The Experimental Study Of Growth Inhibition Induced By Dual Shrnas To Silence Bcl-2and Survivin Gene In Human Cervical Carcinoma Hela Cells

AIM: RNAi-mediated gene silencing is widely used in laboratories for gene functionstudies and also holds a great potential for the treatment of cancer gene therapy. But almostall cancer cells are likely not dependent on a single oncogenic signaling pathway.Moreover, many malignant tumors show striking genetic heterogeneities and phenotypicvariations, suggesting that a single target therapy is not likely to eradicate cancer cells.Coexpression of multiple shRNAs can simultaneously inhibit multiple genes or multipletargeting sites of a single gene. Here we established a simple and efficient dual shRNAsexpression system targeting at Bcl-2and Survivin genes, both of which are closely relatedto cell apoptosis. We transfected the recombinant plasmid vectors into human cervicalcarcinoma Hela cells to observe the influence of Bcl-2and Survivin gene/proteinexpression, cell apoptosis, cell proliferation and sensibility to chemotherapeutic drugcisplatin of Hela cells in vivo and in vitro, which provided an experimental foundation forthe further clinical cervical carcinoma gene therapy.METHODS:①According to the cDNA sequences of Bcl-2and Survivin, the singleshRNA recombinant plasmid pGenesil1.2-Bcl-2, pGenesil1.1-Survivin andpGenesil1.1-HK were constructed;②The dual shRNA recombinant plasmidpGenesil1.1+2-(Bcl-2+Survivin) was constructed according to the constructions ofpGenesil1.2-Bcl-2and pGenesil1.1-Survivin;③The following experiments in vivo and invitro were divided into five groups: pGenesil1.2-Bcl-2group (Bcl for abbreviation),pGenesil1.1-Survivin group (Sur for abbreviation), pGenesil1.1+2-(Bcl-2+Survivin) group(Bcl+Sur for abbreviation), pGenesil1.1-HK group (Neg for abbreviation) and blankcontrol group (Blank for abbreviation);④The relative amounts of Bcl-2and SurvivinmRNA were detected by RT-PCR at24h、48h and72h after transfection respectively. Cellcycle and apoptosis were detected by FCM and Hoechst33258at48h to observe the inhibition effect of Bcl-2and Survivin gene in Hela cells transiently transfected withinterfering recombinant plasmid;⑤Stable Hela cells transfected with respectiverecombinant plasmid were obtained by G418selection. Expression of the mRNA andprotein was estimated by RT-PCR, FCM and Western-blotting. Cell proliferation abilityand sensibility to cisplatin were evaluated by MTT experiment;⑥Models of nude micebearing cancer were established by using BALB/c nude mice inoculated with respectivestable transfection Hela cells. To study the growth inhibition of transplanted humancervical carcinoma induced by dual shRNA, tumorigenic time, tumor size and tumorweight were observed, Bcl-2and Survivin protein expression was detected by usingimmunohistochemistry.RESULTS：①The results of sequencing and restrictive enzyme digestion confirmedthat the single shRNA recombinant plasmid expression vectors were successfullyconstructed.②The results of restrictive enzyme digestion confirmed that the dual shRNArecombinant plasmid expression vector was successfully constructed.③The RT-PCRshowed that both Bcl-2and Survivin mRNA expression were inhibited at24h、48h and72hpost-transfection, and the inhibition rate at48h was most significant. The inhibition rate ofBcl-2and Survivin mRNA in the Bcl+Sur group was85.71％and60.56％, both of whichwere more effective than those of single shRNA groups (P<0.05).④The apoptotic index at48h in the Bcl+Sur group was (17.95±0.45)%, and it was significant higher than those ofthe single shRNA groups (P<0.05), moreover the FCM showed the same results.⑤StableHela cells transfected with various recombinant plasmids were selected with G418. FCMresults showed that the percentage of stable transfection cells was above80%in eachgroup. In the Bcl+Sur group, the inhibition rate of Bcl-2and Survivin mRNA was76.11%and81.33%respectively, the protein inhibition rate was48.01%and51.64%respectively.Both of which were more effective than those of single shRNA groups (P<0.05).⑥TheMTT results showed that the growth of Hela cells treated with dual shRNA was apparentlyinhibited, and these cells were more sensitive to cisplatin.⑦Compared with the singleshRNA treated groups, the average tumorigenic time was significantly delayed, the averagetumor sizes and weights both decreased significantly in the Bcl+Sur group (P<0.05); Theimmunohistochemical results further confirmed the Bcl-2and Survivin protein expressionwere both significantly reduced in the dual shRNA treated group. CONCLUSION: Dual shRNA strategy was more effective than single shRNA ingene silencing and inhibition of cell or even tumor growth.The Hela cells treated with dualshRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treatedcells.