Mechanisms Study On RNAi Inhibition Expression Of Pancreatic Cancer Cell-BxPC-3 Cell' HTERT Gene In Vitro

The pancreatic cancer has been the most common malignant tumor in our country and is leading one of the causes of death in the whole world,which severely enrangers human health and life.Telomerase is a ribonucleoprotion complex that extends telomerases of eukaryotic chromosomes,cell replicative potential and lifespan.Now, maintains and regulates has become cancer an attractive molecular target toward which to direct therapeutic agents.Recently,more and more researchs indicated human telomerase catalytic subunit mRNA(hTERT mRNA)may be the critical rate-limiting step in the activation of telomerase.RNA interference(RNAi)has been shown to interfere the expression of correlating mRNA in various species.RNAi has been an important tool for understanding gene function and attempting gene therapy.Objective This study were constructed the recombinant plasmid pSilencer4.1-CMV-hTERT siRNA as vector,carrying the hTERT siRNA gene into pancreatic cancer BxPC-3 cell to inhibit the expression of human telomerase reverse transcriptase(hTERT)in cell.We investigate the effect of gene hTERT siRNA transfection into BxPC-3 cells,such as cells growth,proliferation,and apoptosis. Using RNAi directed against hTERT gene in our study,we explore to determine potential of hTERT as a therapeutic target for pancreatic cancer by potent highly sequence-specific RNAi technique,and to investigate the effect of hTERT gene silencing on the growth of BxPC-3 cell in vitro.To evaluate practical value of small interfering RNA targeting hTERT gene may be becom therapeutic applications in treating pancreatic cancer.Methods Constructing the recombinant plasmid pSilencer4.1-CMV-hTERT siRNA as vector,it carried the hTERT siRNA gene into BxPC-3 cell in vitro.The change of cell cycle distribution and apoptosis of BxPC-3 cells was detected by FCM (flow-cytometry),electron microscopeand,and gel electrophoresis.Cell proliferation was measured by MTT method.The hTERT protein level of BxPC-3 cell was checked by Western blot methods and the hTERT mRNA expression in BxPC-3 cell was detected by RT-PCR methods.Results The recombinant plasmid pSilencer4.1-CMV-hTERT siRNA as vector were constructed successfully,carrying the hTERT siRNA gene into BxPC-3 cells and silencing the expression of hTERT mRNA.Compared with control group, transfected cell significantly decreased the expression of hTERT,at protein level in vitro(p＜0.01).The growth graph showed that the group of hTERT siRNA transfected BxPC-3 cells were slower than of control group,but apoptosis on the contrary of two group(p＜0.01).More BxPC-3 cells accumulated at G/M phase and proliferated slowlier(p＜0.01)with hTERT siRNA.And then inhibited cell growth effectively and induced apoptosis of pancreatic cancer cells effectively under electron microscopeand,and gel electrophoresis.Conclusions The specific small hair RNA target hTERT gene could inhibit the expression of hTERT mRNA and the proliferation of BxPC-3 cells.The hTERT siRNA can suppressed the telomerase activity notably.It indicated that hTERT mRNA play important in the telomerase activity,and we could cured the pancreatic cancer from depressing the telomerase activity used RNAi in future.