[Construction of miRNA expression vector dual-targeting on HIF-1α/survivin genes and its effects on proliferation of pancreatic cancer Panc-1 cell line].

OBJECTIVE: To design and construct miRNA expression vector dual-targeting on HIF-1α and survivin genes and to investigate its effects on proliferation of human pancreatic cancer cells. METHODS: The specific pre-miRNA single strand DNA oligos for HIF-1 α and survivin genes were designed and synthesized, then via annealing and ligating with pcDNA6.2-GW/EmGFP-miR plasmids in order, two kinds (eight in total) of miRNA expression vectors for HIF-1α and survivin genes were constructed. The vectors, which were most effective to knockdown target genes, were screened with real-time RT-PCR and combined by chaining technology to construct dual-targeting plasmid. The recombined dual-targeting plasmid, mono-targeting plasmids and negative plasmid were transfected into Panc-1 cells, the suppression effect on two genes was identified by real-time RT-PCR, Western blot and MTT assays. RESULTS: The miRNA expression plasmids anti-H, anti-S and anti-H+S were successfully constructed by identification of sequencing analysis, and they were able to effectively inhibit the target genes expressing. MTT assays showed that the inhibition effect of dual-targeting vector anti-H+S was higher than that of mono-targeting vectors anti-H and anti-S 72 h after transfection (P<0.05). CONCLUSION: The effective miRNA expression vector dual-targeting on HIF-1α and survivin genes has been successfully constructed. The inhibition effect on proliferation of pancreatic cancer Panc-1 cells by dual-targeting plasmid was higher than that by mono-target plasmids.