An Experimental Study On Lentivirus-mediated Target-silenced Gankyrin Expression In The Treatment Of Hepatocellular Carcinoma

BackgroundHepatocullular carcinoma(HCC),one of the common carcinomas,contributes to the higher morbility and mortality among caner patients because of its highest malignancy.HCC endangers the lives and health of Chinese people because each year the newly developed hepatic cancer patients in the inland of China account for 45 percent of the global total.In current perspective,surgical therapy and liver transplantation are effective,but very few patients are indicative of surgical operations due to high malignancy,fast growth,susceptibility for relapse and metabasis and a great portion of them are tentatively treated with chemotherapy, radiotherapy as well as other alleviafive treatments.Therefore,innovations for hepatic cancer treatments await our medical researchers and doctors in this field.Recently,the developments in oncomolecularbiology and immunology have brought our sight into deep understanding of oncogene.What is more,transgenic technology has been on its track to perfection,which opens a window for gene therapy of hepatic cancer.So far,the recently detected Gankyrin has been found to an oncogene comprehensively activated in HCC.Other findings indicate Gankyrin is over-expressed in the cancer tissues of nearly all HCC patients and participates in the carcinogenesis and development of HCC by way of systematically mediating multiple cell life cycle modulins and transcription factors,and degrading tumor suppression proteins like p53 and p16.As a result,Gankyrin has its clinical prospect when it is used as a target for diagnosis and treatment of HCC.Therefore,we aim at studying the effect of silencing Gankyrin in vivo and in vitro via lentiviral mediation on the biological behaviors of hepatoma carcinoma cell lines and exploring the relevant mechanism so as to build a scientific and experimental basis for its final application in the clinical treatment of hepatic cancer.Aims1.To prepare rabbit-anti-Gankyrin polyclonal antibody used in the quantitative and qualitative detection of Gankyrin expression.2.To construct and prepare lentiviral vectors of target-silenced Gankyrin shRNA for the following in vivo and in vitro experiments on the effectiveness of HCC gene therapy based on Gankyrin expression.3.To explore the effect of lentivirus-mediated target-silenced Gankyrin expression on the bionomics of hepatoma carcinoma cells and construct a theoretical and experimental basis for its use in the clinical treatment of HCC.Methods1.Preparation,purification and identification of Rabbit-anti-Gankyrin polycional antibody.1.1 pET30a(+)-Gankyrin expression vector plasmid was constructed using gene cloning and identification of enzyme digestion.1.2 His6-Gankyrin fusion protein was harvested using IPTG to induce Gankyrin prokaryotic expression.1.3 Rabit-anti-Gankyrin polyclonal antibody was prepared using purified His6-Gankyrin fusion protein as antigen to conduct immune reaction and antibody specificity and reactance were detected.2.Construction and preparation of lentiviral vectors of target-silenced Gankyrin shRNA2.1 Three segments of 19nt oligonucleotide sequence were selected as shRNA segment sequence of targeted Gankyrin based on Gankyrin(NM₀02814) CDS exon,which was used to synthesize three pairs of DNA segments of long primers,to gain three plasmids by way of renaturation,double enzyme digestion as well as connection and inversion with plasmid pDC316-U6 from the same double enzyme digestion.2.2 The gained plasmids were transfected into SMMC-7721.The plasmid most effectively silencing the Gankyrin expression was screened by PT-PCR and Western blot and used to construct lentiviral packaging plasmid of plenti6/V5-shRNA (Gankyrin)-EGFP using gene cloning.2.3 Lenti-shRNA(Gankyrin)-EGFP expression vectors were prepared using quadri-plasmid contransfection and the related quality test was performed.3.The effect of reconstructed lentivirus-mediated target-silenced Gankyrin on the bionomics of hepatoma carcinoma cells3.1 After the Lenti-shRNA(Gankyrin)-EGFP expression vectors were used to infect SMMC-7721,their reactions on the growth inhibition and apoptosis of hepatoma carcinoma cell,Gankyrin mRNA and protein expressions were detected.3.2 SMMC-7721 transplantation tumor model was constructed.After tumor generation,Lenti-shRNA(Gankyrin)-EGFP expression vectors were injected into the tumor.Their reactions on the growth of the tumors transplanted into the cancer-bearing mice as well as on Gankyrin mRNA of transplanted tumor were detected and the morphologic changes of the transplanted tumors were observed.Results1.Preparation,purification and identification of Rabbit-anti-Gankyrin polyclonal antibody.1.1 Enzyme digestion identification showed the constructed pET30a(+)-Gankyrin expression vector plasmids could induce Gankyrin protein via IPTG after inversion into the host of Bacillus coli.1.2 ELISA and Western blot verified better reactivity and specificity of the rabbit-anti-Gankyrin polyclonal antibody prepared from the purified His6-Gankyrin infusion protein as antigen,which met the demands of following experiments.2.Construction and preparation of lentiviral vectors of target-silenced Gankyrin shRNA2.1 The three target-silenced Gankyrin shRNA plasmids were satisfactorily constructed,all carrying CMV promoters for EGFP transcription and U6 promoters for shRNA sequence transcription.By RT-PCR and Western blot,pDC316-EGFP-U6-shRNA (Gankyrin) 1 plasmid knock clown SMMC-7721cells Gankyrin,based on which the pLenti6/V5-shRNA(Gankyrin)-EGFP,the lentiviral packaging plasmid, was accurately constructed.2.2 Detection by PCR and EGFP activity observation demonstrated Lenti-shRNA (Gankyrin)-EGFP with high titer and high infectious activity,was satisfactorily constructed by using quadri-plasmid contransfection system. 3.The effect of reconstructed lentivirus-mediated target silenced Gankyrin on the bionomics of hepatoma carcinoma cells3.1 By MTT,the growth rates of the in vitro cultured SMMC-7721 growth cells were insignificantly different between the blank control group and the lentivirus control group(P＞0.05),while the differences were significant when MOI at various values was added into Lenti-shRNA(Gankyrin)-EGFP-infected SMMC-7721 cells (P＞0.05).Lenti-shRNA(Gankyrin)-EGFP inhibited the growth of SMMC-7721 cells(P＜0.05) and the inhibitive escalated with the increase of MOI value until MOI went up to a certain value.3.2 Detection under flow cytometry showed infected by Lenti-shRNA(Gankyrin)-EGFP for 48 hours,the in vitro cultured SMMC-7721 growth cells had higher apoptosis rate than those in the blank control group and in the lentivirus control group,the differences significant statistically(P＜0.05).3.3 Expressions of Lenti-shRNA(Gankrin)-EGFP on Gankyrin mRNA and Gankyrin protein were of no difference between the blank control and lentivirus control group statistically(P＞0.05).After SMMC-7721 growth cells were infected,the expressions on Gankyrin mRNA and Gankyrin proteins were both significantly repressed compared to the above-mentioned two control groups(P＜0.05).3.4 Transplanted with SMMC-7721 cells suspension solution into the athymic mice, the SMMC-7721 cells grew and subcutaneous transplantation tumors formed.3.5 The transplantation tumor-carrying athymic mice received Lenti-shRNA (Gankyrin)-EGFP injection into the subcutaneous transplantation tumors and sacrificed at the third week.The subcutaneous transplantation tumor growth was notably inhibited by Lenti-shRNA(Gankyrin)-EGFP,their sizes and weights significantly smaller and lower than those of the control groups(P＜0.05).3.6 Three weeks following Lenti-shRNA(Gankyrin)-EGFP injection into the subcutaneous transplantation tumors,Gankyrin mRNA levels were detected to be significantly lower than those at the control groups(P＜0.05),indicating that Lenti-shRNA(Gankyrin)-EGFP repressed the Gankyrin expressions on liver cell carcinoma cells.3.7 HE and immunohistochemical staining on the transplantation carcinomas from the tumor-carrying mice showed Lenti-shRNA(Gankyrin)-EGFP injected into the transplantation carcinomas repressed the Gankyrin protein expressions on liver cell carcinoma cells.Conclusions1.In this study,pET30a(+)-Gankyrin prokaryotic expression vectors were successfully constructed.The reconstructed His6-tagged-Gankyrin infusion protein was gained by inverting plasmids into Bacillus coli and inducing by IPTG.2.The rabbit anti-Gankyrin antiserum prepared with purified His6-tagged-Gankyrin infusion protein as antigen was detected by Western blot to be reactive and specific against the reconstructed His6-tagged-Gankyrin infusion protein and Gankyrin proteins from SMMC-7721.The antiserum can be used as an antibody for immune response to detect the expressions of Gankyrin protein.3.Lenti-shRNA(Gankyrin)-EGFP with high titer,which was prepared and reconstructed from plenti6/V5-shRNA(Gankyrin)-EGFP contransfected with quadri-plasmid,was detected to be of satisfaction and with high titer and high activity for infection,which provides a necessary experimental basis for further clinical research on targeted Gankyrin gene therapy of hepatic cancer.4.Lenti-shRNA(Gankyrin)-EGFE as a satisfactory gene transfer vector,is effective in transfection of hepatic cancer cells.Gene transfer therapy based on Lenti-shRNA(Gankyrin)-EGFP may be a possibly effective choice for gene therapy of hepatic cancer.5.Lenti-shRNA(Gankyrin)-EGFP may repress the proliferation of SMMC-7721 and knock down inhibit Gankyrin mRNA and Gankyrin expressions in vitro by way of RNA interference(RNAi).6.Lenti-shRNA(Gankyrin)-EGFP injected into the transplantation tumors can suppress the growth of SMMC-7721-induced subcutaneous transplantation tumors and inhibit the expression Gankyrin mRNA and protein,indicating the lentiviral vector of target-silenced Gankyrin may be a prospective therapy for hepatic cancer.The innovations in this study include:1.From the perspective of gene therapy,we have explored the effect of Lenti-shRNA(Gankyrin)-EGFP from lentivirus-mediated target-silenced Gankyrin on hepatic cellular cancer,as well as the curative significance,which provides necessary experimental data for the Gankyrin-targeted gene therapy of HCC and the products for gene therapy into clinical treatment of HCC.2.This study has suggested a general strategy for gene therapy for liver-involved diseases by lentivirus-mediated RNAi.3.Finally,our study has provided an experimental basis for constructing animal models of hepatic diseases using lentivirus-mediated transgenic viral vectors.