Analysis of the interference efficiency of EGFR siRNA and its effect on the proliferation of hepatoma cells
Objective To construct the recombinant eukaryotic expression vector of EGFR shR-NA and screen the EGFR shRNA sequence with the best inhibitory effect.Methods An online tool was used to design the human EGFR siRNA sequence and synthesize the corresponding EGFR shRNA.The eukaryotic expression plasmid pcDNA3.1(+)was cleaved by the restriction endonucloenzymes BamHI and HindIII to construct the siRNA interference vector of three kinds of human EGFR(psilencer 4.1-CMV neo-EGFR siR-NA)and transform the recombinant plasmid vector into the Escherichia coli DH5α receptor state.The positive clones were screened,and the recombinant plasmids were identified by DNA sequencing.HepG2 cells were transfected with the three siRNA interference sequence vectors of human EGFR using lipo2000.Fluorescence microscopy was used to observe the transfection efficiency,real-time quantitative PCR was used to detect the mRNA level of EGFR,and MTT assay was used to detect the cell activity.Results The psilencer 4.1-CMV neo-EGFR siRNA recombinant plasmid has been successfully cloned.The knockdown of EGFR shRNA-1 on EGFR mRNA was 80%,while EGFR shRNA-2 had a knockdown efficiency of 60%,and EGFR shRNA-3 had a knockdown efficiency of over 70%.Both shRNA-2 and shRNA-3 reduced cell activity by 50%(P<0.05),but shRNA-1 did not have a significant effect on cell activity(P>0.05).Conclusion The recombinant psilencer 4.1-CMV neo-EGFRsiRNA plasmid was able to downregulate the expression of EGFR and reduce the activity of hepatocellular carcinoma cell lines.Out of the three EGFR shRNA sequences tested,EGFR SHRNA-3 ex-hibited the most potent inhibitory effect on HepG2 cells.
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