The Effect Of RNAi Targeting HIF-1α Gene On The Expression Of Vasculogenic Mimicry Associated Genes In Esophageal Squamous Cancer Cells

Objective:To investigate the effect of siRNA targeting HIF-1αgene on the expression of vasculogenic mimicry associated genes in esophageal squamous cancer cells.Methods:1.Esophageal Squamous Cancer Cells Eca-109 were routine cultured under hypoxia condition.And for the culture under hypoxia condition we arranged different time(0,12,24,36,48,60,72,96h).The expression of HIF-1αprotein was detected by Western blotting,and we selected a proper culture time as the most suitable culture condition.2.Four groups of cells were cultured under hypoxia condition for 24h.GroupⅠwas ESCC Eca-109,and groupⅡto groupⅣwere constructed cloned ESCC strain with silenced HIF-1αgene by RNA interference(H2/20,H3/10,H3/15 cells,named and preserved by our laboratory).Then,the transfection efficiency of siRNA-HIF-1αwas measured on mRNA and protein level by RT-PCR and Western blotting. We also detected the expression of vasculogenic mimicry associated genes as VE-cadherin,LN5γ2,EphA2 and MMP-2 by Western blotting at the same time.Results:1.The expression of HIF-1αmRNA and protein in Eca-109 cellsWhen Eca-109 cells were cultured under normoxia and hypoxia condition,there was no significance in the expression of HIF-1αmRNA (P＞0.05);but the protein expression increased after hypoxia,and HIF-1αprotein expression rose obviously after hypoxia for 24～48h, reduced after hypoxia for 60h,gradually decreased after hypoxia for 72～96h.Eca-109 cell and the other three HIF-1αsilenced cells were assessed after cultured under normoxia and hypoxia condition for 24h, and there was no significance among them in the mRNA expression(P＞0.05);HIF-1αprotein expression gradually decreased in HIF-1αsilenced Eca-109 cells,and did not rise after hypoxia.We selected 24h under hypoxia condition as subsequent experimental condition.2.The expression of vasculogenic mimicry associated genesFour groups of cells were assessed after cultured under normoxia and hypoxia condition for 24h,protein expression of LN5γ2 and EphA2 gradually decreased in HIF-1αsilenced Eca-109 cells,and did not rise after hypoxia.Eca-109/shRNA cells expressed less VE-cadherin and MMP-2 protein than Eca-109 cells in normoxia,and the protein expression slightly rose when cultured hypoxia for 24h,but there was no significance(P＞0.05).Conclusions:1.HIF-1αprotein expression is related to exposure time under hypoxia condition.There was no significant difference in HIF-1αmRNA expression in all groups.Hypoxia induced HIF-1αexpression happened at protein level,not transcription level.2.All the three RNAi cells have good interference effect,and the HIF-1αprotein expression was obviously inhibited after RNA interference,and did not rise when hypoxia.As a special gene silencing technique,RNAi has better stability and inhibitory effect.3.VE-cadherin,LN5γ2,EphA2 and MMP-2 are intimate to the formation of VM.The protein expression of LN5γ2 and EphA2 gradually decreased in HIF-1αsilenced Eca-109 cells,and did not rise after hypoxia. Eca-109/shRNA cells expressed less VE-cadherin and MMP-2 protein than Eca-109 cells in normoxia,and the protein expression slightly rose when cultured hypoxia for 24h,but there was no significance.The result indicated that HIF-1αsilencing may block the formation of VM by down regulated the expression of LN5γ2 and EphA2.