甲基化调控SLIT3和SPARCL1基因在吸烟致肺腺癌中的表达及其对患者预后的影响

Expression and prognosis effect of methylation-regulated SLIT3 and SPARCL1 genes in smoking-related lung adenocarcinoma

目的探讨甲基化调控SLIT3和SPARCL1基因在吸烟致肺腺癌中的表达及其对患者预后的影响。方法通过实时定量聚合酶链式反应(qPCR)技术检测SLIT3和SPARCL1的mRNA在染烟30代恶性转化细胞(S30)以及肺腺癌细胞系中的相对表达量,并与正常人永生化支气管上皮细胞(BEAS-2B)进行比较;提取癌症基因组图谱(TCGA)计划数据库中mRNA表达、DNA甲基化和患者信息资料数据;验证SLIT3和SPARCL1的mRNA在肺腺癌组织中的表达,分析不同吸烟史患者肺腺癌组织中SLIT3和SPARCL1的mRNA表达差异并绘制患者的10年生存曲线;对TCGA数据库中肺腺癌组织中SLIT3和SPARCL1的mRNA表达和DNA甲基化高通量数据进行相关性分析,同时用甲基化转移酶抑制剂(5-aza)处理S30细胞,分析DNA甲基化对SLIT3和SPARCL1表达的调控作用。结果S30细胞和4种肺腺癌细胞系中的SLIT3 mRNA(0.493±0.134和0.041±0.014、0.161±0.023、0.277±0.055、0.035±0.005)及SPARCL1 mRNA(0.507±0.131和0.453±0.045、0.420±0.040、0.153±0.035、0.430±0.050)相对表达量均显著低于正常BEAS-2B细胞(均P<0.01);肺腺癌组织中SLIT3和SPARCL1的mRNA相对表达量均显著低于癌旁正常肺组织(8.12±1.58比10.84±0.69和11.46±1.06比13.57±0.67)(均P<0.001),并且二者均与吸烟史显著相关(均P<0.001);在非吸烟患者中SLIT3和SPARCL1的mRNA高表达均有很好的预后意义;两基因启动子区域甲基化与表达呈现显著负相关(r=-0.208、-0.574:均P<0.001);5-aza处理可使S30细胞SLIT3和SPARCL1的mRNA表达显著上调(2.137±0.281和3.657±0.882,均P<0.01)。结论SLIT3和SPARCL1基因能够受到DNA甲基化调控并且在吸烟所致的肺腺癌中表达下调并对患者的预后产生影响。

Objective To investigate the expression and prognosis effect of methylation-regulated SLIT3 and SPRCL1 genes in smoking-related lung adenocarcinoma. Methods The expression levels of SLIT3 and SPARCL1 in cigarette smoke-induced malignant transformed cell (S30) and lung adenocarcinoma (LUAD) cell lines were measured by real-time fluorescence quantitative PCR (qPCR). Datasets of mRNA expression, DNA methylation and patient information data were obtained from The Cancer Genome Altas (TCGA) database. The mRNA expression levels of SLIT3 and SPARCL1 were validated in LUAD tissues. The 10-year survival curve of LUAD patients with different smoking history was plotted, and the correlation between mRNA expression level and DNA methylation level of LUAD patients was further analyzed. S30 cells were treated with 5-azacytidine (5-aza), an inhibitor of DNA methyltransferase, to analyze the methylation regulatory mechanism of SLIT3 and SPRCL1. Results The qPCR results showed the significant down-regulation of SLIT3 and SPARCL1 in S30 cell and four LUAD cell lines (SLIT3: 0.493±0.134 and 0.041±0.014, 0.161±0.023, 0.277±0.055, 0.035±0.005;SPARCL1: 0.507±0.131 and 0.453±0.045, 0.420±0.040, 0.153±0.035, 0.430±0.050;all P<0.01). Bioinformatics analysis showed that SLIT3 and SPARCL1 were low expressed in LUAD tissue (8.12±1.58 vs 10.84±0.69 and 11.46±1.06 vs 13.57±0.67;both P<0.001) compared with adjacent peritumoral tissues, and expression levels of SLIT3 and SPARCL1 were significantly correlated with smoking history (both P<0.001). Non-smoker with high expression of SLIT3 and SPARCL1 was associated with better prognosis among LUAD patients. There was a significant negative correlation between promoter methylation and mRNA expression level of the two genes (r=-0.208,-0.574;both P<0.001). 5-aza treatment significantly up-regulated the expression levels of SLIT3 and SPARCL1 genes in S30 cells (2.137±0.281, 3.657±0.882;both P<0.01). Conclusion SLIT3 and SPARCL1 can be regulated by DNA methylation and down-regulated in LUAD tissue, which has important prognostic significance on the smoking-induced LUAD patients.