目的:基于miR-451a靶向磷酸甘油酸变位酶(PGAM5)-线粒体动力相关蛋白1(Drp1)轴调控线粒体动力学,探讨温肺降浊方对氧糖剥夺/复氧(OGD/R)诱导HT22模型的效应机制。方法:建立OGD/R诱导HT22细胞模型,设立正常对照组、OGD/R组、miR-451a过...目的:基于miR-451a靶向磷酸甘油酸变位酶(PGAM5)-线粒体动力相关蛋白1(Drp1)轴调控线粒体动力学,探讨温肺降浊方对氧糖剥夺/复氧(OGD/R)诱导HT22模型的效应机制。方法:建立OGD/R诱导HT22细胞模型,设立正常对照组、OGD/R组、miR-451a过表达组、miR-451a敲低组、温肺降浊方含药血清(WFJZF)组、miR-451a过表达+WFJZF组、miR-451a敲低+WFJZF组和miR-451a空载体组,双荧光素酶确定miR-451a和PGAM5的转录关系,免疫蛋白共沉淀确定PGAM5和Drp1的调控作用,慢病毒转染miR-451a于HT22细胞中,qRT-PCR确定转染效率,CCK-8测定缺糖缺氧状态下细胞活性的最佳时间点,qRT-PCR和Westem Blot检测HT22模型细胞内DGAM5-Drp1轴蛋白及mRNA的表达水平。结果:OGD/R诱导HT22细胞持续2 h为最佳时间点,miR-451a可以靶向调控PGAM5基因,PGAM5和Drp1具有相互调控作用。与正常对照组比较,OGD/R组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达上升,p-Drp1 Ser 616磷酸化表达上升(P<0.05),OPA1蛋白及mRNA表达下降,p-Drp1 Ser 637去磷酸化表达下降(P<0.05);与OGD/R组比较,miR-451a过表达+WFJZF组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达下降,p-Drp1 Ser 616磷酸化表达下降(P<0.05),OPA1蛋白及mRNA表达上升,p-Drp1 Ser 637去磷酸化表达上升(P<0.05)。结论:miR-451a可以靶向调控PGAM5-Drp1轴,miR-451a过表达+WFJZF组可以改善线粒体失衡状态,减少神经元过度损伤,发挥脑保护效应。展开更多
BACKGROUND Aberrant microRNAs expression and associated pathways have been proved participate in regulation vast various physiologic and pathologic processed of different human cancers including liver cancer.While,the...BACKGROUND Aberrant microRNAs expression and associated pathways have been proved participate in regulation vast various physiologic and pathologic processed of different human cancers including liver cancer.While,the function of miR-451a in liver cancer still indistinct.AIM To study the effect of miR-451a in liver cancer development.METHODS GeneChip microarray analysis performed to detect miR-451a expression in liver cancer tissues and normal liver tissues.Reverse transcription-polymerase chain reaction was used to validate the expression of miR-451a in liver cancer cell and other tumor cell lines.Construction of liver cancer cell lines that stably overexpressed miR-451a by transfecting Lentivirus produced by Genechem company.Methylthiazolyldiphenyl-tetrazolium(MTT)bromide assay and colony formation assay to determine the effect of miR-451a in liver cancer cell proliferation.Flow cytometry used to investigate whether miR-451a involved in liver cancer cell apoptosis.Cell migration ability was measured via wound scratch assay.Target gene was explored by bioinformatic analysis,and downstream molecule of miR-451a in liver cancer identified by rescue experiments.RESULTS MiR-451a expression significantly downregulation in liver cancer tissues compared with that in normal liver tissue.MiR-451a also obviously low-expressed in liver cancer cell,colorectal carcinoma cell and esophageal carcinoma cell lines.Human hepatoblastoma G2(HepG2)and BEL-7404 cell lines that stably overexpressed miR-451a by transfecting lentivirus constructed successfully.MTT bromide assay and colony formation assay showed that the overexpressed miR-451a inhibit HepG2 cell proliferation viability,but not BEL-7404 cell.Flow cytometry determined that miR-451a regulating proliferation not through inducing apoptosis.Wound scratch assay revealed that miR-451a overexpression suppressed HepG2 cell migration.Furthermore,mex-3 RNA binding family member C was predicted as the target gene by bioinformatic analysis,and rescue experiments confirmed the hypothesis.CONCLUSION Therefore,miR-451a may be candidate miRNA for understanding molecular mechanisms of liver cancer development and novel target in liver cancer cell.展开更多
目的探索血清外泌体miR-451a在弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)中的水平及其在治疗监测中的价值。方法本研究共纳入56例DLBCL患者,56例健康对照者。收集新发DLBCL患者治疗前、化疗2~4疗程及化疗结束后血清样本...目的探索血清外泌体miR-451a在弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)中的水平及其在治疗监测中的价值。方法本研究共纳入56例DLBCL患者,56例健康对照者。收集新发DLBCL患者治疗前、化疗2~4疗程及化疗结束后血清样本,并同时收集健康人血液标本,提取血清中的外泌体RNA,并进行实时荧光定量PCR(quantitative real time polymerase chain reaction,qRT-PCR),用受试者工作特征(receive operator characteristic,ROC)曲线判定miR-451a的诊断效能,用各时点收集的血清样本动态分析血清外泌体miR-451a水平与化疗效果之间的关系。结果 56例DLBCL患者与56例健康对照者相比,DLBCL患者血清外泌体miR-451a水平下降(P<0.000 1),在两组受试者间用miR-451a诊断DLBCL的曲线下面积(AUC)为0.737(95%CI0.645~0.816)。在随访到的43例DLBCL患者中,化疗后获得完全缓解或者部分缓解的患者其血清外泌体miR-451a水平较化疗前有所上升(P<0.05),与配对的健康人水平差异无统计学意义(P>0.05);化疗后未获得缓解的患者,其血清外泌体miR-451a水平较化疗前无明显变化(P>0.05),且仍然低于配对的健康人水平(P<0.05);化疗完成后在未缓解者与缓解者之间进行鉴别,血清miR-451a的AUC为0.867(95%CI 0.728~0.951)。结论血清外泌体miR-451a水平动态监测有助于DLBCL化疗过程中的疗效(是否缓解)判断。展开更多
文摘目的:基于miR-451a靶向磷酸甘油酸变位酶(PGAM5)-线粒体动力相关蛋白1(Drp1)轴调控线粒体动力学,探讨温肺降浊方对氧糖剥夺/复氧(OGD/R)诱导HT22模型的效应机制。方法:建立OGD/R诱导HT22细胞模型,设立正常对照组、OGD/R组、miR-451a过表达组、miR-451a敲低组、温肺降浊方含药血清(WFJZF)组、miR-451a过表达+WFJZF组、miR-451a敲低+WFJZF组和miR-451a空载体组,双荧光素酶确定miR-451a和PGAM5的转录关系,免疫蛋白共沉淀确定PGAM5和Drp1的调控作用,慢病毒转染miR-451a于HT22细胞中,qRT-PCR确定转染效率,CCK-8测定缺糖缺氧状态下细胞活性的最佳时间点,qRT-PCR和Westem Blot检测HT22模型细胞内DGAM5-Drp1轴蛋白及mRNA的表达水平。结果:OGD/R诱导HT22细胞持续2 h为最佳时间点,miR-451a可以靶向调控PGAM5基因,PGAM5和Drp1具有相互调控作用。与正常对照组比较,OGD/R组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达上升,p-Drp1 Ser 616磷酸化表达上升(P<0.05),OPA1蛋白及mRNA表达下降,p-Drp1 Ser 637去磷酸化表达下降(P<0.05);与OGD/R组比较,miR-451a过表达+WFJZF组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达下降,p-Drp1 Ser 616磷酸化表达下降(P<0.05),OPA1蛋白及mRNA表达上升,p-Drp1 Ser 637去磷酸化表达上升(P<0.05)。结论:miR-451a可以靶向调控PGAM5-Drp1轴,miR-451a过表达+WFJZF组可以改善线粒体失衡状态,减少神经元过度损伤,发挥脑保护效应。
基金Supported by National Natural Science Foundation of China,No.31640052and Natural Science Foundation of Shandong Province,China,No.ZR2019BH031.
文摘BACKGROUND Aberrant microRNAs expression and associated pathways have been proved participate in regulation vast various physiologic and pathologic processed of different human cancers including liver cancer.While,the function of miR-451a in liver cancer still indistinct.AIM To study the effect of miR-451a in liver cancer development.METHODS GeneChip microarray analysis performed to detect miR-451a expression in liver cancer tissues and normal liver tissues.Reverse transcription-polymerase chain reaction was used to validate the expression of miR-451a in liver cancer cell and other tumor cell lines.Construction of liver cancer cell lines that stably overexpressed miR-451a by transfecting Lentivirus produced by Genechem company.Methylthiazolyldiphenyl-tetrazolium(MTT)bromide assay and colony formation assay to determine the effect of miR-451a in liver cancer cell proliferation.Flow cytometry used to investigate whether miR-451a involved in liver cancer cell apoptosis.Cell migration ability was measured via wound scratch assay.Target gene was explored by bioinformatic analysis,and downstream molecule of miR-451a in liver cancer identified by rescue experiments.RESULTS MiR-451a expression significantly downregulation in liver cancer tissues compared with that in normal liver tissue.MiR-451a also obviously low-expressed in liver cancer cell,colorectal carcinoma cell and esophageal carcinoma cell lines.Human hepatoblastoma G2(HepG2)and BEL-7404 cell lines that stably overexpressed miR-451a by transfecting lentivirus constructed successfully.MTT bromide assay and colony formation assay showed that the overexpressed miR-451a inhibit HepG2 cell proliferation viability,but not BEL-7404 cell.Flow cytometry determined that miR-451a regulating proliferation not through inducing apoptosis.Wound scratch assay revealed that miR-451a overexpression suppressed HepG2 cell migration.Furthermore,mex-3 RNA binding family member C was predicted as the target gene by bioinformatic analysis,and rescue experiments confirmed the hypothesis.CONCLUSION Therefore,miR-451a may be candidate miRNA for understanding molecular mechanisms of liver cancer development and novel target in liver cancer cell.
文摘目的探索血清外泌体miR-451a在弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)中的水平及其在治疗监测中的价值。方法本研究共纳入56例DLBCL患者,56例健康对照者。收集新发DLBCL患者治疗前、化疗2~4疗程及化疗结束后血清样本,并同时收集健康人血液标本,提取血清中的外泌体RNA,并进行实时荧光定量PCR(quantitative real time polymerase chain reaction,qRT-PCR),用受试者工作特征(receive operator characteristic,ROC)曲线判定miR-451a的诊断效能,用各时点收集的血清样本动态分析血清外泌体miR-451a水平与化疗效果之间的关系。结果 56例DLBCL患者与56例健康对照者相比,DLBCL患者血清外泌体miR-451a水平下降(P<0.000 1),在两组受试者间用miR-451a诊断DLBCL的曲线下面积(AUC)为0.737(95%CI0.645~0.816)。在随访到的43例DLBCL患者中,化疗后获得完全缓解或者部分缓解的患者其血清外泌体miR-451a水平较化疗前有所上升(P<0.05),与配对的健康人水平差异无统计学意义(P>0.05);化疗后未获得缓解的患者,其血清外泌体miR-451a水平较化疗前无明显变化(P>0.05),且仍然低于配对的健康人水平(P<0.05);化疗完成后在未缓解者与缓解者之间进行鉴别,血清miR-451a的AUC为0.867(95%CI 0.728~0.951)。结论血清外泌体miR-451a水平动态监测有助于DLBCL化疗过程中的疗效(是否缓解)判断。
