RNAi of HIF-1α Gene in BGC-823 Cells through Lentiviral Approach

Objective,To construct shRNA lentiviral vector targeted to human HIF-1α gene,to package and produce recombinant lentivirial particles,infect a gastric cancer cell line BGC-823 efficiently,and to verify the efficiency of gene silencing.Methods: the gene sequence of HIF-1α was searched in NCBI database,and online siRNA design software was used to design three siRNA sequences targeted gene CDS region and a Negative sequence.Double-stranded complementary Oligo DNA sequences for designed siRNA sequences were designed,and annealed to form double strands,and linked to linear vector to construct recombinant shRNA expression vector.293TN cells were used for shRNA lentiviral particle packaging,and the recombinant virus was used to infect BGC-823.Fluorescent marker GFP was used to determine the infection efficiency,and the cell sample was collected.Real-time PCR and Western blot were used to detect the silencing efficiency of target gene at mRNA and protein levels.Results:recombinant shRNA expression vector sequencing results are consistent with the design sequence,packaged virus titer was 1×104 ifu/μL.Infection of BGC-823 cells by lentivirus produced high gene transduction efficiency.Results of mRNA detection show that siRNA3 has the best silencing effect on target gene,and the mRNA expression was decreased by 92% compared with negative control group.Moreover,Western blotting results are completely consistent with the mRNA results.Conclusion:Lentivirus can inhibit the expression of HIF-1α gene in BGC-823 cells efficiently.