The Construction And Appraisal Of Tumor Susceptibility Gene101-siRNA Piasmids

[Objective]1、To construct the tumor susceptibitity gene101-saran2、This study was aim to obtain and appraisal the gastric cance cell line AGS which are different in the expression of TSG101by the transient transfection of TSG101si RNA, which is advantageous to further build the Stable expression of TSG101in gastric cancer cells and discuss the possible relationship between invasion and metastasis of Stomach cancer and TSG101.[Methods]1、According to the small interference RNA design principles and references, design two TSG101target sequences, recombinant DNA technology respectively to sequences of cloned into a small interference RNA expression vector pGenesil1.1;2、The gastric cance cell line AGS were transfected with TSG101siRNA plasmid (pGenesil1.1-TSG1011、pGenesil1.1-TSG1012) by lipofectamineTM2000reagent (TSG101siRNA group), AGS transfected with negative control sequence HK (negative control group) and blank AGS (non-transfected group) were used as blank control group.24h and48h were selected to observation cells, and collect four groups cells in48h. The expressions of TSG101in the human gastric cancer cell lines AGS were detected at both mRNA and protein leverls by RT-PCR、quantitative fluorescent PCR and Western blot,respectively.[Result]1、Restriction enzyme digestion and DNA sequencing suggests Insert an interference vector pGenesil-1.1DNA fragment size, method, completely correct sequence, vector was constructed successfully;2、The observation of cells in24h and48h:The untransfected group did not appear green fluorescence; pGenesil1.1-TSG1011, pGenesil1.1-TSG1012and negative control sequence HK transfection group24h and48h were part of the AGS cells showed green fluorescence positive; through the rough observing the same view of ordinary light and fluorescence of cells and the number of the many experimental results, a rough calculation of transfection efficiency:pGenesill.1-TSG1011, pGenesil1.1-TSG1012and negative control sequence HK transfection group were:24h35%%, respectively:38%,31%;48h respectively:65%,69%,55%. 3、The detection at both mRNA and protein leverls:(1) RT-PCR and Western blot:In order to housekeeping genes beta actin for internal reference: transfection of siRNA on AGS cell TSG101mRNA and protein expression has obvious inhibition, where TSG101siRNA1inhibition effect relative to the TSG101siRNA2slightly weaker; negative control sequence HK TSG101mRNA and protein expression without obvious inhibition.(2) Quantitative fluorescent PCR:The amplification of TSG101curve and melting curve visible each Ct values of the samples have good linear relationship. On the value of Ct statistic analysis showed:AGS-TSG1011, AGS-TSG1012and AGS-HK and AGS had significant difference between (P<0.05); AGS-TSGG1011, AGS-1012, AGS-HK, AGS were not statistically significant (P>0.05). Using2-ΔCt method quantitative analysis shows the group TSG101mRNA expression:AGS-TSG1011, AGS-TSG1012than AGS-HK and AGS decreased significantly; AGS-TSG1011and AGS-TSG1012are slightly different, the former is somewhat low; AGS-HK and AGS showed no significant difference.(3) The statistical analysis of Western blot:AGS-TSG1011, AGS-TSG1012and AGS-HK and AGS had significant difference between (P<0.05); AGS-TSGG1011, AGS-1012, AGS-HK, AGS were not statistically significant (P>0.05)[Conclusion]1、The tumor susceptibitity gene101-siRNA was constructed and transfected into the gastric cance cell line AGS successfully.2、The transient transfection of TSG101si RNA can reduce the expression of TSG101at both mRNA and protein leverls, obviously, which is advantageous to further build the Stable expression of TSG101in gastric cancer cells and discuss the possible relationship between Occurrence, development and Multidrug resistance of Stomach cancer and TSG101.