背景胰腺腺泡细胞增殖及凋亡与急性胰腺炎(acute pancreatitis,AP)发生密切相关,miRNA可通过调控靶基因表达从而参与细胞增殖及凋亡过程,因而寻找与胰腺腺泡细胞增殖及凋亡相关的miRNA分子标志物对临床诊断及治疗AP具有重要意义.目的探...背景胰腺腺泡细胞增殖及凋亡与急性胰腺炎(acute pancreatitis,AP)发生密切相关,miRNA可通过调控靶基因表达从而参与细胞增殖及凋亡过程,因而寻找与胰腺腺泡细胞增殖及凋亡相关的miRNA分子标志物对临床诊断及治疗AP具有重要意义.目的探讨微小RNA-7a-5p(miR-7a-5p)对AP腺泡细胞增殖、凋亡的影响及机制.方法构建雨蛙肽诱导的AP模型并收集胰腺炎腺泡细胞AR42J(雨蛙肽组),采用qRT-PCR与Western blot分别检测未经雨蛙肽诱导的AR42J细胞(对照组)及雨蛙肽组AR42J细胞中miR-7a-5p相对表达量及活化信号转导和转录激活因子的蛋白抑制因子-1(proteininhibitor of activated signal transducer and activator oftranscription 1,PIAS1)表达.分别将anti-miR-7a-5p、pcDNA-PIAS1转染至雨蛙肽组AR42J细胞,采用MTT法检测AR42J细胞增殖能力,流式细胞术检测AR42J细胞凋亡.荧光素酶报告系统检测miR-7a-5p对PIAS1基因的靶向调控作用,并采用Western blot检测miR-7a-5p对PIAS1蛋白表达的调控作用.采用RNA干扰技术沉默PIAS1表达(si-PIAS1组),分别将si-PIAS1及其阴性对照转染至anti-miR-7a-5p组AR42J细胞,观察AR42J细胞增殖及凋亡能力.结果与对照组相比,雨蛙肽组AR42J细胞中miR-7a-5p表达水平显著升高(P<0.05),PIAS1蛋白表达显著降低(P<0.05);抑制miR-7a-5p表达可促进胰腺炎腺泡细胞AR42J增殖并抑制其凋亡;miR-7a-5p可负向调控靶基因PIAS1表达;PIAS1过表达可促进AR42J细胞增殖并抑制其凋亡;与anti-miR-7a-5p+si-NC组相比,anti-miR-7a-5p+si-PIAS1组AR42J细胞活性显著降低(P<0.05),细胞凋亡率显著升高(P<0.05).结论miR-7a-5p可通过抑制PIAS1表达进而促进AP腺泡细胞凋亡并降低细胞增殖能力.展开更多
This study investigated the effect of etoposide,an anticancer chemotherapy drug,on B7-H1 expression in retinoblastoma(Rb) cells and the role of miR-513a-5p in the process.Rb cells were divided into control and etoposi...This study investigated the effect of etoposide,an anticancer chemotherapy drug,on B7-H1 expression in retinoblastoma(Rb) cells and the role of miR-513a-5p in the process.Rb cells were divided into control and etoposide groups.In the etoposide group,cells were treated with etoposide at different concentrations(2.5,5,10,20 and 40 μg/mL) for 24 h.Those given no treatment of etopside served as controls.Reverse transcription polymerase chain reaction(RT-PCR),fluorescence quantitative PCR and flow cytometry were performed to measure the mRNA and protein expression of B7-H1 in Rb cells.The mRNA expression of miR-513a-5p in Rb cells before and after etoposide treatment was also detected by fluorescence quantitative PCR.The miR-513a-5p mimics and the miR-513a-5p inhibitor were transfected into Rb cells separately,and fluorescence quantitative PCR and flow cytometry were used to detect the effect of the miR-513a-5p mimics or inhibitor on B7-H1 expression.TargetScan5.2 was employed to predict the miR-513a-5p binding sites in the 3’-untranslated region of B7-H1 mRNA.Luciferase reporter plasmids carrying this site were prepared and transfected into Rb cells and luciferase activity analyzed.The results showed that etoposide stimulated the mRNA and protein expression of B7-H1 in Rb cells,which reached a maximal level after treatment with 5 μg/mL etoposide(P<0.05).However,miR-513a-5p expression was decreased in Rb cells after etoposide treatment.When the miR-513a-5p inhibitor was added,B7-H1 expression was increased with the concentration of the miR-513a-5p inhibitor(P<0.05).Moreover,B7-H1 expression was decreased gradually with the concentration of the miR-513a-5p mimics increased(P<0.01).Additionally,the miR-513a-5p mimics were found to inhibit the luciferase activity.It was concluded that etoposide can promote B7-H1 expression in Rb cells,which may be associated with chemoresistance.The promoting effect of etoposide on B7-H1 expression can be reversed by miR-513a-5p mimics.MiR-513a-5p inhibits the mRNA and protein expression of B7-H1 via binding to the 3’-UTR of B7-H1 mRNA.展开更多
文摘背景胰腺腺泡细胞增殖及凋亡与急性胰腺炎(acute pancreatitis,AP)发生密切相关,miRNA可通过调控靶基因表达从而参与细胞增殖及凋亡过程,因而寻找与胰腺腺泡细胞增殖及凋亡相关的miRNA分子标志物对临床诊断及治疗AP具有重要意义.目的探讨微小RNA-7a-5p(miR-7a-5p)对AP腺泡细胞增殖、凋亡的影响及机制.方法构建雨蛙肽诱导的AP模型并收集胰腺炎腺泡细胞AR42J(雨蛙肽组),采用qRT-PCR与Western blot分别检测未经雨蛙肽诱导的AR42J细胞(对照组)及雨蛙肽组AR42J细胞中miR-7a-5p相对表达量及活化信号转导和转录激活因子的蛋白抑制因子-1(proteininhibitor of activated signal transducer and activator oftranscription 1,PIAS1)表达.分别将anti-miR-7a-5p、pcDNA-PIAS1转染至雨蛙肽组AR42J细胞,采用MTT法检测AR42J细胞增殖能力,流式细胞术检测AR42J细胞凋亡.荧光素酶报告系统检测miR-7a-5p对PIAS1基因的靶向调控作用,并采用Western blot检测miR-7a-5p对PIAS1蛋白表达的调控作用.采用RNA干扰技术沉默PIAS1表达(si-PIAS1组),分别将si-PIAS1及其阴性对照转染至anti-miR-7a-5p组AR42J细胞,观察AR42J细胞增殖及凋亡能力.结果与对照组相比,雨蛙肽组AR42J细胞中miR-7a-5p表达水平显著升高(P<0.05),PIAS1蛋白表达显著降低(P<0.05);抑制miR-7a-5p表达可促进胰腺炎腺泡细胞AR42J增殖并抑制其凋亡;miR-7a-5p可负向调控靶基因PIAS1表达;PIAS1过表达可促进AR42J细胞增殖并抑制其凋亡;与anti-miR-7a-5p+si-NC组相比,anti-miR-7a-5p+si-PIAS1组AR42J细胞活性显著降低(P<0.05),细胞凋亡率显著升高(P<0.05).结论miR-7a-5p可通过抑制PIAS1表达进而促进AP腺泡细胞凋亡并降低细胞增殖能力.
文摘This study investigated the effect of etoposide,an anticancer chemotherapy drug,on B7-H1 expression in retinoblastoma(Rb) cells and the role of miR-513a-5p in the process.Rb cells were divided into control and etoposide groups.In the etoposide group,cells were treated with etoposide at different concentrations(2.5,5,10,20 and 40 μg/mL) for 24 h.Those given no treatment of etopside served as controls.Reverse transcription polymerase chain reaction(RT-PCR),fluorescence quantitative PCR and flow cytometry were performed to measure the mRNA and protein expression of B7-H1 in Rb cells.The mRNA expression of miR-513a-5p in Rb cells before and after etoposide treatment was also detected by fluorescence quantitative PCR.The miR-513a-5p mimics and the miR-513a-5p inhibitor were transfected into Rb cells separately,and fluorescence quantitative PCR and flow cytometry were used to detect the effect of the miR-513a-5p mimics or inhibitor on B7-H1 expression.TargetScan5.2 was employed to predict the miR-513a-5p binding sites in the 3’-untranslated region of B7-H1 mRNA.Luciferase reporter plasmids carrying this site were prepared and transfected into Rb cells and luciferase activity analyzed.The results showed that etoposide stimulated the mRNA and protein expression of B7-H1 in Rb cells,which reached a maximal level after treatment with 5 μg/mL etoposide(P<0.05).However,miR-513a-5p expression was decreased in Rb cells after etoposide treatment.When the miR-513a-5p inhibitor was added,B7-H1 expression was increased with the concentration of the miR-513a-5p inhibitor(P<0.05).Moreover,B7-H1 expression was decreased gradually with the concentration of the miR-513a-5p mimics increased(P<0.01).Additionally,the miR-513a-5p mimics were found to inhibit the luciferase activity.It was concluded that etoposide can promote B7-H1 expression in Rb cells,which may be associated with chemoresistance.The promoting effect of etoposide on B7-H1 expression can be reversed by miR-513a-5p mimics.MiR-513a-5p inhibits the mRNA and protein expression of B7-H1 via binding to the 3’-UTR of B7-H1 mRNA.
