Inhibitory Effect Of SiRNA Targeting HIF-1α On Esophageal Squamous Cancer Cells Eca-109 In Vitro And In Vivo

Background:Esophageal cancer is one of the diseases threatening to people's health. The mortality of esophageal cancer in China is top two, which has poor therapeutic effect and prognosis. In China, Esophageal Squamous Cell Cancers (ESCC) account for approximately 95% in all esophageal cancer. Transcription factor hypoxia inducible factor-1 alpha (HIF-let) is a key determinant of oxygen-dependent gene regulation in angiogenesis. Suppression of HIF-1αis important for exploring HIF-1-dependent processes and hypoxia-induced pathophysiologic events.Objective:To investigate the inhibitory effect of siRNA targeting HIF-1αon ESCC in vitro and in vivo. Methods:1. At different time (0, 12, 24, 48, 72h), using different density of cobalt chloride (0, 200, 400, 600, 800μM) to mimic hypoxia condition, Esophageal Squamous Cancer Cells Eca-109 were routine cultured. The expressions of HIF-1αprotein were detected by Western blot, and we selected a properly cobalt chloride density and culture time as the most suitable culture condition.2. Four groups of cells were cultured, groupⅠwas ESCC Eca-109, groupⅡto groupⅣwere constructed cloned ESCC strain with silenced HIF-1αgene by RNA interference (H2/14, H3/15, H2/20 cells, named and preserved by our laboratory), using 400μM cobalt chloride for 24h, Then, the transfection efficiency of siRNA- HIF-1αwas measured on mRNA and protein level by RT-PCR, Western blot. We also detected HIF-1 control target genes as HO-1, MMP-2, GLUT-1, p53 genes expression by Western blot at the same time.3. Six-week-old BALB/c nude mice were divided into three groups (n=10 per group). Group A were injected subcutaneously with Eca-109 cells, group B were injected Eca-109 cells transfected with empty expression vector(Eca-109/Neo), and group C were injected Eca-109/shRNA cells (stably transfected with small hairpin RNA eukaryotic expression vector targeting HIF-1αgene). Tumor growth and volume were measured, tumor volume = (lenth×width~2)/2. All nude mice were killed after 28 days, weighed the tumors, drew the tumors growth curves.4. The expression of HIF-1α, VEGF, and bcl-2, survivin (apoptosis inhibit proteins) was determined by Western blot. The expression of bcl-2, bax, surviving, p21 genes, which were related to apoptosis in tumors were analyzed by immunocytochemistry, and the relationship of HIF-1αgene and apoptosis were explored.5. The Eca-109 and Eca-109/shRNA cells in 400μM cobalt chloride were cultured for 48h, and the cell cycle was detected by Flowcytometry. The apoptosis rate was assessed by Annexin V-PE/7-AAD double staining.Results:1. HIF-1αprotein expression in Eca-109 cellsThe HIF-1αprotein expression in Eca-109 cells increased accoding to cobalt chloride density, and HIF-1αprotein expression raised obviously after hypoxia for 12h, reduced after hypoxia for 24h, gradually decreased after hypoxia for 48～72h. We selected 400μM cobalt chloride density and culture for 24h as subsequent experimental condition.2. HIF-1αprotein and related genes expression after HIF-1αsilenced by RNA interferenceFour groups cells were assessed after in normoxia or in 400μM cobalt chloride for 24h, H3/15 cells expressed the minimum HIF-1αprotein than the others, with significant differences (P<0.01). Eca-109/shRNA cells expressed less HO-1, GLUT-1, MMP-2, p53 protein than Eca-109 cells in normoxia, and HO-1, p53 protein were up-regulated after in 400μM cobalt chloride for 24h, while MMP-2, GLUT-1 protein had no obvius change. Aggregate analysis, H3/15 cells has the best effeciency of RNA interference targeting HIF-1α.3. HIF-1αmRNA expression after HIF-1αsilenced by RNA interferenceFour groups cells were assessed after in norrnoxia or in 400μM cobalt chloride for 24h, H3/15 cells expressed less HIF-1αmRNA than the others, but there were no significance (P＞0.05)4. Effect of HIF-1αsilenced by RNA interference on nude mice tumor's growthXenograft tumors were observed after cells inoculation about 1 week in group A and B, while about 2 weeks in group C. Xenograft tumors in group C grew delay and slow. Compared the volumes and weights with two control after 28 days, group C had statistical significance (P＜0.01). The inhibitory rate reached to 70%.5. The expressions of HIF-1α, VEGF and apoptosis related genes in xenograft tumorsThe expressions of HIF-1α, VEGF, bcl-2, survivin proteins were down-regulated significantly in group C than in controls (P＜0.01).The results of immunocytochemistry showed that less bcl-2, survivin, p21 and more bax were expressed in group C tumors than in control groups.6. Cell cycle and apoptosis rate assessed by FlowcytometryAfter 48 hours of exposure to hypoxia, Eca-109/shRNA cells cycle were arrested in G2 stage. Annexin V double staining assay showed apoptosis rate in the Eca-109/shRNA cells was 21.32 4-1.12% after exposure to hypoxia, significantly higher than in those 2 control groups (both P＜0.01).Conclusions:1. HIF-1αprotein expression is related to hypoxia degree mimicked by cobalt chloride and related to exposure time. HIF-1αmRNA expression had no significant difference in all groups. Cobalt chloride induced HIF-1αexpression is at protein level, no transcription level.2. Screened out H3/15 cells after normoxia or hypoxia, which shows the best effeciency of RNA interference. HIF-la protein expression in H3/15 cells increased by hypoxia degrees, the result indicated that RNA interference was not similar to knockout the gene.3. HO-1, MMP-1, GLUT-1 are HIF-1 targeting genes. HO-1, MMP -2, GLUT-1 were down-regulated obviously when ShRNA interferencing HIF-1αgene. The result indicated that inhibiting HIF-1αcould suppress angiogenesis, shutdown glycolysis, inhibit invasion and transfusion, and delay tumor growth.4. Xenograft tumors injected with SiRNA Eca-109 grew slowly. The mechanism maybe suppresses angiogenesis and energy support, restrain cell cycle, and promote cell apoptosis.