Hepatocellular Carcinoma Therapy Mediated By ShRNA Targeting Mcl-1 Gene And Apoptin

Hepatocellular carcinoma (HCC) is the 5th most common cancer and the 3^rd mostcommon cause of cancer-related mortality world wide. The tumour might be curable byresection or liver transplantation.Most patients with HCC show dvanced-stage tumor at thetime of diagnosis, and surgical treatment can only be achieved in a minority of patients.Inaddation, systemic chemotherapy has never been shown to improve survival in patientswith HCC. Therefore, new treatment regimens for patients with HCC are needed badly.Myeloid cell leukemia-1 (mcl-1) is a member of bcl-2 family. The main function ofmcl-1 is to promot cell survival and protect cells from apoptosis. Mcl-1 is overexpressed intissues of HCC and contributes to the developmen of HCC.Apoptin, a protein encoded by thechicken anemia virus (CAV) VP3 gene, has the ability to selectively induce apoptosis intransformed and malignant cells but not in normal cells.RNA interference(RNAi) is a potent molecular biological tool in degrading expressionof aimed genes and has been applied extensively in studies of anti- tumor and gene function.In our researches,we constructed three recombination plasmids encoding shorthairpinRNA(shRNA) targeting mcl-1 gene and selected one that supresses the expressing ofmcl-1 gene most efficiently.The apoptosis of HepG2 cells induced by the plasmid wasinvestigated.Then the effcet of combination of the plasmid with apoptin on the proliferationand apoptosis of HepG2 cells was studied. Therefore, an effective therapeutic strategy forhepatocellular carcinoma will be initiated. PartⅠConstruction and identification of eukaryotic expression plasmidsencoding the short hairpin RNA targeting Mcl-1 geneObjective To design and construct short hairpin RNA(shRNA) eukaryotic expressionplasmids containing EGFP gene and targeting mcl-1 gene. Methods According to theprinciple of shRNA design and the restriction enzyme cutting site of pGenesil-1.1 vector,3pairs of shRNA oligonucleotide fragments were designed and synthesized based on thesequence of mcl-1mRNA in GenBank. Double strands were formed after annealing andinserted into pGenesil-1.1 vetor. The recombinant were named as mcl-1.1～mcl-1.3. Therecombinant plasmid were transformed into DH5α,The plasmids were extracted and identificated by restrictive enzyme digestion andsequencing analysis.Results The restriction enzyme digestion demonstrated that shRNAwas inserted into vector correctly, and sequencing analysis demonstrated that theirsequences were the same as the design. Conclusion The shRNA eukaryotic expressionplasmids targeting mcl-1 gene are constructed successfully. This lays a solid foundation forthe further research on the therapy of hepatocellular carcinoma with shRNA expressionplasmids targeting mcl-1 gene.PartⅡThe screening of shRNA targeting mcl-1 gene and it's effect onapoptosis of hepatoeellular carcinimaObjective To select the plasmid that inhibits the expressing of mcl-1 gene mostefficiently,and to investigate the effect of the plasmid on the apoptosis of HepG2 cells.Methods Plasmids mcl-1.1～mcl-1.3 and negative control plasmid HK weretransfected into HepG2 cells via Lipofectamine 2000. Fourty-eight hours after transfection,the transfection rate of the plasmids was determined by using fluorescence microscopy.Theexpression levels of mcl-1 mRNA and protein were assayed by reverse transcriptase-polymerasechain reaction and Western blotting.The plasmid that silence mcl-1 gene mostefficiently was selected according to the levels of mcl-1 mRNA and protein. Fourty-eighthours after the selected plasmid transfected into HepG2, karyomorphological diversify ofapoptotic cells was detected by Hoechst staining. The apoptosis condition of the cells wasanalyzed by flow cytometry with PI single staining. Results The transfection rate of theplasmids mcl-1.1～mcl-1.3 and HK in HepG2 cells was 64.00%±3.77%, 64.20%±2.10%,63.70%±3.34% and62.5%±2.42% respectively (P=0.59).The mcl-1 mRNA and proteinlevels of mcl-1.1～mcl-1.3 group (mRNA: 0.61±0.02, 0.56±0.02 and 0.46±0.01, protein:0.54±0.01,0.48±0.03,0.36±0.01, respectively) were significantly lower than that of theblank control (mRNA: 0.97±0.01;protein: 0.90±0.03, all P=0.00) and negative control(mRNA: 0.95±0.00, protein: 0.88±0.01, all P=0.00). Compared with mcl-1.1 and mcl-1.2,mcl-1.3 had the strongest inhibitory effect on mcl-1 mRNA (52.27±0.00% vs 36.26±0.12%,41.47±0.13%,both P=0.00) and protein (58.98±0.01% vs 38.80±0.02%, 45.70±0.02 %, bothP=0.00). Karyomorphological diversify of apoptotic cells were obviously observed inmcl-1.3 group by hoechst33258 staining. The apoptosis rate of mcl-1.3 group was higherthan that of negative control group (7.74%±0.97% vs 2.81%±0.46%,P=0.00).ConclusionThe shRNA eukaryotic expression plasmid targeting at mcl-1 gene is selected successfully.The mcl-1 gene expression is suppressed significantly by the plasmid. The apoptosis ofHepG2 is promoted by the plasmid through silencing the expressing of mcl-1 gene.Silencing mcl-1 gene by shRNA maybe a potential approach for HCC gene therapy. PartⅢThe effect of combination of the shRNA targeting mcl-1 gene withapoptin on proliferation and apoptosis of hepatocellular carcinomaObjective To investigate the effect of combination of the shRNA targeting mcl-1 genewith apoptin on proliferation and apoptosis of hepatocellular carcinoma. Methods Plasmidpmcl-1 targeting mcl-1 gene, without EGFP gene, was constructed based on the plasmidsmcl-1.3 and pGensil-1.2. The plasmids pmcl-1 and pcDNA3.0-vp3 were transfected intoHepG2 cells.The mRNA and protein levels of mcl-1 and vp3 were analyzed by RT-PCRand western blot respectively. Cytochrome C was also assayed by western blot. Theproliferation of cells was measured by methyl thiazolyl tetrazolium(MTT), the apoptosiscondition of the cells was analyzed by flow cytometry with Annexin V V-FITC/PI doublestaining. Results The restriction enzyme digestion and DNA sequencing analysisdemonstrated that the recombinant plasmid pmcl-1 was constructed successfully. Analyzedby RT-PCR and western blot, The mRNA and protein of vp3 were positive in the group ofpmcl-1+pcDNA3.0-vp3 and pcDNA3.0-vp3,while negative in the group of pmcl-1,negative control and blank control. The vp3 mRNA and protein levels ofpmcl-1+pcDNA3.0-vp3 group was no difference with that of pcDNA3.0-vp3 group(mRNA:0.83±0.02 vs 0.78±0.37,P=0.20; protein:1.48±0.06 vs 1.40±0.08, P=0.81).The plasmidspmcl-1,pcDNA3.0-vp3 and pmcl-1+ pcDNA3.0-vp3 all had the potential to down-regulatemRNA and protein levels of mcl-1.They also promoted cytochrome C release frommitochondrion. The mcl-1 mRNA and protein level of pmcl-1+ pcDNA3.0-vp3 group(mRNA: 0.34±0.05,protein: 0.38±0.02) were significantly lower than that of pmcl-1 group(mRNA:0.46±0.03,protein:0.44±0.03) and pcDNA3.0-vp3 group (mRNA:0.43±0.01,protein: 0.43±0.01)(all P＜0.05).The cytochrome C level of pmcl-1+ pcDNA3.0-vp3group(1.21±0.09) was higher than that of pmcl-1 group (0.96±0.03,P=0.01) and pcDNA3.0-vp3 group(0.98±0.02, P=0.01). Compaired with negative and blank control, theproliferation of HepG2 at 1～5 d after transfection was significantly supressed by pmcl-1,vp3 and pmcl-1+vp3 (P＜0.05). Viable cells at 1～5d after treated with pcDNA3.0-vp3 wereless than that treated with pmcl-1 and more than that treated with pmcl-1+pcDNA3.0-vp3.Compaired with negative and blank control, the apoptosis of HepG2 cells was promoted byplasmid pmcl-1, pcDNA3.0-vp3 and pmcl-1+ pcDNA3.0-vp3(P＜0.05). The apoptosis at24h,48h and 72h after transfected with pcDNA3.0-vp3 (12.00±1.57%, 22.1±2.82% and41.80±3.37% respectively) was higher than that transfected with pmc1-1(8.60±0.78%,P=0.03; 11.07±0.95%, P=0.03;15.00±1.44%, P=0.00 respectively).While the apoptosis at24h,48h and 72h of pmcl-1+ pcDNA3.0-vp3 group (17.10±1.04%, 29.87±4.59% and52.40±1.73% respectively) was higher than that of pmcl-1 group andpcDNA3.0-vp3(P＜0.05). Conclusion The proliferation of HepG2 is suppressed and theapoptosis of HepG2 is enhanced by the shRNA targeting mcl-1 gene combined withapoptin. The shRNA targeting mcl-1 gene combined with apoptin is a potential approachfor HCC therapy.The apoptosis induced by apoptin is correlate with the inhibition of mcl-1gene expressing and the releasing of cytochrome C from mitochondrion.