肝再生磷酸酶-3上调缺氧诱导因子-1,α亚基促进结肠癌细胞血管生成的研究

Liver regeneration phosphatase 3 promotes angiogenesis in colorectal cancer cells via upregulating hypoxia inducible factor 1-α

目的观察结肠癌细胞中肝再生磷酸酶-3(PRL-3)上调缺氧诱导因子-1(HIF1)-α的表达。方法构建稳定转染的上调PRL-3(LoVo-P/LoVo-NC)及下调PRL-3的结肠癌细胞株(HT29-P/HT29-NC),通过蛋白质印迹法(Western blot)及定量聚合酶链反应(qPCR)检测结肠癌细胞中HIF1-α及血管内皮生长因子(VEGF)的表达。取各株细胞上清液,与人脐静脉内皮细胞(HUVECs)行血管生成试验。将细胞注入小鼠皮下行小鼠成瘤实验后,瘤体切片行免疫组织化学(IHC)观察VEGF及HIF1-α表达差异。应用SPSS 15.0统计软件进行分析。结果Western blot及qPCR结果显示PRL-3上调后结肠癌细胞中的HIF1-α[LoVo-P/LoVo-NC:4]及VEGF[LoVo-P/LoVo-NC:5.8]表达升高(P<0.05),而敲低PRL-3后HIF1-α[HT29-P/HT29-NC:0.3]及VEGF[HT29-P/HT29-NC:0.2]亦随之表达降低(P<0.05)。血管生成试验提示,PRL-3水平高的细胞培养上清有更好的促血管生成作用[血管生成数为LoVo-P/LoVo-NC:(8±2)条比(2±1)条,HT29-P/HT29-NC:(3±1)条比(11±3)条,(P<0.05]。免疫组化显示结肠癌细胞LoVo-P及HT29-NC分别相对于LoVo-NC及HT29-P在HIF1-α、VEGF的表达上都有明显升高[LoVo-P/LoVo-NC,HIF1-α:89%比33%,VEGF:83%比25%。HT29-P/HT29-NC,HIF1-α:8%比67%,VEGF:12%比82%,t=9.934、17.890、19.190、106.000、P<0.05]。结论结肠癌细胞中PRL-3上调HIF1-α的表达从而促进血管生成。

Objective To investigate whether the liver regeneration phosphatase 3(PRL-3)upregulates the expression of hypoxia inducible factor 1-α(HIF1-α),promoting angiogenesis.Methods LoVo cells were selected for constructing PRL-3-overexpression lines,and HT29 cells were chosen for the knockdown groups.We performed qPCR and Western blotting to identify the expression of HIF1-αand VEGF in cell lines mentioned above compared with their negative control group respectively.Besides,their supernatants were collected and used to carry out angiogenesis assay in human umbilical vein endothelial cells(HUVECs).In addition,immunohistochemistry was used to confirm the status of HIF1-αand VEGF in xenografts of mice injected with cells mentioned above.Statisticalanalysis of data using the statistical product and service solutions 15.0 software.Results Western blotting and qPCR showed that overexpression of PRL-3 corelated with high levels of HIF1-α[LoVo-P vs.LoVo-NC:4]and VEGF[LoVo-P vs.LoVo-NC:5.8]in colorectal cancer cells.Similarly,knocking down PRL-3 showed the opposite trend[HT29-P vs.HT29-NC,HIF1-α:0.3,VEGF:0.2,P<0.05].Also,the angiogenesis assay presented that PRL-3 promoted the formation of new vessels[LoVo-P/LoVo-NC:8±2 vs.2±1,HT29-P vs.HT29-NC:3±1 vs.11±3(P<0.05)].Results of immunohistochemistry were consistent with those in Western blotting and qPCR[LoVo-P vs.LoVo-NC,HIF1-α:89%vs.33%,VEGF:83%vs.25%,HT29-P/HT29-NC,HIF1-α:8%vs.67%,VEGF:12%vs.82%,P<0.05].Conclusion PRL-3 in colorectal cancer cells promotes angiogenesis by increasing the expression of HIF1-α.