Construction Of ShRNA Expression Vector Against Different Oncogenes And The Antitumor Efficacy Of Combinations

Cancer is a genomic functional disease with features of oncogene activation and tumor suppressor inactivation.RNA interference(RNAi) technology is considered as a very promising cancer therapy.Inhibition the expression of oncogenes by RNAi reversed the malignant morphology of tumors.However,the malignant phenotype of tumor is partially reversed by RNAi.Two parts are needed to study in pursuit of better anti-cancer effect.The first one is RNAi combined with chemotherapy agents.And the second one is targeting numerous oncogenes.Part 1 Antitumor efficacy of combination of Ras shRNA and VCRLiver cancer is one of the commonest malignancies in the world.Although there are surgery,chemotherapy,radiation therapy and liver transplantation,liver cancer is still one of the most fatal cancers,with five-year relative survival rates less than 11%. It has been reported the growth of liver cancer can be effectively controlled by specific gene silencing by RNAi.In pursuit of better anticancer effect,combination of RNAi therapy and chemotherapy may be promising.In the present study,the effect and mechanism of Ras shRNA combined with vincristine(VCR) were investigated.1.Anti-liver cancer activity of combination of pCSH1-shNR and VCR in vitroGrowth inhibition of HepG2 cells was determined by SRB assay.The results showed that the cell survival rates were 77.24%for 1.5nM VCR,55.0%for pCSH1-shNR and 34.60%for pCSH1-shNR combined with 1.5nM VCR,and coefficient of drug interaction(CDI) was 0.76.The cell survival rates were 46.18% for 2.5nM VCR,17.32%for pCSH1-shNR combined with 2.5nM VCR,and CDI was 0.64.These results indicated that the combination of pCSH1-shNR and VCR had remarkable synergistic activity against HepG2 cells.The inhibition of colony formation was induced by combination of pCSH1-shNR and VCR.The rates of colony formation were 50%,13.75%and 6.67%after the treatments of pCSH1-shNR,2.5nM VCR and the combination,respectively. 2.Alteration of proliferation-related proteins by various treatmentsThe effects of various treatments on the protein kinases ERK1/2 and Akt.Both pCSH1-shNR and VCR were down-regulated the levels of phosphorylated ERK1/2 and Akt.And the lowest level was occurred in the treatment of pCSH1-shNR and VCR.The data indicated that the combination of pCSH1-shNR and VCR inhibited the activities of Ras/Raf/MEK/ERK1/2 and Ras/PI3K/Akt pathways.NF-κB is a kind of important nuclear transcription factor.Over expression and activation of NF-κB are connected with tumorigenesis.Both ERK1/2 and Akt participate in the regulation of NF-κB.Down-regulation of NF-κB induces chemosensitivity to VCR in human multiple myeloma.Our results showed that the combination of pCSH1-shNR and VCR down-regulated the activity of NF-κB,and its inhibitory ability was stronger than that ofpCSH1-shNR or VCR alone,respectively.Epidermal growth factor receptor(EGFR) participates in the regulation of Ras signaling pathway.VCR stimulates the phosphorylation of EGFR.Constitutively expression of phosphrylated EGFR is in HepG2 cells.Western blot analysis showed that the treatment of pCSH1-shNR and VCR down-regulated the expression and phosphorylaiton of EGFR,indicating that N-Ras suppression decreased VCRactivated EGFR.3.Effect of pCSH1-shNR and VCR on cell cycle distribution in HepG2 cellsFlow cytometry analysis showed that pCSH1-shNR had less effect of cell cycle distribution in HepG2 cells.Furthermore,1.5nM VCR induced S phase arrest and 2.5nM VCR induced G2/M phase arrest,indicating that VCR-induced cell cycle arrest was in dose-dependent manner.However,pCSH1-shNR abrogated VCR-induced S and G2/M arrest in combination-treated HepG2 cells.Alteration of cyclin and cyclin dependent kinase(CDK) was determined by Western Blot.Results showed that combination of pCSH1-shNR and VCR was markedly down-regulated the expression of cyclin D1,Cdk2,Cdk4 and Cdk6 compared with pCSH1-shNR or VCR alone.Moreover,pCSH1-shNR reversed VCR-induced up-regulations of cyclin E and cyclin B1,and VCR inhibited the expression of cyclin B1 regulated by pCSH1-shNR.Therefore,combination of pCSH1-shNR and VCR decreased the general expressions of Cyclin and Cdk levels.Western blot analysis showed that pCSH1-shNR decreased phosphorylation of Rb and expression of E2F-1.VCR remarkably increased phosphorylation of Rb and decreased expression of E2F-1.Phosphorylation of Rb was obviously higher in the combination-treated cells compared with control cells,but was significantly lower in VCR-treated cells.Expression of E2F-1 was mostly decreased in the combinationtreated cells.These results indicated that pCSH1-shNR inhibited VCR-induced phosphorylation of Rb.4.Combination of pCSH1-shNR and VCR induced apoptosis of HepG2 cellsFlow cytometry analysis showed that the combination of pCSH1-shNR and VCR induced apoptosis.Apoptosis rates of pCSH1-shNR,VCR and the combination were 6.07%,12.29%,and 22.82%,respectively,indicating that pCSH1-shNR and VCR synergistically induced apoptosis of HepG2 cells.Results of Western blot indicated that Bax was not necessary for combinationinduced apoptosis.Survivin is a member of the family of inhibitor of apoptosis proteins(IAPs).pCSH1-shNR remarkably down-regulated the expression of survivin and suppressed the increase of survivin expression in response to VCR treatment.This was a reason why the combination treatment induced apoptosis synergistically.5.Combination of pCSH1-shNR and VCR induced senescence of HepG2 cellsSA-β-gal staining results showed that rates of senescence cells induced by transfected pCSH1-shNR,VCR and the combination were 11.32%,8.23%,and 18.76%respectively,indicating that the combination treatment had stronger senescence-induced ability.6.Effect of pCSH1-shNR on VCR-induced MDR1 expressionThe resistance of tumor cell to VCR is connected with over expression of P-glycoprotein(P-gp) produced by MDR1 gene,and MDR1 gene expression can be regulated by oncogene ras at the same time.Our results showed that the expression of P-gp in HepG2 cells was up-regulated after VCR treatment.However,the up-regulation of P-gp was impaired in the combination-treated cells,indicating that pCSH1-shNR prevented VCR-induced up-regulation of P-gp. In conclusion,the combination of pCSH1-shNR and VCR synergistically inhibited growth of HepG2 cells and enhanced apoptosis through down-regulating the expression and/or activation of EGFR,E2F-1,NF-κB,phosphorylated ERK1/2,Akt, cyclin,Cdk,survivin and P-gp.Above data suggested that RNAi technology combined with appropriate chemotherapeutic agents seems a promising approach for the treatment of human hepatocellular carcinoma.Part 2 Construction and the Antitumor Efficacy of shRNA Expression Vector against MDM2 and Pin1Over expression of MDM2 suppresses function of tumor suppressor p53 and promotes tumorigenesis.Pin1 is considered as a critical catalyst that amplifies and translates multiple oncogenic signaling mechanisms during oncogenesis.It has been reported that suppression of MDM2 or Pin1 could inhibit proliferation and malignant phenotype of tumor.In the present study,a constructed vector with the ability to express several shRNAs was used.We constructed a shRNA expression vector against MDM2 and Pinl,and investigated its anti-cancer activity.1.Construction of MDM2 shRNA and Pin1 shRNA co-expressing vectorThe sequence of MDM2 shRNA with effective inhibition of MDM2 expression was designed in our laboratory.The reported sequence of Pin1 shRNA could inhibit expression of Pin1 effectively.The two sequences were constructed in pCSH1-1 to form a new vector named as pCSH1-shPM.2.pCSH1-shPM inhibited the expression of MDM2 and Pin1Human fibrosarcoma HT1080 cells were used in this study,which was over expressions of MDM2 and Pin1.Western blot analysis showed that pCSH1-shPin1 inhibited not only the expression of Pin1 but also the expression of MDM2. pCSH1-shPM inhibited the expression of both MDM2 and Pin1.Meanwhile,the inhibition of MDM2 expression in pCSH1-shPM-transfected cells was stronger than that in the pCSH1-shMDM2-transfected cells,suggesting the inhibition of Pin1 might down-regulate the expression of MDM2.Furthermore,the expression of MDM2 was significantly reduced in Pin1 knockdown HT1080/shPin1 cells,supporting that inhibition of Pin1 down-regulated the expression of MDM2 in HT1080 cells.3.The effect of pCSH1-shPM on cell growthThe effects of pCSH1-shMDM2,pCSH1-shPin1 and pCSH1-shPM on cell growth were determined by SRB assay.The results showed that pCSH1-shMDM2, pCSHl-shPinl and pCSHI-shPM suppressed the proliferation of HT1080 cells respectively,and pCSH1-shPM was more effective.These data indicated that inhibition of MDM2 and Pin1 at the same time was more effective to suppress the proliferation of HT 1080 cells.4.The effect of pCSH1-shPM on cell mobilityWound healing assay showed that the mobility of HT 1080 cells was reduced by transfection of pCSH1-shMDM2,pCSH1-shPin1 and pCSH1-shPM.The efficacy of mobility inhibition was no difference among pCSH1-shMDM2,pCSH1-shPin1 and pCSH1-shPM,indicating that both MDM2 and Pin1 inhibitions did not augment the inhibition of cell mobility.Taken together,our data suggested that the dual-interfering vector pCSH1-shPM exhibited a stronger inhibition of HT1080 cell growth,and its mechanism might be investigated in the further experiments.