The Effects And Mechanism In Tumor Progression Of ADAM17, A Member Of The Zinc-dependent Metalloproteinase Superfamily

Objective To construct the shRNA eukaryotic expression vectors of TACE geneand to investigate their inhibiting in cellular functions related to tumor malignancy.Methods A single vector containing four shRNAs that target four regions of theTACE gene was constructed.After transfection with our multiple shRNAs vector,Realtime quantitative PCR and Western blot analysis were used to measure the TACEmRNA and protein expression.Four shRNA vectors each containing one shRNA werealso constructed and compared their ability to silence TACE gene expression with thatof the multiple shRNAs vector.The level of proliferation in HeLa cells transfectedwith different shRNA vectors were compared using the MTT assay.The FlowCytometry was used to examine apoptosis.The adhesive and invasive ability weretested by plated adhesion model and Transwell assay.At last,ELISA kit and Westernblot analysis respectively evaluated sTGF—αand the activation of EGFRs aftertransfection of TACE shRNA vectors.Results We found that in HeLa cells our multiple shRNAs vector produced a higherlevel of TACE knockdown than any single shRNA vector containing only one TACEshRNA.Silencing TACE expression in HeLa cells decreased their malignancy bydecreasing the proliferation,adhesion and migration,as well as inducing apoptosis inthese cells.Furthermore,our data suggest that the effects of TACE on the malignancyof HeLa cells may be mediated via activation of the EGFR(epidermal growth factorreceptor)signaling pathway.Conclusion Our findings suggest that using a combination of shRNAs within one vector to silence the expression of TACE might be a potential therapeutic strategy fortumors. Objective:To explore effects ofdisintegrin domain of TACE on the proliferation,adhesion and invasion potential of tumor cells in vitroMethods:(1)The construction,expression and purification of prokaryoticexpression plasmids of TACE:The total RNA wasisolated from THP1 cell.TACEcDNA was amplified by RT-PCR and subcloned into PMD18-T vector to constructPMD18T—TACE vector.Primers were designed and synthesized according to thepublished cDNA sequence of TACE.The amplified PCR products(T1300,T591)were ligated into the pET-28a(+)prokaryotic expression vector digested with the samerestriction enzymes to create pET-28a-T300,pET-28a-T800 and pET-28a(+)-T1300,which were transformed into E.coli BL21(DE3).After induced by IPTG,therecombinant proteins were purified using a Ni²⁺-chelating resin affinity column.Thepurified productions were analyzed by SDS-PAGE and Western blot.(2)Thebiological activites of the proteins in vitro.The adhesive and invasive ability wereexamined by plated adhesion model and Transwell assay.(3)The full-length TACEcDNA fragment was amplified by PCR from the human THP1 cell cDNA andsubcloned into pMD18T vector to construct PMD18T-TACE vector.The cDNAfragment of disâ–³-TACE and metâ–³-TACE were amplified from plasmidPMD18T-TACE by using the overlap extension PCR and cloned into pIRES2.0-EGFPto construct the expression vector pIRES2.0-EGFP/FL-TACE andpIRES2.0EGFP/disâ–³-TACE.The full-length of TACE was obtained by doubledigesting the plasmid pMD18T-TACE and then inserted into pIRES2.0-EGFP.Thesethree eukaryotic expressing vectors were transfected into HeLa cells by Lipofectamine 2000 after identification of digestion and sequencing.The stabletransfected HeLa cell lines were then established by screening culture withG418.And the transcription and expression of TACE and disâ–³-TACE wereidentified by Western blot and immunofl uorescence.The proliferation,adhesion andmigration ability of stable transfected HeLa cells were detected by MTT,adhesionand migration test.Results:(1)SDS-PAGE and Western blotting analysis revealed that there proteinsof about 18KD,37KD and 55KD were expressed and recognized by anti-His Abrespectively,and the purity of the proteins purified by Ni²⁺-NTA resin was more than90%.(2)The protein pET28a(+)-T300 and pET28a(+)-T1300 can reduce the adhesionand invasion ability of the human lung carcinoma cell A549 in vitro,but otherwisethe pmtein pET-28a(+)-T800 had not shown the inhibitive function.(3)Theeukaryotic expression vector pIRES2.0-EGFP/FL-TACE andpIRES2.0-EGFP/disâ–³-TACE were constructed successfully,stable transfected HeLaeelllines were established,and the aim proteins were expressed successfully.Conclusion:The disintegrin domain of TACE have the similar biological function toother disintegrins,which can be used for further research on function of TACE ininflammation and tumor. Objective To investigate the influence on growth of transplanted tumor byshRNA expression vector PG4-ADAM10/PC7 inhibiting expression of ADAM10 andPC7 gene in vitro.Methods The vector was transfected into H22 and the mRNA expression ofADAM10 and PC7 of H22 before and after transfection was detected byRT-PCR.The tumor—beating mice were treated by PG4-ADAM10/PC7.Results The RNA expressions of ADAM 10 and PC7 gene were down-regulated in thetransfected group of RT-PCR assay.The transplanted tumors were grown slowly intransfected PG4-ADAM10/PC7 mice and comparing with the volume of controlgroup,there was significant difference(P＜0.05).Using immuno-histochemistrymethod to detect injected PG4-ADAM10/PC7 goup,PG4-ADAM10/PC7 expressionwere down-regulated(P＜0.05).Conclusions PG4-ADAM10/PC7 can inhibitory action for the growth of transplantedtumors.