过表达hnRNPAB基因对结直肠癌细胞上皮-间质转化的影响及其分子机制

Overexpression of hnRNPAB promotes epithelial mesenchymal transformation in colorectal cancer cells and its mechanism

目的探讨过表达hnRNPAB基因对人结直肠癌SW480、HT29细胞上皮-间质转化(EMT)的影响及其分子机制。方法分别用过表达hnRNPAB的慢病毒和阴性对照病毒转染SW480、HT29细胞,设置实验组(SW480-hnRNPAB、HT29-hnRNPAB)与对照组(SW480-NC、HT29-NC)。采用RT-qPCR和Western blotting检测hnRNPAB及EMT相关指标(E-cadherin、N-cadherin及波形蛋白)mRNA和蛋白的表达水平,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力,Western blotting检测Wnt/β-catenin信号通路关键蛋白WNT3A、WNT5A及β-catenin的表达水平。结果与对照组(SW480-NC、HT29-NC)相比,实验组(SW480-hnRNPAB、HT29-hnRNPAB)hnRNPAB mRNA和蛋白表达水平明显增高,差异有统计学意义(P<0.01)。实验组划痕愈合率明显高于对照组(SW480:19.6%±0.90%vs.2.23%±0.80%,P<0.01;HT29:40.30%±0.70%vs.6.40%±0.06%,P<0.01),穿膜细胞数明显多于对照组[SW480:(315.0±6.7)个vs.(62.0±4.3)个,P<0.01;HT29:(289.0±7.2)个vs.(54.0±5.2)个,P<0.01]。与对照组相比,实验组上皮细胞标志物E-cadherin mRNA和蛋白表达水平明显降低,间质细胞标志物N-cadherin和波形蛋白mRNA和蛋白表达水平明显升高,差异有统计学意义(P<0.01)。与对照组相比,实验组WNT3A、WNT5A和β-catenin蛋白表达水平明显增高,差异有统计学意义(P<0.01)。结论过表达hnRNPAB基因可诱导结直肠癌SW480和HT29细胞发生EMT,增强其迁移和侵袭能力,其机制可能与激活Wnt/β-catenin信号通路有关。

Objective To investigate the effect of hnRNPAB overexpression on epithelial-mesenchymal transformation(EMT)and its molecular mechanism using human colorectal cancer line SW480 and HT29.Methods SW480 and HT29 cells were infected with lentivirus overexpressing hnRNPAB and negative control virus to generate the corresponding cell lines in experimental group(SW480-hnRNPAB,HT29-hnRNPAB)and control group(SW480-NC,HT29-NC).To quantify the levels of hnRNPAB and EMT-related indicators(E-cadherin,N-cadherin and vimentin),RT-qPCR and Western blotting were used.Cell migration was measured using the scratch assay.Cell invasion was evaluated using the Transwell assay.Western blotting was used to detect the expression levels of Wnt/β-catenin signaling key indicators WNT3A,WNT5A andβ-catenin.Results Compared with control group,the expression levels of hnRNPAB mRNA and protein in experimental group significantly increased,and the differences were statistically significant(P<0.01).The scratch healing rates in experimental group were significantly higher than that in control group(SW480:19.6%±0.90%vs.2.23%±0.80%,P<0.01;HT29:40.30%±0.70%vs.6.40%±0.06%,P<0.01).The number of Transwell cells was significantly higher than that in control group[SW480:(315.0±6.7)cells vs.(62.0±4.3)cells,P<0.01;HT29:(289.0±7.2)cells vs.(54.0±5.2)cells,P<0.01].Compared with control group,the epithelial cell marker E-cadherin mRNA and protein expression levels in experimental group significantly decreased.As expected,the mesenchymal cell markers N-cadherin and vimentin mRNA and protein levels significantly increased,and the differences were statistically significant(P<0.01).Compared with control group,the expressions of WNT3A,WNT5A andβ-catenin protein in experimental group significantly increased(P<0.01).Conclusions The overexpression of hnRNPAB could induce EMT of human colorectal cancer SW480 and HT29,demonstrated by enhanced cell migration and invasion ability.The mechanism may be related to the activation of the Wnt/β-catenin signaling pathway.