基于miRNA-132信号通路探究胡椒碱对肝癌血管形成及肿瘤抑制作用

Piperine inhibits angiogenesis and tumor growth in hepatocellular carcinoma through miRNA-132 signaling pathway

目的:基于miRNA-132信号通路探究胡椒碱对肝癌血管形成及肿瘤抑制作用。方法:制备肝癌细胞转移瘤,将裸鼠分为空白组(不制备肝癌裸鼠模型)、模型组(肝癌小鼠模型组)、胡椒碱组(肝癌小鼠模型给予胡椒碱干预组)、miR-132组(肝癌小鼠模型给予miR-132类似物组)、联合组(肝癌小鼠模型给予胡椒碱联合miR-132类似物组)。比较各组裸鼠的瘤体积和肿瘤抑制率;比较肿瘤组织病理学变化;用ELISA检测血清中血管内皮生长因子(VEGF)含量;比较微血管密度(MVD)数量;用蛋白质印迹法检测肝组织中miR-132、GP73表达。结果:和模型组相比,miR-132组、胡椒碱组和联合组裸鼠的瘤体积均显著降低,且联合组瘤体积较胡椒碱组和miR-132组降低更明显(P<0.05)。和模型组相比,miR-132组、胡椒碱组和联合组裸鼠肿瘤生长抑制率显著增加(P<0.05);联合组裸鼠的肿瘤生长抑制率明显高于miR-132组和胡椒碱组(P<0.05)。模型组裸鼠血清中VEGF含量和MVD数量和空白组相比显著增加(P<0.05);和模型组相比,miR-132组、胡椒碱组和联合组裸鼠血清中VEGF含量和MVD数量均显著减少,且联合组血清中VEGF含量和MVD显著低于胡椒碱组(P<0.05)。和空白组相比,模型组裸鼠肝组织中miR-132表达显著降低,GP73蛋白表达显著增加(P<0.05);和模型组相比,miR-132组、胡椒碱组和联合组裸鼠肝组织中miR-132表达均明显增加,GP73蛋白表达均显著降低,且联合组中miR-132和GP73表达变化最大(P<0.05)。双荧光素酶检测报告结果显示,过表达miR-132可降低GP73活性(P<0.05)。结论:胡椒碱可通过调控miR-132负向调控GP73抑制肝癌血管形成,同时也能显著抑制肿瘤的生长。

Objective:To explore the inhibitory effect of piperine on hepatocellular carcinoma angiogenesis and tumor based on miRNA-132 signaling pathway.Methods:Liver cancer cell metastases were prepared and nude mice were divided into blank group(no liver cancer model was prepared),model group(liver cancer mouse model group),piperine group(liver cancer mouse model treated with piperine intervention group),miR-132 group(liver cancer mouse model treated with miR-132 analogue group)and combination group(liver cancer mouse model treated with piperine combined with miR-132 analogue group).The tumor volume,inhibition rate of tumor and pathological changes of tumor tissues were compared.Serum vascular endothelial growth factor(VEGF)was detected by ELISA.The number of microvascular density(MVD)was compared.Using western blot method in the detection of liver tissue miR-132,GP73 expression.Results:Compared with model group,the tumor volume in miR-132 group,piperine group and combined group was significantly decreased,and the tumor volume in combined group was higher than that in piperine group and miR-132 group(P<0.05).Compared with model group,tumor growth inhibition rate of miR-132 group,piperine group and combined group was significantly increased(P<0.05).The tumor growth inhibition rate in combined group was significantly higher than that in miR-132 group and piperine group(P<0.05).The serum VEGF content and MVD quantity in model group were significantly increased compared with blank group(P<0.05).Compared with the model group,the levels of VEGF and MVD in serum of the miR-132,piperine and combination groups were significantly decreased,and the levels of VEGF and MVD in serum of the combination group were significantly lower than those of the piperine group(P<0.05).Compared with blank group,the expression of miR-132 was significantly decreased and GP73 protein was significantly increased in model group(P<0.05).Compared with the model group,the expression of miR-132 in the liver tissue of nude mice in the miR-132 group,piperine group and the combined group was significantly increased,and the expression of GP73 protein was significantly decreased.The changes in the expressions of miR-132 and GP73 were the most significant in the combined group(P<0.05).Dual-luciferase reporter assay showed that over-expression of miR-132 reduced the activity of GP73(P<0.05).Conclusion:Piperine can negatively regulate GP73 by regulating miR-132 and inhibit the angiogenesis of hepatocellular carcinoma,which also significantly inhibit tumor growth.