Study On Adenovirus With HTERT Promoter Interfering Expression Of ERCC1 To Reverse Cisplatin Resistance In Ovarian Cancer Cell Lines

Ovarian cancer is the most common malignant cancer seriously threatening women's health.Althouth operation plays a key role in the treatment of ovarian cancer,chemotherapy based on cisplatin and radiotherapy are very important as well.Clinical survey has confirmed that 80%ovarian cancer patients were sensitive to chemotherapy based on cisplatin,howerer 80%of them apperaed resistance to cisplatin finally which blocked efficiency of the antitumor agent,and 5-year survival limited to 25%～30%.Increasing expression of excision repair cross complementation group 1(ERCC1)is a most important factor to cause cisplatin resistance.RNA interfering(RNAi)has been an effective technique in silencing target gene expression specialy in recent years which has played an important role in gene function research and antitumor therapy.ERCC1 plays an important role in protecting cell from antitumor agents and radioactive injury.If adenovirus-mediated gene transfection can not be prohibited expressing in somatic cells,serious side effect will arise.So the very critical solution is exploring a key to limit ERCC1 short hairpin interfering RNA to express only in cancer cells.Tolomerase is a special ribonucleoprotein complex,activation of which has close relationship with tumorigenisis,development and aging and is the critical limit step in cell cancerization.It has been found that telomerase are negative in most somatic cells,positive in germ cell and stem cell,but its activity is low.It is active in over 90% immorality cells and malignant cancer cells including ovarian cancer Cells.Human tolomerase reverse transcriptase(hTERT)is the limit speed enzyme of telomerase which expresses only in cancer cells,and plays a critical role in tolomerase regulation.Tolomerase has close relationship with hTERT and hTERT promoter.It has been confirmed that hTERT promoter is activated in tolomerase positive ovarian cancer cell lines and its activity has positive correlation with that of telomerase.It suggests that hTERT promoter could be regarded as target promoter in gene therapy of telomerase positive ovarian cancer.The aim of this study is to construct recombinant adenovirus with hTERT promoter and ERCC1 shRNA,then observe adenovirus mediated RNA interfering ERCC1 expression to reverse cisplatin resistance in vitro which provide new way to reverse cisplatin resistance in gene therapy of ovarian cancer.Part one Construction of plasmid pGL3-hTERT promoter-Luc and evaluation of activity of hTERT promoter in cellsObjective:to construct recombinant plasmid with hTERT promoter,and evaluate the expression specialty of hTERT promoter.Methods:1.Cleave pGL3 Basic Vector and CTS883P-4 with hTERT promoter with restriction endonuclease KpnⅠ/HindⅢ,then subclone hTERT promoter into pGL3 linear vector;evaluate recombinant plasmid with enzyme cleavage and sequencing.2.Transfect ovarian cell line SKOV3, SKOV3/DDP and umbilical vein endothelial cell ECV 304 with recombinant plasmid pGL3-hTERT promoter-Luc,negative control plasmid pGL3 Basic and positive control plasmid pGL3 Control with lipofectamine respectively,then observe fluorescence under fluorescent microscope 24 hour post-transfection.Results:1.Recombinant plasmid pGL3-hTERT promoter-Luc was constructed successfully,enzyme cleavage and sequencing confirm its correction.2.Fluorescence can be observed in SKOV3,SKOV3/DDP transfected with recombinant plasmid,but not in umbilical vein endothelial cell ECV 304.Conclusions:Activation of hTERT promoter exists in cancer cells only,but not in somatic cells.Part two Construction of plasmid vector with hTERT promoter and ERCC1 shRNA and evaluation of effect of RNA interferingObjective:to construct plasmid vector with hTERT promoter and ERCC1 shRNA and evaluate the effect of ERCC1 mRNA and protein silencing in ovarian cancer cells.Methods:1.Construct PMD 18 T-hTERT promoter plasmid.2.Construct pDNR-1r-ERCC1 shRNA plasmid vector.3.Construct pDNR-1r-hTERT promoter-ERCC1 shRNA plasmid vector.4.Ovarian cell lines SKOV3,SKOV3/DDP and somatic cell ECV304 are transfected with recombinant plasmid pGL3-hTERT promoter-ERCC1 shRNA by lipofectamine respectively,then observe effect of ERCC1 mRNA silencing.5.Detect effect of RNA interfering on protein level.Results:1.Construct PMD18 T-hTERT promoter plasmid vector successfully, enzyme cleavage and sequencing confirm the exogenous insert gene is correct.2.Construct pDNR-1r-ERCC1 shRNA plasmid vector successfully,enzyme cleavage and sequencing confirmed it was correct.3.Construct pDNR-1r-hTERT promoter-ERCC1 ShRNA plasmid vector successfully,enzyme cleavage and sequencing confirmed it was correct.4.RT-PCR indicated that post-transfected with recombinant plasmid 24,48,72 hours,ERCC1 mRNA of SKOV3 decreased(35.02±7.27)%,(49.28±2.46)%,(55.54±2.31)%,which had significant stastistic difference (all P＜0.01);ERCC1 mRNA of SKOV3/DDP decreased(37.83±13.01)%,(59.14±13.68)%,(61.46±12.28)%respectively compared with those transfected with negative plasmid,which had significant stastistic difference(P＜0.05,P＜0.01,P＜0.01); ERCC1 mRNA of normal somatic cell did not decrease compared with negative plasmid(all P＞0.05).5.Western Blot indicated that post-transfected with recombinant plasmid 24,48,72 hours ERCC1 protein of SKOV3 decreased(29.76±5.55)%,(52.56±9.83)%,(56.07±10.09)%respectively compared with those transfected with negative plasmid(all P＜0.01);ERCC1 protein of SKOV3/DDP decreased(22.01±8.89)%,(37.30±7.63)%,(42.31±6.90)%respectively(all P＜0.01);ERCC1 protein of normal somatic cell did not decrease compared with negative plasmid which are consistent with those of RT-PCR(all P＞0.05).Conclusions:hTERT promoter expresses in cancer cells only while not in somatic cells which may avoid causing serious side effect to somatic cells while using RNA interfering to siiience ovarian cancer cells ERCC1,and indicate the feasibility of treating hTERT promoter as regulation key.Part three Construction of recombinant adenovirus expression vector with hTERT promoter and ERCC1 shRNAObjective:to construct recombinant adenovirus expression vector with hTERT promoter and ERCC1 shRNA.Methods:1.pDNR-1r-hTERT promoter-ERCC1 shRNA and adenovirus vector underwent homologous recombinance by use of recombinase.Transform recombinant adenovirus vector into Ecoli.DH10B competent cell,then electrotransform,harvest bacterial,PCR method select positive clone,harvest positive clone,extract plasmid and enzyme cleavage.2.After linearization of recombinant adenovirus vector,it is packaged,augmented in HEK293 cell,finally yield high titers of recombinant adenovirus.3.Using the same method to yield blank adenovirus.Results:1.Construct recombinant adenovirus expression vector successfully and confirmed correctby using enzyme cleavage.2.The titer of recombinant adenovirus is 8×10~9 pfu/ml.3.The titer of blank recombinant adenovirus is 3×10~9 pfu/ml.Conclusions:Recombinant adenovirus expression vector Ad-hTERT promoter-ERCC1 shRNA was constructed successfully which was stable and easy to be augmented and purified,and titer of which was high.Part fourIn vitro study of recombinant adenovirus mediated RNA interfering silencing expression of ERCC1 to reverse cisplatin resistance in ovarian cancer cellsObjective:On basis of successful constrction of recombinant adenovirus expression vector,in vitro study to proceed to explore adenovirus mediated RNA interfering silencing expression of ERCC1 to reverse cisplatin resistance in ovarian cancer cells.Methods:1.Infect SKOV3,SKOV3/DDP and normal somatic cell ECV304 with 1,10,50,80MOI recombinant adenovirus and blank adenovirus,and regard cells pre-infection as control,ERCC1 mRNA and protein in ovarian cancer cells post-infection are detected by RT-PCR and Western Blot.2.Recombinant adenovirus infect SKOV3/DDP combine with cisplatin in vitro,then MTT test detected the sensitivity of SKOV3/DDP to cisplatin.3.Flow cytometry detected the effect of cisplatin combining with different titer recombinant adenovirus on viabilily of SKOV3/DDP and sensitivity of which to cisplatin.Results:1.ERCC1 mRNA of SKOV3 infected with 1,10,50,80MOI recombinant adenovirus decreased(31.91±5.32)%,(42.01±5.34)%,(57.37±11.80)%,(60.49±11.80)%respectively,which had significant statistic difference(P＜0.05,P＜0.01,P＜0.01,P＜0.01);ERCC1 protein decreased(17.76±6.71)%,(26.28±3.86)%,(44.67±8.38)%,(48.11±8.50)%,which had significant statistic difference(P＜0.05,P＜0.01,P＜0.01,P＜0.01);ERCC1 mRNA and protein of SKOV3 did not decrease infected with individual titer blank adenovirus(all P＞0.05).2.ERCC1 mRNA of SKOV3/DDP infected with 1,10,50,80MOI recombinant adenovirus decreased(31.07±3.55)%,(50.67±2.71)%,(63.86±3.63)%,(68.92±1.50)%respectively,which had significant statistic difference(P＜0.05,P＜0.01,P＜0.01,P＜0.01);ERCC1 protein decreased(17.43±8.84)%,(27.12±12.40)%,(34.21±15.34)%,(38.11±17.08)%respectively,which had significant statistic difference(all P＜0.01);ERCC1 mRNA and protein of SKOV3 did not decrease infected with individual titer blank adenovirus(all P＞0.05).3.ERCC1 mRNA and protein of normal somatic cell did not decreased infected with recombinant adenovirus(all P＞0.05).4.MTT test confirmed that SKOV3/DDP infected with 1,10,50,80 MOI recombinant adenovirus,sensitivity of which to cisplatin enhanced 11.1%(t=9.76,P＜0.05),16.7%(t=15.07,P＜0.05),26.4%(t=22.18,P＜0.05)and 33.5%(t=24.92,P＜0.05)respectively.