干细胞标志Musashi2基因RNAi腺病毒载体的构建及鉴定

Construction and identification of adenoviral vectors of RNA interference of stem cell marker Musashi2 gene

目的:构建人干细胞标志分子Musashi2(Msi2)RNAi腺病毒载体,并检测其在膀胱癌细胞株BIU87中的干扰效果及对细胞增殖的影响。方法:将2对干扰片段msi2-1和msi2-2及阴性对照scramble片段克隆入pSES-HUS穿梭载体,经酶切鉴定及测序后,将测序正确的克隆经PmeⅠ酶切后与pAdeasy-1骨架质粒在BJ5183菌内重组,重组质粒经PacⅠ酶切后转染入293A细胞,进行腺病毒的包装和扩增。Real-time PCR和Western blotting法分别检测腺病毒感染BIU87细胞后Msi2的mRNA和蛋白表达水平,MTT实验检测干扰Msi2对BIU87细胞增殖的影响。结果:成功将干扰片段和scramble片段克隆入pSES-HUS穿梭载体,构建出pAdeasy-1-pSES-HUS-msi2和pAdeasy-1-pSES-HUS-scramble重组腺病毒载体并包装扩增出腺病毒。与scramble组比较,msi2-1和msi2-2组的Msi2mRNA和蛋白表达水平均明显下降(P<0.05)。干扰Msi2后,BIU87细胞增殖能力下降(P<0.05)。结论:成功构建Msi2RNAi腺病毒载体,其可有效抑制膀胱癌细胞株BIU87内Msi2的表达并抑制细胞增殖。

Objective To construct the adenoviral vectors of RNA interference of a stem cell marker Musashi2（Msi2） gene and to detect the interference effect and the influence on the proliferation of bladder cancer cell line BIU87.Methods Two interference DNA fragments named msi2-1 and msi2-2 and scramble as negative control were cloned into pSES-HUS shuttle vector and sent to sequence.After being digested and identified correctly the clones digested by PmeⅠwere recombinated with the bone plasmid pAdeasy-1 in the BJ5183 bacteria.After being digested by PacⅠ the recombinant plasmids were transfected into 293A cells to package and amplify adenovirus.Real-time PCR and Western blotting were used to test Msi2 mRNA and protein expression respectively in BIU87 cells infected by adenovirus.MTT was employed to detect whether knockdown of Msi2 affected the proliferation of BIU87 cells.Results Two interference and the scramble fragments were cloned into pSES-HUS shuttle vectors correctly.The recombinant vectors named pAdeasy-1-pSES-HUS-msi2 and pAdeasy-1-pSES-HUS-scramble were constructed and also the adenovirus were packaged and amplified successfully.Compared with scramble group,both of the mRNA and protein expression levels of Msi2 in msi2-1 and msi2-2 groups were decreased apparently（P0.05）;the growth of BIU87 cells was reduced after knockdown of Msi2（P0.05）.Conclusion The adenoviral vectors of Msi2 RNA interference which could inhibit the expression of Msi2 and cellular proliferation effectively in bladder cancer cell line BIU87 is successfully constructed.