Effect Of RNA Interference Targeting HIF-1α Gene On Biological Behavior Of Human Esophageal Squamous Cancer Cells And Gastric Cancer Cells In Vitro

Backgroud:Esophageal squmaous cell carcinoma (ESCC) and gastric cancer are commonly seen in China, with high mortality and fatality in the world. As a key regulator to adapt hypoxia environment, hypoxia-inducible factor-1 alpha (HIF-1α) is involved in ESCC and gastric cancer. HIF-1αplays an important role in tumor angiogenesis, invasion and metastasis. Vasculogenic mimicry (VM) is a capillary structure formed by aggressive tumor cells instead of endothelial cells. It is a novel pathway to generate tumor microcirculation. VM is closely correlated with tumor invasion and metastasis and prognosis of patients. It brings bad effect on the traditional antiangiogenic therapy of tumors. Overexpression of HIF-1αgene may be related to the VM formation.Objectives:1. To observe whether Eca-109, TE13 and SGC-7901 cell lines could form the VM structures or not.2. To investigate the inhibitory effect of siRNA targeting HIF-1αon proliferation, migration and VM formation of ESCC and gastric cancer cells in vitro.Methods:1. The recombinant plasmid pGCsi-shHIF-1αwas transfected into human gastric cancer cells SGC-7901. 2. Eca-109, Eca-109/Neo, Eca-109/shRNA, TE13, TE13/Neo, TE13/shRNA, SGC- 7901, SGC-7901/Neo and SGC-7901/shRNA cells were incubated under normoxic and hypoxic conditions for 12h. Then the inhibitory effect of HIF-1αwas measured on protein level by Western blot under normoxia and hypoxia.3. Each group cells were cultured under normoxia. The cell proliferation was detected by colony formation assay and MTT assay.4. Migrations were measured by transwell chamber. 1×105 cells of each group were seeded into the upper chamber with 100μl FBS free DMEM, and 600μl 10%FBS DMEM was added to the base well. After incubation for 24h, the migrated cells were counted under a light microscope.5. 300μl matrigel was added into every well of 24-well plate. After the matrigel had solidified, each group cells were seeded into the 24-well plate by 5×105 per well, then incubate for 12 hours. Observe and count the number of capillary like structures of each group cells.Results:1. The expression of HIF-1αprotein in each group of recombinant plasmid transfected cells was suppressed significantly under normoxia and hypoxia (P<0.01).2. Compared with the control groups, colony formation of the recombinant plasmid transfected cells was greatly decreased (P<0.05), the cell proliferation of the transfected cells was inhibited significantly (P<0.05).3. The migration of the recombinant plasmid transfected cells was significantly suppressed under normoxia and hypoxia (P<0.01). The number of migrated cells of each untreated group was incresed greatly under hypoxia (P<0.05).4. Eca-109, TE13 and SGC-7901 cells could all form capillary tube like structures in vitro, and the number of tubules was obviously increased in hypoxia (P<0.05). The tubule-forming ability of transfected groups was significantly inhibited in normoxia and hypoxia(P<0.01).Conclusions:1. The recombinant plasmid pGCsi-shHIF-1αcan suppress the protein expression of HIF-1αin Eca-109, TE13 and SGC-7901 cells.2. RNA interference targeting HIF-1αcan significantly inhibite the colony formation and cell proliferation of the cell lines Eca-109, TE13 and SGC-7901.3. Hypoxia can enhance the migration of Eca-109, TE13 and SGC-7901 cells. Silenceing HIF-1αgene can inhibit the migration of tumor cells efficiently.4. Esophageal squamous cancer cell lines Eca-109, TE13 and gastric cancer cell line SGC-7901 are all capable of forming VM structures in vitro. Silenceing HIF-1αgene can suppress their vasculogenic mimicry formation efficiently.