缺氧诱导因子-1α介导和调控肝癌相关血管生成因子的表达

Hypoxia-inducible factor-1αmediates and regulates angiogenesis-related factors expression in hepatocellular carcinoma

目的探讨肝病进展过程中缺氧诱导因子(HIF)-1α、血管内皮生长因子(VEGF)和血管生成素-2(Ang-2)的表达及HIF-1α调控肝细胞癌(HCC)血管生成因子表达的分子机制。方法收集住院肝癌、肝硬化、慢性肝炎患者血清标本,以健康人群为对照;以鼠肝癌发生模型检测肝细胞癌变过程中HIF-1α、VEGF和Ang-2动态表达情况;以酶联免疫吸附法定量分析肝病患者及鼠血清HIF-1α、VEGF和Ang-2水平。构建HIF-1α特异miRNA表达质粒转染人肝癌HepG2细胞,干预HIF-1αmRNA转录,分析其对人肝癌细胞生物学行为、VEGF和Ang-2表达以及对上皮间充质转换(EMT)的影响。多组样本均数间比较用方差分析,q检验行两两比较;以χ2检验比较样本率。结果慢性肝病患者血清HIF-1α、VEGF和Ang-2表达情况,肝癌组分别为(145.6±32.6)μg/L、(458.9±125.3)μg/L和(42.9±5.1)μg/L,均显著高于(P<0.001)肝硬化组的(79.5±28.4)μg/L、(206.8±56.8)μg/L和(26.2±6.1)μg/L及慢性肝炎组的(60.1±18.8)μg/L、(178.1±85.4)μg/L和(21.8±6.9)μg/L,且HIF-1α和VEGF(r=0.937,P<0.001)、HIF-1α和Ang-2(r=0.933,P<0.001)及VEGF和Ang-2(r=0.910,P<0.001)呈显著正相关。鼠肝细胞癌变模型证实,从正常肝细胞、肝细胞变性、癌前病变至癌变的恶性转化过程中,HIF-1α、VEGF和Ang-2呈进行性增高表达。以HIF-1αmiRNA干预质粒转化HepG2细胞,在72 h与空白组比较:HIF-1αmRNA、HIF-1α、VEGF和Ang-2分别下降88.1%、59.8%、54.0%和36.0%;EMT相关蛋白Snail(0.26±0.02与0.67±0.09,q=6.75,P<0.003)、波形蛋白(0.27±0.08与0.73±0.04,q=10.35,P<0.001)和Twist(0.24±0.07与0.73±0.02,q=12.08,P<0.001)表达水平均显著降低,但E-钙黏素(0.76±0.08与0.27±0.09,q=7.05,P<0.002)表达水平显著升高。结论HIF-1α介导和调控肝癌相关血管生成因子VEGF和Ang-2的表达,干预HIF-1α转录可显著影响肝癌细胞的生物学特征和EMT转化。

Objective To investigate the expression of hypoxia-inducible factor(HIF)-1α,vascular endothelial growth factor(VEGF)and angiopoietin-2(Ang-2)during the progression of liver diseases and the molecular mechanism of HIF-1α in regulating the expression of angiogenic factors in hepatocellular carcinoma(HCC).Methods Serum samples from hospitalized patients with liver cancer,liver cirrhosis,and chronic hepatitis were collected,and healthy people were used as controls.Mouse models of hepatocarcinogenesis were used to detect the dynamic expression of HIF-1α,VEGF and Ang-2.Enzyme-linked immunosorbent assay was used to quantitatively analyze the serum levels of HIF-1α,VEGF and Ang-2 in patients with liver disease and mice.HIF-1α-specific miRNA expression plasmids was constructed to transfect HepG2 human HCC cells.HIF-1α mRNA transcriptional interference effects were analyzed on biological behavior,VEGF and Ang-2 expression,and epithelial mesenchymal transformation(EMT)in human HCC cell line.The sample means of multiple groups were compared by analysis of variance and q test and the sample rate was compared by χ^2 test.Results In patients with chronic liver disease,the serum expression of HIF-1α,VEGF and Ang-2 in the liver cancer group(145.6±32.6)μg/L,(458.9±125.3)μg/L and(42.9±5.1)μg/L was significantly higher than the liver cirrhosis(P<0.001)(79.5±28.4)μg/L,(206.8±56.8)μg/L and(26.2±6.1)μg/L and chronic hepatitis group(60.1±18.8)μg/L,(178.1±85.4)μg/L and(21.8±6.9)μg/L.In addition,HIF-1α was positively correlated with VEGF(r=0.937,P<0.001),HIF-1α and Ang-2(r=0.933,P<0.001),and VEGF and Ang-2(r=0.910,P<0.001).Mouse models of hepatocarcinogenesis confirmed that HIF-1α,VEGF and Ang-2 had progressively increased during the process of malignant transformation from normal hepatocytes,hepatocyte degeneration,and precancerous lesions to canceration.HIF-1α miRNA intervention plasmid had transformed HepG2 cells.Compared with the blank group,HIF-1αmRNA,HIF-1α,VEGF and Ang-2 were decreased by 88.1%,59.8%,54.0% and 36.0%at 72h,respectively.The expression level of EMT-related protein Snail(0.26±0.02 and 0.67±0.09,q=6.75,P<0.003),VIM(0.27±0.08 and 0.73±0.04,t=10.35,P<0.001)and Twist(0.24±0.07 and 0.73±0.02,q=12.08,P<0.001)was significantly reduced,but the expression level of E-cadherin(0.76±0.08 and 0.27±0.09,q=7.05,P<0.002)was significantly increased.Conclusion HIF-1α mediates and regulates angiogenesis-related factors such as VEGF and Ang-2 expression in hepatocellular carcinoma.Furthermore,HIF-1αtranscriptional interference can significantly affect the biological characteristics and EMT transformation of hepatocellular carcinoma cells.