Sequence-specific Gene Silencing Of Exogenous Integrated Gene And Endogenous Oncogene In Human Pancreatic Cancer Cells By Introduction Of Homologous Promoterless DNA Fragments

Pancreatic cancer is exceptionally aggressive with high mortality,and up to now no effective therapeutic approaches has been found.So it is urgent to find more powerful method to diagnose the disease earlier and treat it more effectively.But even the new powerful research tool siRNAs has its own problem,i.e.the efficiency.In recent years,several researchers reported that delivery of promoterless double-strand DNA molecules into cells could induce sequence-specific gene silencing in plants.The plasmid having the target gene's cDNA inserted alone was sufficient to induce silencing.This simple and efficient method of gene target silencing has not been used in human cells yet,especially in human cancer cells.On one hand,genetically encoded fluorescent proteins have become widely used as markers in living cells.The application of these fluorescent proteins as noninvasive tags revealed new aspects of protein dynamics and the biological processes they regulated.The modification of naturally occurring fluorescent proteins as well as the identification of new fluorescent proteins now provide researchers with a variety of useful fluorescent markers suitable for all kind of investigations in live-cell imaging studies.Enhanced jellyfish green fluorescent protein(EGFP) a mutant of green fluorescent protein(GFP) with reinforced green fluorescent was selected as a visible index.A plasmid containing GFP full-length cDNA was constructed.The silencing efficiency was observed through detecting the GFP expression by transfecting the plasmid into the steady-expressing GFP clone cell or by contransfecting it with GFP expressing plasmid pEGFP-C1.K-RAS is a member of the RAS family of GTP-binding proteins that mediate a wide variety of cellular functions including proliferation,differentiation,and survival.The activation of K-ras oncogene is the most important initial step in the genesis of human pancreatic cancer and most of human pancreatic cancers have high frequency of K-ras point mutation.All these together with the importance of ras in cell function and survival,highlight K-ras being an attractive target for the treatment of pancreatic cancer.K-ras was thus chosen as the target gene in the present study.Aim:The present study aimed to test the efficiency of the gene silencing effect by introducing promoterless double-strand DNA containing coding region of a gene in human pancreatic cancer cells.Material and method:First of all,several plasmids were constructed. Considering the low transfect efficiency of naked DNA,promoterless plasmid puc-19 was used as a vector of target gene's full-length cDNA fragment.To construct plasmids containing promoterless DNA of the target gene,GFP and K-ras full length cDNA were obtained and ligated into puc-19.The constructs were named as puc-GFP and puc-K-ras respectively.Puc-exon,with DNA sequence of K-ras first exon integrated in puc-19,was also constructed to testify if the silencing efficiency had relationship with specific sequence in cDNA.Next,puc-GFP was transfected to GFP transformed human pancreatic cancer cell line Pane-1 cells to visualize the silencing efficiency.Flow cytometry,phase contrast fluorescence microscopy and Western blot were used to detect expression of GFP.Finaly,puc-K-ras and puc-exon were transfected to Panc-1 to observe whether K-ras gene expression could be inhibited.Puc-K-ras was also transfected to two other human pancreatic cancer cell lines Miapaca-2 and Aspc-1 to detect the K-ras gene expression.As compared with Panc-1,the two cell lines have similar or different point mutation of K-ras.Real-time transcriptive PCR and Western blot were used to detect the K-ras gene expression.Results:(1) Panc-1 exogenous integrated gene GFP could be silenced by plasmid puc-GFP,which containing promoterless DNA fragment of GFP full-length cDNA.Puc-GFP also inhibited the transient transfecting efficiency of GFP expressing plasmid.(2) Endogenous gene K-ras of Panc-1,Miapaca-2,Aspc-1 cells also could be silenced by promoterless plasmid having its full-length cDNA integrated.The silencing efficiency was correlated to the length of cDNA fragment.Different K-ras point mutant did not affect the silencing efficiency.Conclusion:The results of the present study indicate that the transfection of human pancreatic cancer cells with plasmids carrying a full length cDNA sequence of the target gene is sufficient to decrease the expression of the genes.It may take effect through reduction of the endogenous transcripts.The effective region of the promoterless DNA fragment is not clear yet and need to study further.The result of present study indicats that the whole length of cDNA fragment rather than the consensus sequences may account for the silencing effects.This study is for the first time to prove the sequence-specific gene silencing method used in plant, also is effective in silencing the target gene in human pancreatic cancer cells.