MKRN1基因siRNA重组腺病毒载体构建及功能鉴定

目的构建MKRN1的siRNA重组腺病毒载体，并在表达MKRN1的HeLa细胞中鉴定其干扰作用和对端粒酶活性的影响，为探讨MKRN1功能及其与肿瘤关系提供有效工具。方法人工合成靶向MKRN1的siRNA干扰序列，用分子克隆的方法克隆到穿梭载体pSES-HUS上得到pSES-HUS-MKRN1 siRNA，并与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组得到pAdeasy-SES-HUS-MKRN1 siRNA；在HEK293细胞中包装成重组腺病毒，感染表达MKRN1的HeLa细胞株，用RT-PCR法及Western印迹技术检测腺病毒对细胞MKRN1表达的影响，用PCR-TRAP法检测细胞中端粒酶活性的变化。结果成功构建了MKRN1的siR-NA重组腺病毒载体；MKRN1的siRNA重组腺病毒能显著抑制HeLa细胞中MKRN1的表达，并显著上调细胞中端粒酶的活性。结论构建的Adeasy-MKRN1 siRNA腺病毒能有效地抑制HeLa细胞中MKRN1的表达并上调细胞端粒酶活性，从而为进一步研究MKRN1的功能及其与肿瘤的关系提供了有效的新工具。

Construction and Functional Identification of Human MKRN1-siRNA Recombinant Adenovirus Vector

AIM To construct recombinant adenovirus vector containing siRNA targeting MKRN1 gene and to identify the siRNA interfering effects on MKRN1 function and telomerase activity in Hela cells expressing MKRN1. Methods The siRNA sequence targeting MKRN1 gene was synthesized and cloned into the shuttle plasmid pSES-HUS to construct the vector pSES-HUS-MKRN1 siRNA. It was homogenously recombined with adenovirus backbone plasmid pAdeasy-1 in E. coli BJ5183. The recombinant adenovirus vector pAdeasy-SES-HUS-MKRN1 siRNA was then transfected into HEK293 cells where adenovirus was packaged and amplified. The Hela cells expressing MKRN1 gene were infected by the produced adenovirus. The effect of siRNA on expression of MKRN1 target gene was detected by RT-PCR and Western blotting. The activities of telomerase in Hela cells were detected by PCR-TRAP. Results The recombinant adenoviral vector containing siRNA targeting MKRN1 gene was successfully constructed. MKRN1 expression in Hela cells was significantly inhibited by MKRN1 siRNA. Meanwhile, the activity of telomerase was notably upregulated. Conclusion The recombinant adenovirus Adeasy-MKRN1 siRNA effectively inhibits MKRN1 expression and increases telomerase activity in Hela cells, thereby providing a valid tool for further studying the function of MKRN1 and its relation to tumor.