多形性腺瘤基因样蛋白2对前列腺癌细胞增殖、转移和上皮-间充质转化的影响

Effect of pleomorphic adenoma gene like-2 on proliferation,metastasis and epithelial-mesenchymal transition of prostate cancer cells

目的观察多形性腺瘤基因样蛋白2(PLAGL2)在前列腺癌细胞中的表达水平及其生物学功能。方法采用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)技术检测正常前列腺上皮细胞和前列腺癌细胞中PLAGL2 mRNA和蛋白的相对表达水平。在前列腺癌PC-3细胞中转染si-PLAGL2, 将细胞分为对照组和转染组, 采用RT-qPCR和Western blot验证转染效率。分别采用细胞克隆形成实验和细胞计数试剂盒(CCK-8)实验检测细胞增殖能力;采用Transwell实验、细胞划痕试验检测细胞迁移和侵袭能力;采用Western blot检测磷酸化蛋白激酶B(p-Akt)、N-钙黏蛋白(N-cadherin)、E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)相对表达水平;结果应用SPSS 26.0统计软件分析。结果 RT-qPCR和Western blot结果表明DU-145和PC-3细胞中PLAGL2相对表达水平高于正常前列腺上皮细胞(t=7.296和15.918、9.180和3.561, P<0.05);另外转染组细胞PLAGL2 mRNA和蛋白相对表达水平均低于对照组(t=11.667、13.321, P<0.05);细胞克隆形成实验结果表明转染组细胞克隆形成数目低于对照组(t=6.786, P<0.05);CCK-8实验中, 转染组细胞在24、48、72 h的增殖活性均低于对照组(t=40.113、3.842、14.698, P<0.05), Transwell迁移和侵袭实验中, 转染组细胞迁移和侵袭数目均低于对照组(t=15.707、14.721, P<0.05);划痕试验结果表明转染组细胞24 h相对迁移率低于对照组(t=6.795, P<0.05);另外Western blot结果表明转染组细胞p-Akt、N-cadherin和Vimentin的表达明显降低(t=10.250、22.380、5.863, P<0.05), 而E-cadherin显著增高(t=8.900, P<0.05), 以上差异均有统计学意义。结论 PLAGL2在前列腺癌细胞中异常高表达, 且可能通过Akt信号通路影响前列腺癌细胞的增殖、转移和上皮-间充质转化过程。

Objective To investigate the expression level and biological function of pleomorphic adenoma gene like-2(PLAGL2)in prostate cancer cells.Methods The expression of PLAGL2 in normal prostate epithelial cells and prostate cancer cells was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting.PC-3 cells were transfected with si-PLAGL2,and the cells were divided into control group and transfection group.The transfection efficiency was verified by RT-qPCR and Western blotting.Cell proliferation ability was detected by cell clone formation assay and cell counting kit-8(CCK-8)assay.The Transwell test and scratch test were used to detect the ability of cell migration and invasion.The phosphorylated protein kinase B(p-Akt),N-cadherin,E-cadherin and Vimentin were detected by Western blotting.SPSS 26.0 statistical software was used for analysis.Results The results of RT-qPCR and Western blotting showed the expression of PLAGL2 in DU-145 and PC-3 cells was higher than in normal prostate epithelial cells(t=7.296 and 15.918,9.180 and 3.561,P<0.05).The expression of PLAGL2 mRNA and protein in transfected cells was lower than in control group(t=11.667,13.321,P<0.05).The result of cell clone formation assay showed the number of cell clone formation in transfection group was lower than in control group(t=6.786,P<0.05).The CCK-8 assay revealed that the proliferation activity of transfected cells at 24,48 and 72 h was lower than in control group(t=40.113,3.842,14.698,P<0.05).The Transwell migration and invasion assays indicated that the number of migrating and invasive cells in transfected group was less than in control group(t=15.707,14.721,P<0.05).The scratch test showed that the 24 h relative mobility of cells in transfected group was lower than in control group(t=6.795,P<0.05).In addition,Western blotting results demonstrated that the expression of p-Akt,N-cadherin and Vimentin in transfected group was significantly decreased(t=10.250,22.380,5.863,P<0.05),while E-cadherin was increased(t=8.900,P<0.05),the differences were statistically significant.Conclusion PLAGL2 is abnormally overexpressed in prostate cancer cells and may influence the proliferation,metastasis,and epithelial-mesenchymal transition via the Akt signaling pathway.