内源性反转录病毒-H长末端重复关联蛋白2在人肾透明细胞癌细胞株中的表达及其生物学功能

Human endogenous retrovirus subfamily H long tenninal repeat associating protein 2 expression and its contribution to the regulation of biological function of human clear cell renal cell carcinoma cell line

目的探讨内源性反转录病毒-H长末端重复关联蛋白2(HHLA2)在人肾透明细胞癌(ccRCC)细胞株786-O和ACHN中的表达及生物学功能.方法通过使用RNA干扰(RNAi)技术构建HHLA2稳定下调表达的人肾癌细胞株786-O和ACHN,通过细胞增殖实验、划痕实验及Transwell实验观察HHLA2下调表达对人肾癌细胞株生物学行为的影响;基因芯片分析探讨人肾癌细胞株中HHLA2敲低后的差异表达基因.结果细胞计数试剂盒(CCK-8)增殖实验结果显示,LV-HHLA2-sh1组和LV-HHLA2-sh2组中肾癌细胞系的细胞增殖能力较LV-NC组显著降低;划痕试验结果显示划痕48 h后,LV-HHLA2-sh1及LV-HHLA2-sh2组的无细胞区域显著宽于LV-NC组(LV-HHLA2-sh1:786-O:F=99.340,P<0.01,ACHN:F=113.5,P<0.01;LV-HHLA2-sh2:786-O:F=35.32,P<0.01,ACHN:F=26.47,P<0.01);Transwell侵袭试验结果显示LV-HHLA2-sh1组和LV-HHLA2-sh2组中肾癌细胞系的细胞侵袭能力较LV-NC组显著降低;细胞周期检测结果显示HHLA2下调表达后细胞周期阻滞于G1期,同时细胞周期蛋白(Cyclin)D1、c-Myc和细胞周期蛋白E1表达减少,Cyclin D1表达水平在LV-HHLA2-sh1与LV-HHLA2-sh2组中较LV-NC组显著降低;c-Myc表达水平在LV-HHLA2-sh1与LV-HHLA2-sh2组中较LV-NC组显著降低;Cyclin E1表达水平在LV-HHLA2-sh1与LV-HHLA2-sh2组中较LV-NC组显著降低.此外,基因芯片检测结果显示,肾癌细胞系下调HHLA2表达后,上皮细胞-间充质转换标志物E-钙粘蛋白表达显著增加,N-钙粘蛋白和波形蛋白的表达显著降低.结论HHLA2在人肾透明细胞癌细胞株786-O和ACHN中下调表达可显著抑制细胞的增殖、迁移和侵袭能力.

Objective To investigate the clinical significance and biological function of human endogenous retrovirus subfamily H long terminal repeat associating protein 2(HHLA2)in human clear cell renal cell carcinoma(ccRCC).Methods Down-regulation of HHLA2 was performed by using RNA interference(RNAi)method to investigate the role of HHLA2 in regulation of biological behaviors in human clear cell renal cell carcinoma cell lines 786-0 and ACHN.The cell counting kit-8(CCK-8)assay,wound healing assay,and transwell invasion assay were used to examine the cellular function after HHLA2 knockdown of these cells.We also identified the differentially expressed genes upon HHLA2 knockdown in ccRCC cell lines by using gene microarray analysis.Results To investigate the functions of HHLA2 in human ccRCC,we successfully constructed HHLA2 knockdown expression in human ccRCC cell lines by using the RNAi method.CCK-8 assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell proliferation.The wound healing assay showed that decreased HHLA2 expression in ccRCC cell lines 786-0 and ACHN significantly attenuated cell migration(LV-HHLA2-shl:786-0:F=99.340,PvO.Ol,ACHN:F=113.500,P<0.01;LV-HHLA2-sh2:786-O:F=35.320,P<0.01,ACHN:F=26.470,P<0.01).Trans well invasion assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell invasion.The cell cycle arrest at Gphase was induced and the expressions of Cyclin DI,c-Myc and Cyclin El were decreased.In addition,according to the microarray data,the expressions of epithelia-to-mesenchymal transition markers,such as E-cadherin,N-cadherin and Vimentin,were significantly changed after knockdown of HHLA2 expression.After knockdown of HHLA2 in 786-0 and ACHN,the expression of E-cadherin was significantly increased,meanwhile the expression of N-cadherin and Vimentin were significantly decreased.Conclusion Our findings indicated that HHLA2 was involved in the progression of human ccRCC and could be used as an important prognostic predictor for this malignancy.