下游靶基因KRAS的靶向结合位点,双荧光素酶报告基因实验验证两者之间的靶向调控关系。蛋白免疫印迹检测siRNA沉默CNE2细胞中circROBO1表达对KRAS的影响。 结果鼻咽癌组织中circROBO1表达高于癌旁组织(P＜0.05)。与转染si-circNC的对照组相比,si-circROBO1组鼻咽癌CNE2细胞侵袭与体内肺转移能力均显著降低(P＜0.05)。circROBO1下游miR-217与KRAS之间存在靶向结合位点,并且circROBO1可影响KRAS的蛋白和mRNA表达量。 结论沉默circROBO1通过miR-217下调KRAS抑制鼻咽癌CNE2细胞侵袭与肺转移。     

脓毒症患者外周血单核细胞miR-147b的表达及其与病情严重程度和预后的关系

目的 探讨脓毒症患者外周血单核细胞miR-147b的表达水平及其与病情严重程度及预后的相关性。方法 选择2019年1月至2021年6月于安徽医科大学第一附属医院就诊的46例脓毒症患者(脓毒症组)和50例普通感染患者(普通感染组)作为研究对象。查阅患者病历,记录年龄、性别、感染部位等一般资料。采用酶联免疫吸附试验试剂盒检测研究对象血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和C反应蛋白(C-reactive protein, CRP)的浓度。计算序贯性器官衰竭评分(sequential organ failure score, SOFA)和急性生理学与慢性健康状况评分Ⅱ(acute physiology and chronic health scoreⅡ,APACHEⅡ)。观察或随访脓毒症患者的28 d预后情况,分为生存组和死亡组。应用逆转录聚合酶链反应测定外周血单核细胞中miR-147b的表达水平。结果 脓毒症组和普通感染组的年龄、性别、感染部位分布比较差异无统计学意义(P>0.05)。脓毒症组患者的TNF-α、CRP水平、SOFA评分和A...     

Overexpression of TONSL might be an independent unfavorable prognostic indicator in hepatocellular carcinoma

Background

TONSL has been suggested to function as an oncogene in lung, esophageal and cervical cancer. This study was aimed to identify the expression of TONSL and its role in hepatocellular carcinoma (HCC).

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-107抑制胶质瘤细胞的体外增殖、迁移和侵袭

目的探讨miR-107对胶质瘤细胞增殖、迁移和侵袭的调控机制。方法运用RT-qPCR检测人正常的星形胶质细胞系NHA、神经胶质瘤细胞系U87、A172、U251中miR-107和FOXK1的表达;将细胞分为miR-NC组(转染miR-NC)、miR-107组(转染miR-107 mimics)、si-NC组(转染si-NC)、si-FOXK1组(转染si-FOXK1)、miR-107+pcDNA3.1组(共转染miR-107 mimics和pcDNA3.1)和miR-107+pcDNA3.1-FOXK1组(共转染miR-107 mimics和pcDNA3.1-FOXK1);用脂质体法分别转染至U87细胞;CCK-8法检测细胞的增殖;Transwell小室实验检测细胞的迁移和侵袭;Western blot检测细胞中FOXK1的蛋白表达;双荧光素酶报告基因检测实验检测细胞的荧光活性。结果与正常的星形胶质细胞NHA相比,神经胶质瘤细胞U87、A172、U251中miR-107表达明显下调,FOXK1表达明显上调(P<0.05);过表达miR-107、敲减FOXK1均可抑制U87细胞的增殖、迁移和侵袭;miR-107可抑制野生型FOXK1的细胞荧光活性,并负向调控FOXK1的表达;过表达FOXK1可逆转miR-107对U87细胞增殖迁移侵袭的抑制作用。结论 miR-107抑制胶质瘤细胞增殖、迁移和侵袭的作用机制可能与靶向负调控FOXK1有关,将可为胶质瘤的诊断和治疗提供靶向治疗的依据。     

Long non-coding RNA LINC01116 accelerates the progression of keloid formation by regulating miR-203/SMAD5 axis

BackgroundEmerging evidence reveals the importance of long non-coding RNAs (lncRNAs) in the development and progression of keloid formation. However, the roles and molecular mechanism of lncRNA LINC01116 in the progression of keloid formation remain largely unknown.MethodsThe expression levels of LINC01116, microRNA-203 (miR-203) and SMAD family member 5 (SMAD5) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation, migration and invasion were detected by Cell counting Kit-8 (CCK-8) assay and transwell assay. Flow cytometry and western blot assay were used to examine cell apoptosis and extracellular matrix (ECM) production. The interaction between miR-203 and LINC01116 or SMAD5 was predicted by bioinformatics analysis and verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) and RNA pull-down assays.ResultsLINC01116 and SMAD5 were upregulated while miR-203 was downregulated in keloid tissues and keloid fibroblasts. LINC01116 knockdown suppressed the proliferation, migration, invasion, and ECM production but induced apoptosis in keloid fibroblasts through enhancing miR-203 and inhibiting SMAD5. Moreover, SMAD5 was identified as a direct target of miR-203 and miR-203 could directly bind to LINC01116. Besides, LINC01116 regulated SMAD5 expression by targeting miR-203.ConclusionDownregulation of LINC01116 inhibited the progression of keloid formation by regulating miR-203/SMAD5 axis, which might provide a novel target for keloid therapy.     

人参皂苷Rg1对心肌细胞氧化应激损伤的抑制作用

目的 通过构建H₂O₂诱导的H9c2细胞氧化应激模型,观察人参皂苷Rg₁对氧化应激的抑制作用,并探讨mi R-499c是否参与人参皂苷Rg₁抑制氧化应激的作用机制。方法 采用600μmol/L的H₂O₂诱导H9c2细胞氧化应激模型,给予人参皂苷Rg₁预处理24 h,检测乳酸脱氢酶(lactate dehydrogenase,LDH)、超氧化物歧化酶(superoxide dismutase,SOD)活性及丙二醛(malondialdehyde,MDA)、活性氧(reactive oxygen species,ROS)水平和线粒体膜电位(mitochondrial membrane potential,MMP)变化;采用Western blotting检测凋亡相关蛋白表达。采用Lipofiter 3.0将miR-499c转染至H9c2细胞再制备H₂O₂诱导的氧化应激模型,给予人参皂苷Rg     

miR-21对骨质疏松小鼠骨髓基质细胞增殖的影响

目的探讨miR-21对骨质疏松小鼠骨髓基质细胞(BMSCs)增殖的影响。方法采用双侧卵巢切除法构建骨质疏松小鼠模型(VOX),分离、培养、纯化小鼠BMSCs并采用si PORT Neo FX转染pre-miR-21、pre-miR-negative control(pre-miR-NC)、antmiR-21、ant-miR-negative control(ant-miR-NC)并进行RT-PCR验证,MTT法检测小鼠BMSCs增殖情况、茜素红与碱性磷酸酶染色法检测小鼠BMSCs成骨能力、Western-blotting检测细胞增殖、成骨分化相关蛋白水平。结果骨质疏松症小鼠BMSCs中miR-21相对表达水平低于Ctrl组(P0.05),OVX-pre-miR-21组BMSCs中miR-21相对表达水平、细胞增殖、PCNA水平、Ki-67水平、ALP染色程度、ALP活性、茜素红染色程度、Runx2水平、Osterix水平均高于OVX-pre-miR-NC组(P0.05),OVX-premiR-NC组BMSCs中miR-21相对表达水平、细胞增殖、PCNA水平、Ki-67水平、ALP染色程度、ALP活性、茜素红染色程度、Runx2水平、Osterix水平均显著低于Ctrl-pre-miR-NC(P0.05); OVX-ant-miR-21组BMSCs中miR-21相对表达水平、细胞增殖、PCNA水平、Ki-67水平、ALP染色程度、ALP活性、茜素红染色程度、Runx2水平、Osterix水平均显著低于OVX-ant-miR-NC组(P0.05),OVX-ant-miR-NC组BMSCs中miR-21相对表达水平、细胞增殖、PCNA水平、Ki-67水平、ALP染色程度、ALP活性、茜素红染色程度、Runx2水平、Osterix水平均显著低于Ctrl-ant-miR-NC(P0.05)。结论提高miR-21表达水平可促进骨质疏松小鼠BMSCs增殖能力与成骨分化能力。     

miR-146a-5p regulates autophagy and NLRP3 inflammasome activation in epithelial barrier damage in the in vitro cell model of ulcerative colitis through the RNF8/Notch1/mTORC1 pathway

Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon that can be influenced by microRNAs (miRNAs). This study aims to investigate the impact of miR-146a-5p on lipopolysaccharide (LPS)-induced Caco-2/HT-29 cell autophagy and NLRP3 inflammasome activation and the underlying mechanism, with the aim of identifying potential therapeutic targets. We used LPS to establish Caco-2/HT-29 cell models and measured cell viability by CCK-8. The levels of miR-146a-5p, RNF8, markers of NLRP3 inflammasome activation and autophagy, proteins involved in the Notch1/mTORC1 pathway, and inflammatory factors were assessed by RT-qPCR, Western blot, and ELISA. Intestinal epithelial barrier function was evaluated by measuring transepithelial electrical resistance. Autophagic flux was measured using tandem fluorescent-labeled LC3. miR-146a-5p was highly-expressed in LPS-induced Caco-2/HT-29 cells, and autophagy flux was blocked at the autolysosomal stage after LPS induction. Inhibition of miR-146a-5p suppressed NLRP3 inflammasome activation, reduced intestinal epithelial barrier damage, and facilitated autophagy inhibition in LPS-induced Caco-2/HT-29 cells. The autophagy inhibitor NH4Cl partially nullified the inhibitory effects of miR-146a-5p inhibition on NLRP3 inflammation activation. miR-146a-5p targeted RNF8, and silencing RNF8 partly abrogated the action of miR-146a-5p inhibition on promoting autophagy and inhibiting NLRP3 inflammasome activation. miR-146a-5p inhibition suppressed the Notch1/mTORC1 pathway activation by upregulating RNF8. Inhibition of the Notch1/mTORC1 pathway partially nullified the function of silencing RNF8 on inhibiting autophagy and bolstering NLRP3 inflammasome activation. In conclusion, miR-146a-5p inhibition may be a potential therapeutic approach for UC, as it facilitates autophagy of LPS-stimulated Caco-2/HT-29 cells, inhibits NLRP3 inflammasome activation, and reduces intestinal epithelial barrier damage by upregulating RNF8 and suppressing the Notch1/mTORC1 pathway.