Relative Research On Synthesized Eucaryotic Expression Vector And Succeeded In Transfecng NK4 Into Cervical Carcinomacell Strain

IntroductionHepatocyte growth factor(HGF) was,seperated from the serum of the rats with the partly-cut liver,a very strong mitogen stimulating hepatocyte proliferation.A grown-up HGF molecule is a heterodimer made ofαchain(69kD) andβchain(34kD) connected with a disulfide bond.αchain has four Kringle regions,whose N end has a hairpin structure.The hairpin structure and the former two Kringle regions are responsible for the biological effect of HGF.Bchain has the structure of serine proteinase but doesn't have catalysis effect due to the substitution of the two amino acids at the activated region.HGF brings its biological effect into full play through the specific receptor c-Met on the cell membrane. HGF-c-Met signal transmission pathway,widely existing in various cells,plays a very important role in regulating the growth and development of lots of tissue and organs.But overexpression of HGF or c-Met may often lead to cancer,and has an important effect on the growth,invasion and metastasis of various kinds of tumor cells.Through the digestion and lysis of recombinant human HGF Date and his colleagues found a new HGF antagonist in 1997,which was composed of 447 amino acids at the N end of HGFαchain and 4 Kringle regions,then named as N K4 with the molecular weight being 50kD。The structure was quite similar to angiostatin.N K4 can be combined with c-Met,competitively controlling the combination of HGF with c-Met,impairing the signal transmission of HGF/ c-Met pathway,then suppressing the proliferation,movement and morphology formation induced by HGF.N K4,however,cannot induce c-Met tyrosine phosphoserine。N K4 could suppress the cellular migration and invasion of gallbladder carcinoma,cholangiocarcinoma,breast cancer,Prostate Cancer and pancreatic carcinoma induec by HGF in vitro。 Cervical cancer,as one of the three malignant gynecological carcinoma,has a great harm on the women health.With the spreading and dissemination of human papilloma virus,the prevalence of cervical cancer is gong up.So it is very important for the treatment of this malignant gynecological carcinoma.Gene therapy is the fourth therapy of carcinoma exept for operation,radiotherapy and chemotherapy.With the recent rapid development of molecular biology,immunology and gene engineering science,scientists can make konwlege of the molecular occurrence and development of carcinoma and the huma anti-cancer mechanism and then developed lots of new cancer therapies.With the use of gene engineering method,antioncogene and suicide gene can be transported into cancer cells,inducing gene backward shifting and apoptosis,then developing a new region for the treatment of cancer.Recent research has proved that NK4 could be regarded as cancerherapy due to its cutting up of HGF-c-Met pathway and inhibiting the formation of carcinoma vessels.An pathological analysis of cancer tussue indicated that N K4 markedly inhibited the formation of mirovessels of cancer tussue and the proliferation of cancer cells and promoted apoptosis of cancer cells.Recently Saga and his colleagues transplanted plasmid expressing NK4 into ovarian cancer cell strain HRA and found that NK4 markedly inhibited the migration of HRA cells.Also NK4 significantly suppressed the growth of carcinoma of mouse and the accumulation of serous fluid in peritoneal cavity and prolonged the mouse life span.Therefore,NK4 is not only antagonist of HGF but also an inhibitor of formation of mirovessels.Nk4 may play an important role in controlling the growth,invasion and metastasis of carcinoma.Fewer reports at abroad and no reports at home appeared upon the effect of NK4 on the gynecological carcinoma such as cervical carcinoma.ObjectiveThe objective of this subject was to study the effect of NK4 on the growth and migration of cervical carcinoma through the building up of pBudCE4.1-EGFP/NK4 eucaryotic cell expression carrier and its transfection and expression in cervical carcinoma cell strain.That will provide with evidence for turmor-resisting effect of NK4 in tumor transplanting models.Then transfection of NK4 gene will become a new effective method of tumor treatment,hence inreasing the therapeutic effect of comprehensive treatment of carcinoma. Materials and MethodsMaterialsRNA abstraction kit,RT-PCR kit,agarose gel reclaiming kit,restriction enzyme KpnⅠ,BglⅡ,Xbal-Ⅰand HindⅢ,T4 DNA connecting enzyme from Takara company; competent cell Mach1-T1,cloning vector pEasy-Blunt Simple from Beijing TransGen Biotech,pEGFP-N1 plasmid,pBudCE4.1 vector,Escherichia coli DH5α,plamid abstraction kit,TaqDNA polymerase Lipofectamine2000 from Guangzhou Taikang Biotech;Annexin V-FITC/PI double transfection kit(B620033,BIPEC BIOPHARMA,U.S.A)。DNA sequencing was completed by Shanghai Yingjun Company.Main methodsNK4 cDNA was finished by RT-PCR with the use of double man-made primers the model of the total RNA abstracted from human fetal liver.Then restriction enzyme was indrafted.Competent plasmid was first transferred,and then transformed Escherichia coli to amplify.Selected positive cloning colony.NK4 gene was then proved to have been transferred through enzyme and sequencing.Then connected with pBudCE4.1-EGFP vector to produce GFP flourescent labelled double gene vector.NK4 plasmid was transfected into cervical cancer cells with the method of liposome.Selected the positive cloning to exam NK4 mRNA and protein expression in transfected cells and to study the inhibitive effect on the growth and migration of Hela cells.1.designed a double of primers according to the NK4 cDNA sequence2.RT-PCR products were tested by 1.0%agarose gelelectrophoresis image analysis system.3.Cloned the objective fragment of NK4 and made determination.4.building up and determination of recombinant plasmid:pBudCE4.1 -EGFP/HGF double gene expression vector。5.cell culture and transfection:NK4 plasmid was transfected into Hela cells of cervical carcinom with the method of liposome. 6.Selected the positive cloning:tested the NK4 mRNA expression in transfected cells(real-time flourescent quantitative PCR);tested the NK4 protein expression in transfected cells(western blot)7.Observed the transfected cells under flourescent microscope and tested the growth curveThe cells was divided into the following groups:normal control, vacant vector transfection group,stable NK4 transfection group.8.tested apoptosis:Apoptosis was tested by flow cytometry.CELLQuest software was used to make an analysis of apoptosis expressed as the percentage of apoptosis cells.9.studied the inhibitive effect of transfected NK4 gene on migration of Hela cells:observed the effect of movement and migration of Hela cells with the method of Transwell small chamber.Tested the FN,MMP-2,MMP-9 expression in transfected Hela cells by real-time flourescent quantitative PCR.Results1.Synthesizing,cloning and determining the objective NK4 fragment About 1326bp NK4 fragment was observed by agarose gelelectrophoresis.Synthesized NK4 gene-contalning eucaryotic expression vector(flourescent labelled)pBudCE4.1-e GFP/NK4 plasmid.Both NK4 PCR amplication and recombinant plasmid enzyme determniation produced 1326 bp frament,thus proving NK4 fragment has inserted pBudCE4.1-EGFP vector,finally producing pBudCE4.1-EGFP/NK4 vector.The sequencing results were the same as Genebank.2.The positive expression of NK4 mRNA in NK4 plasmid transfected cervical carcinoma Hela cell strain was observed and the amount of NK4 mRNA expression was 3.84 times more than controls.The results of NK4 protein level by Western blot indicated that a specific strand,whose molicule weight was about 50Kd and similar to the expected that of NK4 protein,was tested in supernatant culture.That indicated that the recombinant plasmid can effectively induce the expression of the objective NK4 gene.3.The change of growth curve of the transfected cellsTested the growth conditions before and after the NK4 transfection with the direct counting method.During first 5 and 10 days,population multiplying lasted for 22h and 24 h in Hela cells.Logarithmic growth stage came from the third day with no significant difference with vacant vector group.During first 5 and 10 days,population multiplying lasted for 30h and 28.2 h in Hela/NK4 cells.Logarithmic growth stage came from the third day.The largest cell number at the tenth day was 1.9×10~5,which was slightly lower than that of non transfection group(2.5×10~5)with no significant difference (P＞0.05).72h apoptosis percentage of transfection group was 30.4%,which was higher than that of vacant vector group(22.1%) and controls(11.4%,P＜0.05)4.Effect of transfected NK4 on the movement and migration of Hela cells:no effect of vacant or NK4 transfected vector was found(P＞0.05) on the movement and migration of Hela cells without HGF.When compared with controls without HGF,the movement and migration indexes in HGF-adding Hela cell group and transfected vacant vector group were significantly higher(P＜0.05).The transfected NK4 markedly suppressed the movement and migration Hela cells induced by HGF(P＜0.01) except for the transfected vacant vector group(P＞0.05).The expression of(MMP) -2 and -9 in transfected NK4 Hela cells was reduced,esp.MMP-9 with the significant difference(P＜0.05)when comparing with controls.Conclusions1.Synthesized pBudCE4.1-EGFP/NK4 eucaryotic expression vector and made determination2.Succeeded in transfecting NK4 into cervical carcinoma cell strain.The expression of NK4 mRNA and protein in transfected cells was poistive.Hela/NK4 cells promoted apoptosis.3.Transfected NK4 inhibited the movement and migration of Hela cells,which may play an important role in resisting metastasis of cervical carcinoma.