The Impact Of Rna Interference On The Line Panc-1 Pancreatic Cancer Cells Htert Gene Expression

Objects:To produces two recombinant plasmids hTERT-S370 and hTERT-S371, this can express siRNA to inhibit hTERT gene expression.Methods:Design the hTERT-siRNA, apply the designed siRNA targeting hTERT to BLASTTM analysis in GenBank and select the sequence; synthesize the expression template of siRNA; construct the expression carrier of hTERT-siRNA; conduct the enzyme cutting and sequencing evaluating of recombinant plasmid.Results:The synthetic S370 and S371 which are two pairs of oligonucleotide single strands form double strands by renaturation.Conduct electrophoresis in 2%agarose gel. The result shows the two fragments both have a length of 55bps and display the clear straps in the agarose gel electropherogram.Use HindⅢand BamHI to doubledigest the recombinant plasmid, and the agarose gel electrophoresis shows:In accord with the experiment prediction, recombinant plasmid takes on the positive strap at the expected site in the agarose gel electropherogram, which proves the initial identification is true. The result of sequencing shows that the expression template of siRNA has been successfully constructed on the pSilence4.1CMV carrier and the sequence is completely correct.Conclusions:The sequence design of siRNA targeting hTERT gene is reasonable and successfully produces two fragments hTERT-S370 and hTERT-S371 both of which have a length of 55bps. Result from enzyme cutting and sequence detecting proves expression templates of hTERT-S370 and hTERT-S371 have been successfully constructed in pSilence4.1CMV neo carrier, which produces two recombinant plasmids Silencer4.1-CMV neo-hTERT-S370 and Silencer4.1-CMV neo-hTERT-S371 with correct sequence. Objects:Transfer the pSilencer4.1-CMV neo-hTERT-siRNA to PANC-1 cells and to see certain anti-pancreatic cancer effect.Methods:Use lipofectamine2000 to transfer the pSilencer 4.1-CMV neo-hTERT-siRNA into PANC-1 cells and selects the positive cells using G418; Use dilution method to select cell clone. Detect the hTERT gene expression of cell clones by RT-PCR;Results:The result of RT-PCR shows:the hTERT-mRNA expression of PANC-1 cells in group of S370-A1,S371,S370/371-D4,S370/371-B5,S371ΔC. S371ΔC-E5. S371ΔC-A2,S371ΔC-C1,S371ΔC-C2 has significantly declined compared with the expression in group Nc and cell.No obvious strap displays in the group GAPDH.Conclusions:Recombinant plasmid Silencer4.1-CMV neo -hTERT-siRNA can cause degradation targeting PANC-1 pancreatic cancer cells and down regulation of hTERT mRNA and have favorable RNAi silence effect.