The Growth Inhibition Effect Of Dual ShRNAs Targeting IGF-1R And HPV18-E6Genes On Hela Cell Line In Vitro And In Vivo

AIM: Cervical cancer is one of the most common types of gynecological malignanttumors, its incidence has more than colorectal cancer, next to breast cancer, and keepincreasing recently. Surgery, radiotherapy, chemotherapy and other traditional methods arenot ideal. With the developmrnt of RNA interference (RNAi) technology, tumorgene-therapy shows bright future. The relationship between insulin-like growth factorsystem (IGFs) and tumor become a research hotspot. IGF-1R could promote tumor cellgrowth and proliferation, inhibit apoptosis of tumor cells by stimulating the malignanttransformation of a normal cell phenotype. Our previous studies showed thatIGF-1R-shRNA could inhibit expression of IGF-1R and induce apoptosis of Hela cells.High-risk HPV16/18-E6gene is one of the major oncogenes causing cervical cancer.Studies have shown that HPVE6-siRNA was able to reduce the expression of E6gene andinhibit cell proliferation on HPV-positive cervical cancer cells. But the formation of humantumor is a process of multi-genes and multi-steps, almost all cancer cells are likely notdependent on a single oncogenic signaling pathway. Coexpression of multiple shRNAs ormultiple targeting sites of a single gene has been concerned by many researchers.Therefore we constructed a efficient dual shRNAs expression vector targeting IGF-1R andHPV18-E6genes, observed the influence of co-silencing of multiple genes, comparing theinterference of a single gene. While, we detected the sensitivity changes of Hela cellscombining with chemotherapy drug cisplatin stimulation by shRNA, to explore thepossibility to improve cancer treatment methods.METHODS:1. Construction of Plasmid vector expression shRNA①According tothe cDNA sequences of IGF-1R、HPV18-E6and negative control, the single shRNArecombinant plasmid for pGenesil1.1-IGF-1R、 pGenesil1.2-HPV18-E6andpGenesil1.1-HK was constructed respectively;②The dual shRNA recombinant plasmidpGenesil1.1+2-(IGF-1R+HPV18-E6) was constructed according to the constructions of pGenesil1.1-IGF-1R and pGenesil1.2-HPV18-E6by molecular subcloning techniques;2.Experimental studies in vitro①The following experiments were divided into five groups:pGenesil1.1-IGF-1R group (IGF-1R for abbreviation), pGenesil1.2-HPV18-E6group (E6for abbreviation), pGenesil1.1+2-(IGF-1R+HPV18-E6) group (IGF-1R+E6forabbreviation), pGenesil1.1-HK group (NC for abbreviation) and blank control group(Blank for abbreviation);②Expression of IGF-1R and HPV18-E6mRNA was detected byRT-PCR at24h,48h and72h after transfection respectively.③Cell apoptosis wasanalyzed by Hoechst33258at48h after transfection;④Hela cells stably transfected withrespective recombinant plasmid were obtained by G418selection. Expression of theIGF-1R and HPV18-E6mRNA and protein were estimated by RT-PCR andWestern-blotting.⑤Cell proliferation ability and sensibility to cisplatin were evaluated byMTT experiment.3. Experimental studies in vivo①To establish xenograft model ofcervical cancer, BALB/c nude mice were vaccinated with respective Hela cells stablytransfected with recombinant plasmid vectors.②To study the growth inhibition oftransplanted human cervical carcinoma induced by dual shRNA, tumorigenic time, tumorsize and tumor weight were observed.③IGF-1R, HPV18-E6and CD34protein expressionwere detected by immunohistochemistry.④The content of SCC-Ag, TSGF and TPS tumormarkers in BALB/c nude mice serum was measured by ELISA.RESULTS：1. The recombinant shRNA expression vectors were successfullyconstructed.①shRNA plasmid expression vectors for pGenesil1.1-IGF-1R,pGenesil1.2-HPV18-E6and pGenesil1.1-HK which were verified by restriction enzymesand DNA sequencing were successfully constructed.②The results of restrictive enzymesdigestion confirmed that the dual shRNA recombinant plasmid expression vector wassuccessfully constructed.2. Studies in vitro①The results of RT-PCR illustrated thatexpression of both IGF-1R and HPV18-E6mRNA was inhibited at24h,48h and72hpost-transfection in all interference groups. The inhibition rate of IGF-1R and HPV18-E6mRNAin the IGF-1R+E6group was63.01％and78.46％at48h, both of which were moreeffective than those of single shRNA groups (P<0.05).②The apoptosis rate at48h in theIGF-1R+E6group was (32.81±1.07)%, higher than (21.65±0.34)%and (19.73±1.62)%ofthe IGF-1R and E6groups (P<0.05).③Hela cells stablely transfected with various recombinant plasmids were selected by G418. FCM results showed that transfectionefficiency was above80%in each group. In the IGF-1R+E6group, the inhibition rate ofIGF-1R and HPV18-E6mRNA was71.30%and78.52%respectively, the proteininhibition rate was67.88%and81.14%respectively.④The MTT results demonstrated thatthe growth of Hela cells in IGF-1R+E6group was apparently inhibited, and these cellswere more sensitive to cisplatin（P<0.05）compared with either IGF-1R group or E6group.3.Studies in vivo①Compared with the single shRNA treated groups, the average tumorsizes and weights both decreased significantly in the IGF-1R+E6group (P<0.05).②Theimmunohistochemical results further confirmed the IGF-1R and HPV18-E6proteinexpression were both significantly reduced in the dual shRNA treated group and CD34protein expression had a similar effect.③ELISA detection showed that the amount of TPSwas significantly lower than other groups.CONCLUSION: pGenesil1.1+2-(IGF-1R+HPV18-E6)-shRNA plasmid expressionvector transfected into human cervical cancer Hela cells line positive for humanpapillomavirus type18was more effective than pGenesil1.1-IGF-1R andpGenesil1.2-HPV18-E6single shRNA groups on gene silencing, growth inhibition,induction of apoptosis and was also more helpful to improve the sensitivity ofchemotherapeutic drugs.