干细胞标志Musashi2基因RNAi腺病毒载体的构建及鉴定

Abstract: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（P<0.05）；the growth of BIU87 cells was reduced after knockdown of Msi2（P<0.05）. 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.

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

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(P<0.05);the growth of BIU87 cells was reduced after knockdown of Msi2(P<0.05).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.