Construction Of Eukaryotic Plasmid And Selection Of Functional SiRNA Target Sites Of Human S100A6

Background:Gastric cancer is one of the most common malignant tumor ofdigestive tract, In addition to surgery, chemotherapy is the most important treatment foradvanced gastric cancer, However, due to multidrug resistance of tumors (multidrugresistance,MDR), often leading to failure of chemotherapy, Therefore, depth studyof theformation mechanism of gastric cancer MDR in order to take measures (such asinhibitors, gene overexpression, RNA interference) reversal of gastric cancer of MDR, itwill be possible to bring a new breakthrough for the treatment of gastric cancer.RNA interference (RNAinterference RNAi) is initiated by double-stranded RNA(double strand RNA, dsRNA) sequence-specific post-transcriptional gene silencing(PTGS) process. In this process,the double-stranded RNA are cut into small interferenceRNA (small interfering RNA,siRNA)by endonuclease enzymes, small interfering RNAand related proteins binding into RNA-induced silencing complexes (siRNA-inducedsilencing complex，RISC), RISC recognize and degradation of target mRNA, resultingin effective gene silencing. RNAi technology has lots of characteristics,such assimple,easy to carry out,short cycle,low cost, specificity and efficiency. Thistechnology has become the important means to study gene function and recognize thenature of disease,and even is likley to be used for targeted gene therapy of certaindisease, the potential clinical application is very broad.In order to find out new MDR related proteins of gastric cancer,two-dimentionalgel electrophoresis(2-DE)was used to separate the total proteins of Adriamycin-resistanthuman gastric cancer cell line SGC7901/ADR,its parental cell line SGC7901,and thereversed cell line by Salvia miltiochiga.PDQuest software was applied to analyze2-DEimages,And the differential protein spots among the three cell lines were identified byHPLC-ESI-MS/MS.We found that,S100A6is highly expressed in SGC7901,down-regulated in SGC7901/ADR,and up-regulated again after the reveral bySalvia miltiochiga.In this study, four S100A6-shRNA plasmid vectors were built andtransfected into the gastric cancer cell line SGC7901Which has a high S100A6expression.Real time PCR was used to detect interference effects and then the bestsilencing effect of plasmid was found out so that we can lay the foundation for the nextstep to continue to deeply explore the role of S100A6in the development of MDR inhuman gastric cancer.Objective: To construct the human S100A6gene shRNA eukaryotic expressionplasmid,to detect its effect on the S100A6gene silencing by real time PCRtechnology,finding out the most effective RNA interference fragment, lay thefoundation for further study of deeply exploring the role of S100A6in the developmentof MDR in human gastric cancer.Methods:According to the Gene Bank sequence of the human S100A6gene, thefour oligonucleotides of shRNA and an unrelated sequence as the negative control(negative control，NC) were designed and synthesized.The four interference fragmentsand the negative control were directionally cloned into plasmid pRNAT-U6.1/GFP/Neowith ampicillin resistance and green fluorescent protein. The recombinant plasmidswere confirmed by positive clone identificationand DNA sequencing. The recombinantplasmids were transfected into SGC7901cell line by LipofectamineTM2000,theexpression of Green fluorescent protein and transfection efficiency were observed byfluorescence microscopy. Total cellular RNA was extracted48hours after transfection,The impaction of four plasmids on the level of mRNA transcription of S100A6genewas detected by Real-time PCR technology. In order to find out the most effective RNAinterference fragment to determine the optimum interference target.Results:Through the identification of positive clone and DNA sequencing,the fourexpression of short hairpin RNA plasmids and its negative control plasmid wereconstructed successfully. These five plasmids were transfected into SGC7901cells,bright green fluorescence can be observed under a fluorescence microscope, thetransfection efficiency is40%. Total cellular RNA was extracted48hours aftertransfection, the inhibitory rates of four shRNA eukaryotic expression plasmid at themRNA level of S100A6was detected by Real-time PCR,they were31.3%、20.6%、27.9%and22%. This shows that the S100A6mRNA expression can be decreased bythe four plasmids, the S100A6-1inhibition is the most obvious and its inhibitory rate is31.3%. The four plasmids RNA interference efficiency was78.25%、51.5%、69.75%、 55%respectively.The efficiency of interference of S100A6-1is the highest which is78.25%, this shows that the S100A6-1is the best screening target.Conclusion:The expression of the human S100A6gene in SGC7901cells can bespecifically inhibited by eukaryotic expression plasmids expressing shRNA sectionstargeting human S100A6gene. S100A6-1eukaryotic expression plasmid as the bestscreening target can be used for follow-up experimental study.