The Construction Of The Specific MUC5AC-siRNA Expression Plasmid And A Study On The Effect Of SiRNA On The Proliferation And Apoptosis In Human Bile Duct Cancer Line

Since the RNA interference phenomenon has been found,RNA interference gene silencing because of their high efficiency, high stability, high specificity, etc. are widely used in gene function research, gene therapy and drug screening and other areas.MUC5AC is a secretory mucin normally expressed in the gastrointestinal tract, respiratory and reproductive tract, its played the role of lubrication and protection. MUC5AC in a variety of tumors have abnormal expression ,probably played a role in the occurrence and development of tumor. It is reported that When the diagnosis of cholangiocarcinoma MUC5AC can be used as a diagnostic tumor marker, but the occurrence of cholangiocarcinoma development must have played a role remains unclear.This experiment using RNA interference technology, for MUC5AC gene sequences of different target to build three express a small hairpin RNA expression plasmid,Observed separately in the HCCC-9810 on the role of MUC5AC gene silencing,and to study the MUC5AC gene inhibition of tumor cells to this growth and apoptosis.This can be studied at MUC5AC associated MUC5AC tumor occurrence and development of the role, and to explore in the MUC5AC gene therapy of tumor-related aspects of the value of potential applications.Objective: To observe specific siRNA silencing effect on MUC5AC gene in human intrahepatic cholangiocarcinoma cell line HCCC-9810 as well as to investigate the influence to tumor cell on the proliferation and apoptosis after silencing the MUC5AC gene.Methods:Three pairs of specific MUC5AC-siRNA was designed and synthesized through transcription in vitro.The the three different siRNA expression plasmids(pRNAT-U6.1/Neo-MUC5AC-siRNA1/2/3) were constructed by gene recombination . Then the three stable expression plasmids and the comparison plasmid (empty plasmid-transfected control) were transfected into HCCC-9810 by liposome-mediated transfection.At the same time, these vectors also expressed a variant of DsRed fluorescent protein. With the DsRed-Express fluorescent marker we can directly monitor the delivery efficiency of the gene silencing construct using fluorescence microscopy at 8h-12h after transfection. The expression of fluorescent protein is not correlated with the expression of siRNA, so it doesn't influence the silencing efficiency of siRNA at all.MUC5AC-mRNA level was detected by RT-PCR.Expression of MUC5AC was investigated by immunohistochemical SABC method.Cell apoptosis and proliferation were analyzed by flow cytometry and MTT,respectively.Results:The results of gene sequencing indicated that the pRNAT-U6.1/Neo-MUC5AC-siRNA1/2/3 were successfully constructed.After the transfection,the efficiency of DsRed fluorescent protein expression reached 28.57%;The results of RT-PCR and immunocytochemistry showed that constructed plasmids down-regulated mRNA and protein of MUC5AC at 48h after transfection .The results of MTT indicated that the growth of HCCC-9810 could be obviously inhibited after silencing the MUC5AC gene.Apoptosis could be induced in the tumor cell after suppressing the express of MUC5AC gene by flow cytometry.Conlusion Constructed three different stable expression plasmids of siRNA specific for MUC5AC gene obviously inhibit the expression at MUC5AC-mRNA and protain level.The blockage of MUC5AC gene expression in HCCC-9810 cells shows significant effect on cell apoptosis and proliferation.These findings suggest that this study may provide theoried basis for the applications of MUC5AC in the gene therapy of MUC5AC related tumors.