A DNA-based Small Interfering RNA Targeting Stat3Inhibits The Growth Of Renal Carcinoma786-O Cells In Vitro And In Vivo

Background: Renal cell carcinoma (RCC) is one of the mosttreatment-resistant malignancies. Though the traditional methods includingsurgical treatment, radiation therapy and chemotherapy have made someachivements, the side effect will accure and the tumor recur usually. Despiteother new therapeutic advances development, almost all patients displayresistance to treatment. As to the metastasis RCC, cure is seldom seen. Signaltransducer and activator of transcription3(Stat3) is the hotspot gene in thecancer gene therapy research. Several articals showed that Stat3is aberrantlyactivated in several types of cancers, including RCC. Stat3plays a key role intumor cell survival and proliferation, angiogenesis. Therefore, the sdutyinvestigated that the effect of inhibition Stat3expression via RNAi technologyon human RCC cell line786-O cell proliferation, apoptosis, angiogenesis invitro and in vivo.Objective:We investigated the effect of a DNA-based small interferingRNA (siRNA) targeting Stat3(pSi-Stat3) on tumor growth, cell proliferation,angiogenesis and apoptosis of the human renal carcinoma cell line786-O bothin vitro and in vivo.Method:We constructed the pSi-Stat3plasmid, transfected it into786-0cells and observed the effect on the cells. We used real-time Reversetranscriptase PCR (RT-PCR) and Western blot to detect the transfectionefficiency. The effects of vector coding small interference RNA (SiRNA)targeting Stat3on the expression of Stat3and downstream gene were evaluatedby Western blot and real-time RT-PCR respectively. The effect of pSi-Stat3onproliferation and apoptosis of786-O cells were detected using MTT andacridine orange (AO) staining respectively in vitro. The effect of pSi-Stat3onStat3target genes Bcl2(apoptosis relative gene) and VEGF (angiogenesis relative gene) were detected by real-time RT-PCR and Western blot. The nudemouse model of RCC was developed by subcutaneously injection of786-0cells into nude mice. We also investigated effect of pSi-Stat3treatment ontumor growth of in mouse model by tumor growth analysis, and then detectedthe mechanism underlying. The effects of Stat3-siRNA treatment on theexpression of Stat3and downstream gene were evaluated by Western blot andreal-time RT-PCR respectively. The Ki-67expression (proliferation specificmarker) in tumor tissue was analyzed using immunohistochemistry (IHC). Thetumor cells apoptosis in tumor tissue was analyzed via TUNEL assay. The Stat3target genes Bcl2and VEGF expressions were detected by real-time RT-PCRand Western blot.Result:Real-time RT-PCR and Western blot showed that Stat3expressionwas heavily prevented after pSi-Stat3trasfection in786-O cells. MTT andacridine orange (AO) staining assay showed pSi-Stat3trasfection significantlyinhibited cell proliferation and induced cell apoptosis (P <0.01). Real-timeRT-PCR and Western blot showed that the expression of Stat3-downstreamgenes Bcl2and VEGF was also suppressed by pSi-Stat3trasfection. In vivo,intratumor injection of pSi-Stat3significantly inhibited tumor growth in786-O-bearing nude mice. Real-time RT-PCR and Western blot showed thatStat3expression was heavily prevented after treatment with pSi-Stat3. Therewas a significant decrease in the expression of Ki-67in xenograft tumors ofpSi-Stat3-treated mice compared with that in the control groups as showed inIHC. TUNEL assay showed the apoptosis cells in the pSi-Stat3group weresignificantly increased. The decreasion of Ki-67expression and the increase ofapoptosis cells inlustrated treatment with pSi-Stat3could inhibite RCC cellproliferation and induce cell apoptosis. We also found that treatment withpSi-Stat3significantly decreased the expression of the Stat3-downstream genesBcl2and VEGF in tumor xenografts, that may increase apoptosis and decrease angiogenesis, respectively.Conclusion: Using a DNA vector-based small interference RNA toinhibit the Stat3expression showed tumor cells proliferation inhibition andtumor cells apoptosis enhancement. It might be a useful therapeutic strategy inRCC and other malignancies.