Study On Influence Of Biological Behavior Of Bladder Cancer T24 Cells Via Silencing Gene DNMT1 And DNNM3b With ShRNA

Part 1.Construction of eukaryotic expression vector carrying shRNAtargeting DNMT1 and its influence on expression of DNMT1Objective To construct eukaryotic expression vector carrying shRNA targeting geneDNMT1,and detect its influence on expression of DNMT1.Methods To design a pair of shRNA sequence targeting DNMT 1 and a pair of shRNAsequence making negative control(HK)respectively,and clone them into pGensil-1 vectorby using technology of gene recombination.The recombinant plasmids were transfectedinto Bacterium E.coli DH5α,and extracted with nucleic acid purification Kits afterscreening.The sequence of recombinant plasmids was evaluated by electrophoresis andDNA sequencing.The expression of DNMT1 mRNA and protein were detected by RT-PCRand western blot.Results The recombinant plasmids were separated two gene fragments by restrictionenzyme SalⅠ,and they are identical to the size of design.The sequence of them wascompletely right by sequencing.The inhibition rates of DNMT1 mRNA were 28.44%,52.48% and 70.91% at 24,48,72 h respectively;The inhibition rates of DNMT1 proteinwere 24.27%,57.79% and 69.74% 24,48,72 h respectively.Conclusion The recombinant plasmids were constructed successfully and caneffectively silence expression of DNMT1 mRNA and protein in T24 cells. Part 2.Construction of eukaryotic expression vectors carrying shRNAtargeting DNMT3b and their influenceon expression of DNMT3bObjective To construct three eukaryotic expression vectors carrying shRNA targetinggene DNMT3b,and select the most effective vector of silencing expression of DNMT3bfrom them.Methods To design three pairs of shRNA sequence targeting DNMT3b mRNA,andclone them into pGensil-1 vectors by using technology of gene recombination,andextracted with nucleic acid purification Kits after screening.The sequence of recombinantplasmids was evaluated by electrophoresis and DNA sequencing.The expression ofDNMT3b mRNA and protein were detected by RT-PCR and western blot.Results The recombinant plasmids were separated two gene fragment by restrictionenzyme SalⅠ,and they are identical to the size of design.The sequence of them wascompletely right by sequencing.The inhibition rates of DNMT3b mRNA of three groups,pGensil-1-DNMT3b-shRNA(1,2,3),were 20.44%,79.91% and 54.48% respectively aftertransfecting 72 h;The inhibition rates of DNMT3b protein of them were 17.27%,77.74%and 56.79% respectively after transfecting 72 h.Conclusion Three eukaryotic expression vectors were constructed successfully,andpGensil-l-DNMT3b-shRNA2 was selected for the further study as the most effectiveplasmid in silencing expression of DNMT3b.Part 3.Construction of eucaryotic expression vector carrying doubleshRNA targeting DNMT1 and DNMT3b respectively and itsinfluence on expression of DNMT1 and DNMT3bObjective To construct eukaryotic expression vector carrying two pairs of shRNAtargeting gene DNMT1 and DNMT3b respectively,and detect its influence on expression of DNMT1 and DNMT3b.Methods To cut pGensil-1-DNMT1-shRNA1 and pGensi1-1.1-DNMT3b-shRNA,which were constructed previously,by using restriction enzyme HindⅢand EcoRI,andconnect the smaller DNA fragment (including DNMT1-shRNA from pGensil-1-DNMT1-shRNA1 and the bigger DNA fragment (including DNMT3b-shRNA from pGensil-1.1-DNMT3b-shRNA).The sequence of the new recombinant plasmids,pGensil-1.1-shRNA1-shRNA3b,was evaluated by electrophoresis and DNA sequencing;Expression of mRNAand protein of DNMT1 and DNMT3b was detected by RT-PCR and western blotrespectively.Results The sequence of the new recombinant plasmid was identical to the designedsequence.The inhibition rates of DNMT1 rnRNA,at 24,48,72h,were 28.79%,57.88%and 80.24% respectively;The inhibition rates of DNMT3b mRNA,at 24,48,72h,were23.88%,52.82% and 81.87% respectively;The inhibition rates of DNMT1 protein,at 24,48,72h,were 31.73%,58.27%,and 82.09% respectively;The inhibition rates of DNMT3bprotein,at 24,48,72h,were 35.83%,59.87%,and 65.84% respectively.Conclusion The eukaryotic expression vectors were successfully constructed andcould effectively silence the expression of two genes mRNA and protein in T24 cells.Part 4.Study on Influence of Biological Behavior of Bladder CancerT24 cells via Silencing gene DNMT1 and DNNM3bwith shRNA in VitroObjective To observe the influence of silencing gene DNMT1 and/or DNMT3b withshRNA on proliferation and apoptosis of bladder cancer T24 cell in vitro,and research theirinter-relation in DNA methylation of CpG islands and tumorigenesis of bladder.Methods There were four groups,blank control,pGensil-1-DNMT1-shRNA1,pGensil-1.1-DNMT3b-shRNA and pGensil-1.1-shRNA1-shRNA3b respectively,in ourstudies;To culture T24 cells regularly and transfect the recombinant plasmids into T24 cellswith lipofectamine 2000^TM;Expression of their (DNMT1 and DNMT3b)mRNA and protein was detected by RT-PCR and Western blot,respectively.Proliferation and apoptosisof T24 cells were detected by MTT assay and flow cytometry with Annexin-V-FITC/PIstaining.Results Each group plasmid was transfected into T24 cells successfully;Therecombinant plasmids could inhibit effectivey expression of the homologous gene mRNAand protein in T24 cells.The growth inhibition rate of pGensil-1.1-shRNA1-shRNA3bgroup were (12.45±1.98)%,(21.76±3.54)% and (37.07±4.23)% respectively at 24,48,72h,and there were statistically significant difference between pGensi1-1.1-shRNA1-shRNA3b group and the other groups at above time;The cell apoptosis rate ofpGensi1-1.1-shRNA1-shRNA3b group were (10.36±1.98)%,(20.23±3.62)% and (32.96±4.82)% respectively at 24,48,72h,and there were statistically significant differencebetween pGensi1-1.1-shRNA1-shRNA3b group and the other groups at above time.Conclusion To silence expression of gene DNMT1 and DNMT3b together can moreremarkably inhibit cell proliferation and promote cell apoptosis than separate use,suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in theCpG island methylation and human bladder tumorigenesis.Part 5.Study on Influence of Biological Behavior of Bladder CancerT24 cells via Silencing gene DNMT1 and DNNM3bwith shRNA in vivoObjective To observe the influence of recombinant plasmids carrying DNMT1-shRNA and/or DNMT3b-shRNA on the growth of nude mice bladder tumor in vivo,andexplore their role on bladder tumorigenesis and inter-relation between them.Methods To construct the nude mice model of bladder cancer T24 cells,and they weredivided into four groups at random after the size of bladder tumor reached 5cm.Injectingtransfection reagent,including pGensil-1-DNMT 1-shRNA 1,pGensil-1.1-DNMT3b-shRNAand pGensi1-1.1-shRNA1-shRNA3b respectively,into the tumor of nude mice,the treatment was repeated every five days and operated four times in all;we drafted thetumor's volume growth curvature during this period;At the third days after the lastinjection,all mice were killed,and tumors were taken out and made into paraffin section;The expression of PCNA of tumor cells was evaluated by immuno-histochemistry;theapoptosis of tumor cells was detected by TUNEL methods.Results The nude model of bladder tumor was established successfully.After injecting,tumor's volume of pGensi1-1.1-DNMT3b-shRNA group continued to grow with timepassing;tumor's volume of pGensi1-1-DNMT1-shRNA group stop growing;tumor'svolume of pGensi1-1-shRNA1-shRNA3b group regressed distinctly.PCNA positive expre-ssion rates of three groups,pGensi1-1-DNMT1-shRNA group,pGensi1-1.1-DNMT3b-shRNA group,and pGensi1-1-shRNA1-shRNA3b group,were 50%,83.36% and 22.73%respectively;Apoptosis cells positive expression rates of three groups,pGensi1-1-DNMT1-shRNA group,pGensil-1.1-DNMT3b-shRNA group,and pGensil-1-shRNA 1-shRNA3bgroup,were 59.09%,22.72% and 81.09% respectively.Conclusion Although DNMT1 plays a major role on biological behavior of bladdertumor,its effects will be reduced obviously if DNMT3b do not have a supporting role.Theresults revealed there have some synergistic function between DNMT1 and DNMT3b inDNA methlylation of CpG islands and tumorigenesis.