MiR-217对小鼠胰腺成体干细胞增殖的调控作用  

Regulation of miR-217 on the proliferation of mouse adult pancreatic stem cells

作　　者：吴琼 卢洲 马东慎[1] 邢芸[1] 金亮[1] WU Qiong;LU Zhou;MA Dongshen;XING Yun;JIN Liang(School of Life Science and Technology,China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国药科大学学报》2018年第2期247-252,共6页Journal of China Pharmaceutical University

基　　金：国家高技术研究发展计划("八六三"计划)资助项目(No.2015AA020314);国家自然科学基金资助项目(No.81570696);江苏高校"青蓝工程"资助项目;高等学校学科创新引智计划("111计划")(B16046);江苏高校优势学科建设工程资助项目(PAPD)~~

摘　　要：为了进一步研究miR-217在小鼠胰腺成体干细胞生长过程中的调控作用,在培养的胰腺成体干细胞中过表达miR-217,用Western blot和免疫荧光的方法检测miR-217对细胞增殖的影响。结果显示,miR-217能够明显抑制胰腺成体干细胞增殖相关蛋白Cyclin D1和Ki-67的表达。使用生物信息学软件预测miR-217的靶基因,并通过双荧光素酶报告基因的方法验证了miR-217对靶点的靶向性,实验结果表明,miR-217能够抑制PMIR-REPORT-Sirt1-3'UTR荧光素酶活性。进一步在二维培养的胰腺成体干细胞中,过表达miR-217检测Sirt1的表达变化,q PCR和Western blot表明在胰腺成体干细胞中miR-217能够抑制Sirt1的蛋白水平的表达,但对m RNA水平没有影响。在胰腺成体干细胞中分别加入Sirt1的抑制剂尼克酰胺和激动剂白藜芦醇,尼克酰胺能够抑制胰腺成体干细胞的增殖,而白藜芦醇促进了胰腺成体干细胞的增殖。以上结果说明,miR-217通过Sirt1调控胰腺成体干细胞的生长,低表达的miR-217有助于维持胰腺成体干细胞的增殖。To further evaluate the effect of miR-217 in the proliferation of mouse adult pancreatic stem cells,we firstly transfected adult pancreatic stem cells with miR-217 mimics and studied the effect of miR-217 on proliferation through Western blot and immunofluorescence.Results showed that during the proliferation of adult pancreatic stem cells,miR-217 inhibited the protein expression of Ki-67 and Cyclin D1,which are related to cell propagation.As well as that,to investigate the target genes of miR-217 and their conserved sites bound by the seed region of miR-217,we used bioinformatic algorithms to find a potential target of miR-217 and verified by dual-luciferase activity assay.Surprisingly,dual-luciferase activity assay revealed that miR-217 could decrease PMIR-REPORT-Sirt1-3′UTR luciferase activity and Sirt1 is a direct target of miR-217.Finally,we verified the function of Sirt1 in the proliferation of pancreatic stem cells.Overexpression of miR-217 in pancreatic stem cells inhibited the level of Sirt1 in protein level but not in mRNA level.Furthermore,activator of Sirt1 played positive effect on colony formation ability and cell proliferation and inhibitor of Sirt1 showed the opposite function.In conclusion,miR-217 inhibits the proliferation of mouse adult pancreatic stem cells through Sirt1 and decreased expression of miR-217 to contribute to the pancreatic stem cells development.

关 键 词：miR-217 胰腺成体干细胞 Sirt1 CYCLIN D1 Ki-67 增殖 

分 类 号：R735[医药卫生—肿瘤]