人内源性反转录病毒-H长末端重复关联蛋白2在人非小细胞肺癌细胞株中的表达及其生物学功能

Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 expression and its biological function in human non-small cell lung cancer cell lines

目的探讨人内源性反转录病毒-H长末端重复关联蛋白2(HHLA2)在人非小细胞肺癌(NSCLC)细胞株A549、H1975和SK-MES-1中的表达及其生物学功能。方法利用癌症基因组图谱(TCGA)数据库采用Log-rank检验分析HHLA2 mRNA在NSCLC组织中的表达水平对患者预后的预测价值。利用RNA干扰(RNAi)技术构建HHLA2表达稳定下调的人NSCLC细胞株,通过细胞增殖实验、划痕实验及Transwell侵袭实验观察HHLA2表达下调对人NSCLC细胞株生物学功能的影响,组间比较采用t检验。结果 TCGA数据库结果显示,HHLA2 mRNA表达水平低的肺癌患者的总生存期(OS)显著高于HHLA2 mRNA表达水平高的患者(肺腺癌:Log-rankP=0.082,肺鳞癌:Log-rankP=0.021)。富集分析结果显示HHLA2可能与NSCLC细胞的增殖、迁移和侵袭能力相关。实时定量聚合酶链反应(Real-time PCR)和蛋白质印迹法(Western blot)结果显示,3种细胞株中LV-shHHLA2组HHLA2 mRNA和蛋白表达水平均显著低于LV-NC组。细胞计数试剂盒(CCK-8)增殖实验显示,下调HHLA2的表达可显著降低3种细胞的增殖能力(A549:48 h:0.475±0.014比0.600±0.030,t=3.813,P<0.05;72 h:0.658±0.044比0.911±0.039,t=4.336P<0.05;H1975:48 h:0.414±0.026比0.550±0.026,t=3.669,P<0.05;72 h:0.645±0.049比0.926±0.026,t=5.076,P<0.01;SK-MES-1:48 h:0.387±0.043比0.606±0.037,t=3.870,P<0.05;72 h:0.675±0.044比0.971±0.045,t=4.717,P<0.01);划痕实验显示,下调HHLA2的表达可显著降低3种细胞的迁移能力(A549:0.489±0.046比1.000±0.033,t=8.975,P<0.001;H1975:0.242±0.060比1.000±0.028,t=11.390,P<0.001;SK-MES-1:0.403±0.071比1.000±0.092,t=5.150,P<0.001);Transwell侵袭实验显示,下调HHLA2的表达可显著降低3种细胞的侵袭能力[A549:(150.333±7.881)个比(226.000±6.429)个,t=7.440P<0.01;H1975:(92.000±11.790)个比(203.333±7.839)个,t=7.864,P<0.01;SK-MES-1:(95.333±14.881)个比(201.000±16.643)个,t=4.733,P<0.01]。结论干扰NSCLC细胞中HHLA2的表达可降低其增殖、迁移和侵袭能力。

Objective To investigate the expression and biological function of Human endogenous retrovirus subfamily H long terminal repeat associating protein 2(HHLA2)in human non-small cell lung cancer(NSCLC)cell lines A549,H1975,and SK-MES-1.Methods We used The Cancer Genome Atlas(TCGA)database to analyze HHLA2 mRNA expression′s prognostic value in NSCLC tissue by Log-rank test.Human NSCLC cell line with stable down-regulation of HHLA2 expression was constructed by RNA interference(RNAi)technique.The effect of down-regulation of HHLA2 expression on the biological function of human NSCLC cell line was observed by cell proliferation assay,wound healing assay and transwell invasion assay,and the comparison between groups was analyzed by t-test.Results TCGA database results showed that the overall survival(OS)of patients with lung cancer with low expression of HHLA2 mRNA was significantly higher than that of patients with high expression of HHLA2 mRNA(Lung adenocarcinoma:Log-rank P=0.082,Lung squamous cell carcinoma:Log-rank P=0.022).The functional enrichment analysis showed that HHLA2 might be related to the proliferation,migration,and invasion ability of NSCLC cells.The results of Real-time polymerase chain reaction(Real-time PCR)and Western blotting showed that the expression levels of HHLA2 mRNA and protein in LV-shHHLA2 group were significantly lower than those in LV-NC group.Cell Counting Kit-8(CCK-8)assay showed that down-regulation of HHLA2 expression could significantly reduce the proliferation ability of the three types of cells(A549:48 h:0.475±0.014 vs.0.600±0.030,t=3.813,P<0.05;72 h:0.658±0.044 vs.0.911±0.039,t=4.336 P<0.05;H1975:48 h:0.414±0.026 vs.0.550±0.026,t=3.669,P<0.05;72 h:0.645±0.049 vs.0.926±0.026,t=5.076,P<0.01;SK-MES-1:48 h:0.387±0.043 vs.0.606±0.037,t=3.870,P<0.05;72 h:0.675±0.044 vs.0.971±0.045,t=4.717,P<0.01).Wound healing assay showed that down-regulation of HHLA2 expression could significantly reduce the migration ability of the three types of cells(A549:0.489±0.046 vs.1.000±0.033,t=8.975,P<0.001;H1975:0.242±0.060 vs.1.000±0.028,t=11.390,P<0.001;SK-MES-1:0.403±0.071 vs.1.000±0.092,t=5.150,P<0.001).Transwell invasion assay showed that down-regulation of HHLA2 expression could significantly reduce the invasion ability of the three types of cells(A549:150.333±7.881 vs.226.000±6.429,t=7.440 P<0.01;H1975:92.000±11.790 vs.203.333±7.839,t=7.864,P<0.01;SK-MES-1:95.333±14.881 vs.201.000±16.643,t=4.733,P<0.01).Conclusion Interfering with the expression of HHLA2 in NSCLC cells can reduce the ability of proliferation,migration,and invasion.