RDH5对肺腺癌恶性生物学行为及预后的影响

Effects of RDH5 on malignant biological behavior andprognosis of lung adenocarcinoma

目的 验证视网膜脱氢酶5(RDH5)在肺腺癌中的功能及其对预后的影响。方法 基于癌症基因组图谱(TCGA)数据库比较RDH5在肺腺癌组织和癌旁正常组织中的表达及对肺腺癌生存的影响。采用实时荧光定量逆转录PCR(RT-qPCR)和Western blot检测细胞中RDH5的表达情况。siRNA转染抑制RDH5的表达,使用CCK-8和克隆形成评估细胞增殖能力,通过流式细胞术对细胞凋亡进行测定。检测2012年1月至2014年12月该院139例肺腺癌术后患者癌组织中RDH5表达情况,分析RDH5表达与临床病理特征之间的相关性,以及RDH5表达与患者无病生存(DFS)和总生存(OS)的关系,COX回归分析DFS、OS的危险因素。结果 TCGA分析结果显示,RDH5在肺腺癌组织表达水平高于正常组织,且RDH5高表达患者的OS率低于低表达患者(P<0.05)。肺腺癌细胞系(A549、H1437)中RDH5 mRNA相对表达水平较支气管上皮细胞系(16HBE)升高,A549、H1437中RDH5蛋白表达增加(P<0.05)。siRNA处理后,A549、H1437中RDH5 mRNA相对表达水平较处理前降低,且RDH5蛋白表达减少(P<0.05)。siRNA转染96 h的A549、H1437细胞增殖率较对照明显降低(P<0.05)。克隆形成实验结果显示,siRNA转染后细胞群落数明显减少。随着siRNA转染,RDH5表达下降,细胞凋亡比例增加,A549、H1437中凋亡细胞分别从9.3%、10.0%增至32.1%、45.6%。siRNA转染后,A549、H1437的抗含半胱氨酸的天冬氨酸蛋白水解酶(caspase)3表达减弱,抗裂解(cleaved)-caspase3表达增强。RDH5高表达与低表达的肿瘤分化程度、淋巴结转移、TNM分期比较,差异有统计学意义(P<0.05)。139例肺腺癌患者术后5年中位DFS时间为33(15,68)个月,中位OS时间为46(24,69)个月。RDH5高表达的DFS、OS时间较低表达缩短,差异有统计学意义(P<0.05)。多因素COX回归分析结果显示,RDH5高表达提示患者的不良预后,DFS、OS时间的HR分别为0.40(95%CI:0.22~0.68)、0.52(95%CI:0.30~0.91),差异有统计学意义(P<0.05)。结论 RDH5在肺腺癌中高表达,其可调节细胞增殖和凋亡。

Objective To verify the function of retinal dehydrogenase 5(RDH5)in lung adenocarcinoma and impact on prognosis.Methods Based on The Cancer Genome Atlas(TCGA)database,the expression of RDH5 in cancer tissues and adjacent tissues and its effect on the survival of lung adenocarcinoma were compared.Real-time fluorescent quantitative reverse transcription PCR(RT-PCR)and western-blot were used to detect the expression of RDH5 in cells.siRNA transfection technology inhibited the expression of RDH5.CCK-8 and clone formation were used to evaluate cell proliferation ability,and cell apoptosis was determined by flow cytometry.The expression of RDH5 in cancer tissues of 139 patients with lung adenocarcinoma after surgery was detected,and the correlation between RDH5 expression and clinicopathological characteristics was evaluated,and the relationship between RDH5 and disease-free survival(DFS)and overall survival(OS)was analyzed.The risk factors of DFS and OS were analyzed by COX regression.Results The results of TCGA analysis showed that the expression level of RDH5 in lung adenocarcinoma tissues was higher than that in normal tissues,and the OS rate in patients with high expression of RDH5 was lower than that in patients with low expression(P<0.05).The relative expression of RDH5 mRNA in lung adenocarcinoma cell lines(A549,H1437)was higher than that in bronchial epithelial cell lines(16HBE),and the expression of RDH5 protein in A549 and H1437 was increased(P<0.05).After siRNA treatment,the relative expression level of RDH5 mRNA in A549 and H1437 was decreased compared with that before treatment,and the expression of RDH5 protein in A549 and H1437 was decreased(P<0.05).The cell proliferation rate of A549 and H1437 transfected with siRNA for 96 h was significantly lower than that of the control group(P<0.05).The results of clone formation experiment showed that the number of cell communities decreased significantly after siRNA transfection.With siRNA transfection,the expression of RDH5 decreased and the apoptosis rate increased.The apoptotic cells in A549 and H1437 increased from 9.3%and 10.0%to 32.1%and 45.6%,respectively.After siRNA transfection,cysteinyl aspartate specific proteinase(caspase)3 expression of A549 and H1437 was decreased,and cleaved caspase3 expression was increased.There were significant differences in tumor differentiation,lymph node metastasis and TNM stage between high and low expression of RDH5(P<0.05).The 5-year median DFS time and median OS time of 139 patients with lung adenocarcinoma were 33(15,68)months and 46(24,69)months respectively.DFS and OS time with high RDH5 expression was shorter than that with low RDH5 expression,the difference was statistically significant(P<0.05).Multivariate COX regression analysis showed that high expression of RDH5 suggested poor prognosis,and the HR of DFS and OS time were 0.40(95%CI:0.22-0.68)and 0.52(95%CI:0.30-0.91),respectively,with statistically significant differences(P<0.05).Conclusion RDH5 is highly expressed in lung adenocarcinoma,regulates cell proliferation and apoptosis.