HIF-1α对食管癌Eca109细胞体内外侵袭转移的影响及其分子机制

Effect of HIF-1α and its mechanism on invasion and metastasis of esophageal carcinoma Eca109 cells in vitro and in vivo

目的:采用RNA干扰技术(RNAi)观察低氧诱导因子-1α(HIF-1α)对食管癌Eca109细胞及其裸鼠移植瘤侵袭转移的影响及其作用机制。方法:CoCl2模拟肿瘤低氧微环境,分别用RT-PCR和免疫细胞化学法检测体外低氧条件下Eca109细胞中HIF-1α、E-钙黏蛋白及基质金属蛋白酶-2(MMP-2)mRNA及蛋白的表达。RT-PCR检测RNAi沉默HIF-1α对E-钙黏蛋白及MMP-2 mRNA表达的影响。通过细胞划痕试验和Transwell试验检测RNAi对Eca109细胞侵袭和转移能力的影响。建立Eca109细胞裸鼠移植瘤模型,观察HIF-1α对肿瘤生长及淋巴结转移的影响;采用Western blot检测各组移植瘤中HIF-1α、E-钙黏蛋白及MMP-2的表达,分析HIF-1α在离体和在体对肿瘤生长、侵袭及转移的影响。结果:低氧诱导Eca109细胞的HIF-1α蛋白表达升高,E-钙黏蛋白的mRNA及蛋白表达降低和MMP-2 mRNA及蛋白表达升高,但对HIF-1αmRNA无明显影响。用RNAi能有效沉默HIF-1α,并可逆转低氧导致的E-钙黏蛋白及MMP-2的降低及升高,使Eca109细胞迁移速度减慢,侵袭穿膜细胞数减少(P<0.05);在体内使移植瘤生长减缓,淋巴结转移率降低(P<0.05)。结论:HIF-1α在体内外通过影响E-钙黏蛋白和MMP-2表达,来影响食管癌Eca109细胞的生长、侵袭和转移。

AIM： To investigate the effects of H IF-1α by RNAi on invasion and metastasis of esophageal carcinoma Ecal09 cells in vitro and in vivo, in order to explore its prob- able mechanism. METHODS： CoCl2 was used to mimic tumor hypoxic microenvironment, mRNA and protein levels of HIF-1α, E-cadherin and MMP-2 under hypoxia were detected by RT-PCR and immunohistochemistry. The effects of silencing HIF-1α by RNAi on HIF-la, E-cadherin and MMP-2 mRNA were detected by RT-PCR. The effect of RNAi on invasion and metastasis were tested by cell scratch assay and transwell chambers. The EcalO9-implanted nude mouse model was established, and the effects of HIF-1α on tumor growth and lymphoid node metastasis were observed. The expressions of HIF-1α, E-cadherin and MMP-2 in transplanted tumors were detected by Western blot, and the effects of HIF-1α on tumor growth, invasion and metastasis in vitro and in vivo were analyzed. RESULTS： Hypoxia up-regulated HIF-1α protein, mRNA and protein levels of Ecadherin down-regulated, and MMP-2 up-regulated, while had no effect on HIF-1α mRNA. RNAi could silencing HIF- 1α effectively, and inhibited E-cadherin or MMP-2 decreased or increased, respectively. The migration and the number of invading cells decreased （ P 〈 0.05） after silencing HIF-1α by RNAi. The tumor volume was much smaller, lymph node metastasis rate lower as well in vivo （P 〈 0.05）. CONCLUSION： Via its effects on E-cadherin and MMP-2, HIF-1α regulate the growth, invasion and metastasis of Ecal09 cells in vitro and in vivo.