The Influence Of WWOX Gene On Biological Behavior And Its Mechanism In Human Hepatic Carcinoma Cell

PART ONEExpression of WWOX gene in human hepatocellularcarcinoma and its significanceObjectives:To investigate the expressional and clinical meaning of WWOX inhepatocellular carcinoma(HCC).Methods:Expression of WWOX protein in HCC and the paired para-carcinoma livertissues from 50 HCC patients was detected by immunohistochemistry(SP method),andWWOX mRNA expression was analyzed by reverse transcription polymerase chainreaction(RT-PCR).The relation ship between expression of WWOX gene andclinicopathologic parameters in patients with HCC was analysed.Results:The positive rates of WWOX protein in HCC were respectively 38.00%(19/50) which were prominently lower than those in paired para-carcinoma liver tissues72.00%(36/50)(p＜0.05),The positive rates of WWOX mRNA were respectively 42.00%(19/50) which were prominently lower than those in paired para-carcinoma liver tissues84.00% (39/50) .Chisquare test analyses revealed that reduced WWOX gene expressionwas correlated with tumor clinical grade and stage,portal cancer embolus(p＜0.05).Conclusions:The WWOX protein deletion may play an important role in thecarcinogenesis of HCC.It indicate that WWOX may be a new molocular biology parameterin evaluating diagnosis,therapy and prognosis for HCC. PART TWOConstruction of eukaryotic expression vectorcontaining the WWOX geneObjectives:To extract total RNA from the normal tissue of human liver,clone cDNAof anti-cancer gene WWOX by RT-PCR,design and achieve plasmid vectorpEGFP-N 1-WWOX which contains anti-cancer gene WWOX.Methods:We extracted from normal human liver,designed the primer referring tosequence of WWOX mRNA from the GeneBank,We cloned cDNA of anti-cancer geneWWOX by RT-PCR.The cDNA of WWOX was 1234bp.The cDNA of WWOX andpEGFP-N1 were cut by endonuclease Hindâ…¢and BamHâ… ,then they were joined together.We gained recombined plasmid vector pEGFP-N1-WWOX containing anti-cancer geneWWOX.Results:We extracted mRNA from normal human liver successfully,gained thecDNA of WWOX by RT-PCR successfully.The cDNA of WWOX was 1234bp onelectrophoresis site.We gained recombined plasmid vector pEGFP-N1-WWOX .ThecDNA of WWOX was right.Conclusions:We can gain the cDNA of WWOX by RT-PCR properly and recombinedplasmid vector pEGFP-N1-WWOX successfully.The recombined plasmid can betransfected into cell and be indentified and screened out. PART THREEExperimental studies of eukaryotic expression vectortransfecting hepatoma cells in vitroObjectives:To copy,reproduce,purify and analyse the recombined plasmidpEGFP-N1-WWOX into SMMC-7721 cell screen out the SMMC-7721 cell with WWOX.To expiore the effect of WWOX on the growth,proliferation,apoptosis and cell growthcycle phase of SMMC-7721 cell,discuss the process mechanism of WWOX inhepatocellular carcinoma.Methods:SMMC-7721 cell was cultured in RPIM-1640 with 10% FCS and glutamine,then mixed with liposome Lipofectamine2000 and plasmid vector pEGFP-N1-WWOX,totransfect recombined plasmid vector pEGFP-N1-WWOX into SMMC-7721 cell Weobserved the growth and proliferation of SMMC-7721 cell containing pEGFP-N1-WWOX,by MTT method,analysed the apoptosis and cell growth cycle phase of SMMC-7721 cellcontaining pEGFP-N1-WWOX,by Flow Cytometry method.Results:We transfected the recombined plasmid vector pEGFP-N1-WWOX,intoSMMC-7721 cell successfully and screen out the signle clone SMMC-7721 cell withWWOX.After transfected WWOX into SMMC-7721 cell,WWOX enhanced the growthsuppression of SMMC-7721 [Suppression ratio=(32.1±4.2)%,p＜0.01],induced theapoptosis of SMMC-7721 cell [Apoptosis ratio=(17.96±1.2)%,p＜0.01],Anti-cancergene WWOX also increased the ratio of G₀/G₁ from (44.24±2.35)% to (64.90±1.03)%(p＜0.05),inhibited the advance of SMMC-7721 cell phase significatively,arrested andstopped SMMC-7721 cell at G₀/G₁ phase.Conclusions:The recombined plasmid vector pEGFP-N1-WWOX can be transfectedinto SMMC-7721 cell with transfection vector liposome Lipofectamine2000.WWOX genecan enhanced the growth suppression and induce apoptosis of SMMC-7721 cell in vitrosignificatively.The mechanism of WWOX on hepatocellular carcinoma may correlate withthe growth suppression,inducement of apoptosis and inhibition and arrestment ofSMMC-7721 cell cycle. PART FOURInhibitory effects of WWOX gene transfection on hepatocellularcarcinoma xenograft in mude miceObjectives:Construct nude mice bearing human hepatocellular carcinoma andobserve the effect of WWOX on tumorigenesis in nude mice.Methods:Construct nude mice bearing human hepatocellular carcinoma.the lengthand short-diameter and weight of the transplantation tumor were measured,so did tumorinhabitation rate.The expressions of Bcl-2,CyclinD₁ protein in nude mice were detected byimmunohistochemsitry.Tumor cell apoptosis were monitored by use of TUNEL.Results:The tumorigenesis capability in nude mice injected with transfectedpEGFP-N1-WWOX cells was lower than in nude mice injiected with nontransfected cells.The tumor volume in transfected nude mice was smaller than in nontransfected nude mice,the inhibition rate was 59.42%.The expressions of Bcl-2,CyclinD₁ protein in nude micewere inhibited by immunohistochemsitry method,and apoptosis was increased by TUNEL.Conclusions:The tumorigenesis capability in SMMC-7721 cells transfected bypEGFP-N 1-WWOX reduced,the tumor growth was inhibited and apoptosis was induced.