RNA干扰HIF-1α对低氧状态下食管鳞癌Eca109细胞放射敏感性的影响

Effect of HIF-1αby RNAi on radiosensitivity in esophageal squamous carcinoma cell line Eca109 under hypoxia

目的:观察低氧状态下食管鳞癌Eca109细胞中低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)及表皮生长因子受体(epidermal growth factor receptor,EGFR)的表达,探讨HIF-1α对Eca109细胞放射敏感性的影响及其可能的分子机制。方法:以常氧组为对照。氯化钴(cobalt chloride,CoCl_2)模拟肿瘤低氧微环境,用RT-PCR检测体外低氧状态下Eca109细胞中HIF-1α和EGFR mRNA表达水平,免疫细胞化学法检测HIF-1α和EGFR蛋白表达水平。Western blot检测RNAi沉默HIF-1α对EGFR蛋白表达的影响、采用流式细胞术检测HIF-1α沉默后,Eca109细胞经放射线照射后的凋亡情况,评价其放射敏感性的变化。结果:与常氧组比较,低氧状态下,Eca109细胞中HIF-1αmRNA表达无明显变化(P>0.05),HIF-1α蛋白表达增加。EGFR mRNA表达明显升高(P<0.05),EGFR蛋白表达也相应增加。与未转染组和对照组比较,siRNA转染Eca109细胞可有效沉默HIF-1α蛋白在低氧状态下的表达(P<0.05),EGFR蛋白的表达水平也明显下调(P<0.05)。Eca109细胞在低氧状态下放射敏感性较常氧下明显降低(P<0.05)。RNAi沉默HIF-1α可部分逆转低氧造成的Eca109细胞的放射耐受。结论:低氧可上调食管鳞癌Eca109细胞HIF-1α蛋白水平;HIF-1α增加Eca109细胞在低氧状态下的放射抗性,可能与HIF-1α上调EGFR mRNA及蛋白表达有关,有效抑制HIF-1α可增加Eca109细胞的放射敏感性。

OBJECTIVE：To investigate the expression of HIF-1αand EGFR in esophageal squamous carcinoma cell line Eca109 under hypoxia,in order to explore the effect of HIF-1αon radiosensitivity and its possible molecular mechanism.METHODS：CoCl_2 was used to mimic hypoxic tumor microcnvironment.mRNA levels of HIF- 1αand EGFR under hypoxia were detected by reverse transcription-polymerase chain reaction（RT-PCR）,and protein levels were measured by immunohistochemistry.The effect silenced of HIF-1αgene on EGFR expression was assayed by Western blot.Radiosensitivity was evaluated via detection of apoptosis after radiation by flow cytometer（FCM）,and then the effect of silencing HIF-1αon radiosensitivity was measured.RESULTS：Under hypoxia,HIF-1αmRNA had no significant change（P0.05）,while its protein increased obviously.EGFR mRNA was up-regulated（P0.05）,and its protein also increased accordingly.The siRNA targeting HIF-1αgene down-regulated HIF-1αin Eeal09 cells efficiently under hypoxia,and EGFR protein levels were down-regulated as well（P0.05）.The radiosensitivity in esophageal carcinoma cell line Eca109 was lower under hypoxia than that in normoxia.The siRNA targeting HIF-1αcould partially reverse radioresistance.CONCLUSION：Hypoxia could increase HIF-1αprotein expression in esophageal squamous carcinoma Eca109 cell.Resistance to radiation enhanced in Eca109 cells maybe correlated with HIF-1αup-regulating the expression of EGFR mRNA and protein levels.Radiosensitivity could be augmented if HIF-1αwas effectively suppressed.