miR-451a调控BAP31诱导结直肠癌细胞凋亡  

MiR-451 aregulated BAP31 in colorectal cancer cells induced apoptosis

作　　者：许可[1] 韩彬[1] 柏杨[1] 周黎明[1] Xu Ke;Han Bing;Bo Yang;Zhou Li-ming(Department of Pharmacology, West China School of Basic Medical Sciences ＆ Forensie Medicine, Sichuan University, Sichuan Chengdu 610041)

机构地区：[1]四川大学华西基础医学与法医学院药理学教研室,四川成都610041

出　　处：《四川生理科学杂志》2017年第4期171-177,共7页Sichuan Journal of Physiological Sciences

摘　　要：目的:探讨miR-451a及靶蛋白BAP31在结直肠癌中的作用。方法:体外培养HCT116、SW620、SW480、DLD细胞,以Real-time PCR法检测结直肠癌细胞系中miR-451a和BAP31的相对表达量;通过建立miR-451a不同表达情况的结直肠癌细胞HCT116模型,应用抑制消减杂交方法建立抑制消减文库,从中筛选miR-451a的调控蛋白,并通过萤光素酶报告基因检测miR-451a的直接调控靶基因;采用MTT法、流式细胞术以及Hoechst染色法评价miR-451a调控的靶蛋白BAP31对结直肠细胞凋亡的调节作用。结果:在抑制杂交消减文库中得到了miR-451a可能调控的相关基因,其中在正向文库中得到BAP31、EEF1A1和CDC20等7个基因,在反向文库中得到DKK1和PSME1等4个基因。在HCT116、HT29、SW480、SW620和DLD结直肠癌细胞中miR-451a的相对表达量是正常结肠上皮细胞NCM460中的0.32、0.44、0.53、0.43和0.73倍,BAP31在DLD、HT29、SW620和HCT116结直肠癌细胞中的表达量是正常结肠上皮细胞NCM460中的1.85、2.84、2.37和3.71倍。双萤光素酶报告基因实验证明,miR-451a可作用于与BAP31开放阅读框上游177的位点,通过miR-451a作用使海肾荧光素酶的活性降低80.3%。在HCT116细胞和SW620细胞中过表达miR-451a后72h抑制率分别为39.50%和39.50%;沉默BAP31后72h抑制率分别为45.32%和53.56%。过表达miR-451a48h后HCT116凋亡增加13.57%,SW620细胞凋亡率增加13.2%;沉默BAP31 48h后HCT116细胞凋亡增加5.62%,SW620细胞凋亡率增加8.68%。结论:miR-451a在结直肠癌细胞中能够直接通过调控BAP31诱导细胞的凋亡从而抑制细胞的增殖。Objective：To investigate the role of miR-451 aand its target gene BAP31 in colorectal cancer cell lines.Methods：The relative expression of miR-451 aand BAP31 in colorectal cancer cell lines was detected by real-time PCR in HCT116,SW620,SW480 and DLD cells.The inhibitory subtractive library of colorectal cancer cell line miR-451 awas established by suppressing subtractive hybridization method by using the different expressing of miR-451 aHCT116 model.The BAP31 protein was selected as the study object of miR-451 a.Luciferase reporter gene was used to determine whether BAP31 is directly regulated target genes of miR-451 aby testing luciferase relative activity.The proliferation of colorectal cancer cells was evaluated by MTT assay.The apoptosis inducted by miR-451 aregulated BAP31 was evaluated by flow cytometry and Hoechst staining.Results：The genes related to the possible regulation of miR-451 awere obtained in the suppression subtractive library.Seven genes,such as BAP31,EEF1 A1 and CDC20,were obtained in the positive library.DKK1,PSME1 and other genes were obtained in the reverse library.The relative expression levels of miR-451 ain HCT116,HT29,SW480,SW620 and DLD colorectal cancer cells were 0.32,0.40,0.53,0.43 and 0.73 times higher than those in normal colon epithelial cells NCM460.The relative expression of BAP31 in DLD,HT29,SW620 and HCT116 in colorectal cancer cells was 1.85,2.84,2.37 and 3.71 times higher than that in normal colon epithelial cells NCM460.Double-luciferase reporter gene experiments show that miR-451 acan act on the upstream of the BAP31 open reading frame at 177 site,and the activity of renilla luciferase is reduced by 80.3%.Over-expressed miR-451 ain the HCT116 and SW920 cells,the inhibitory rates of were39.50% and 39.50% at 72 h,respectively;and the inhibitory rates were 58.32% and 53.56% at 72 hafter silencing BAP31,respectively.The apoptosis of HCT116 and SW620 cells were increased 13.57% and 13.2% after 48 hours over-expressed miR-451 a;the apoptosis of HCT116 and SW620 cells wer

关 键 词：结直肠癌 miR-451a BAP31 凋亡 

分 类 号：R735.3[医药卫生—肿瘤]