Effect Of RNA Interference-based Silencing Of HIF-1 Gene On The Expression Of Glycolysis Associated Genes In Esophageal Squamous Carcinoma Cells

Backgroud:Esophageal squmaous cell carcinoma (ESCC) is commonly seen in China, with highincidence and mortality in the world. The dependency of cancer cells on glycolyticpathway for ATP generation is metabolic basis for hypoxic tolerance,also the causefor the resistance to radiation and chemotherapy. Hypoxia-inducible factor-1(HIF-1)is a transcription factor which present in most of mammal and human tissue in thehypoxia condition. As a key regulator to adapt tumor microcirculation, HIF-1αis overexpressed in ESCC, significantly correlated with depth of tumor invasion, tumorlymph node metastasis, organ metastasis and TNM stage. HIF-1αgene may play animportant role in tumor glycolysis, and it has become the hot point of the researchabout tumorous aetiology and treatment.ObjetivesTo investigate the inhibitory effect of RNA interference targeting HIF-1αand theeffect of RNA interference-based silencing of HIF-1αgene on the expression ofglycolysis associated genes in esophageal squamous carcinoma cells (Eca-109)cultured in either a normoxia or a hypoxia environment, and to explore the relationbetween HIF-1αand tumor glycolysis. MethodMethods1．Esophageal squamous cancer Eca109 cells were incubated under normoxic andhypoxic conditions for different time ( 6,12,24,48). Expression of HIF-1αprotein wasdetected by Western blot, thus the most suitable hypoxic incubated time was selected.2. Eca-109, Eca-109/Neo, Eca-109/shRNA cells were incubated under normoxic andhypoxic conditions for 12h. Then the inhibitory effect of HIF-1αwas measured onprotein level by Western blot.3.Eca-109, Eca-109/Neo, Eca-109/shRNA cells were incubated under normoxic andhypoxic conditions for 12h. Then the expression of glycolysis associated genes(Glut-1,LDH-A,HK-II) was measured on mRNA level by RT-PCR.4. Eca-109, Eca-109/Neo, Eca-109/shRNA cells were incubated under normoxic andhypoxic conditions for 12h. Then the expression of glycolysis associated genes(Glut-1,LDH-A,HK-II) was measured on protein level by Western blot.5. Eca-109, Eca-109/Neo, Eca-109/shRNA cells were incubated under normoxic andhypoxic conditions for 12h. Then spectrophotometry were employed to determine theactivities of the glycolysis enzymes (LDH,HK).6. Eca-109, Eca-109/Neo, Eca-109/shRNA cells were incubated under normoxic andhypoxic conditions for 12h. Then spectrophotometry were employed to determin thecontent of lactic acid (LA) in the culture supernatant.Results1. The expression of HIF-1αincreasd after incubated in hypoxia environment,reaching its peak at 12h, Thus,12h was selected as subsequent hypoxic incubationtime.2. The expression of HIF-1αwas hardly detected in HIF-1αsilenced cells, and couldnot be upregulated in a hypoxia environment.3. The expression of Glut-1,LDH-A,HK-II mRNA was significantly down-regulatedin HIF-1αsilenced cells(P<0.05), and up-regulated in Eca-109, Eca-109/Neo cells under hypoxia, with significant differences(P<0.05).4. The expression of Glut-1,LDH-A,HK-II proteins was significantly down-regulatedin HIF-1αsilenced cells(P<0.05), and up-regulated in Eca-109, Eca-109/Neo cellsunder hypoxia, with significant differences(P<0.05).5. The HK and LDH activities were decreased greatly in HIF-1αsilencedcells(P<0.05), and increased slightly in Eca-109, Eca-109/Neo cells when hypoxia,with no significance(P>0.05).6. The lactic acid content in the culture supernatant was obviously decreased in HIF-1αsilenced cells(P<0.05), and increased slightly in Eca-109, Eca-109/Neo cells whenhypoxia, with significant differences(P<0.05).ConclusionConclusions1. HIF-1αprotein expression is related to exposure time under hypoxia condition,reaching its peak at 12h.2. RNA interference targeting the HIF-1αgene is able to efficiently silence theexpression of HIF-1αgene in human esophageal squamous carcinoma cells (Eca-109).3. The expession of Glut-1,LDH-A,HK-II were down-regulated obviously whenShRNA interferencing HIF-1αgene. Glut-1,LDH-A,HK-II are HIF-1 targetinggenes.4. Silenceing HIF-1αgene can inhibit the expression of glycolysis associated genesand the content of lactic acid in the culture supernatant. Either in normoxia or hypoxia,the expression of HIF-1αwas closely related to tumor glycolysis.