知识与财富的链接
目的 探讨SHOX2和RASSF1A基因启动子区甲基化检测对早期肺腺癌筛查及诊断的价值。 方法 下载癌症基因组图谱（TCGA）数据库中471例肺腺癌患者的mRNA测序数据及相应的413例甲基化数据分别计算两基因启动子区甲基化水平,分析正常肺组织与肿瘤组织的甲基化水平差异。回顾性分析2018年1月至2019年1月南京大学医学院附属鼓楼医院接受手术的54例早期肺腺癌及31例肺良性肿瘤患者临床资料,检测肿瘤组织及正常肺组织（距肿瘤>5 cm）（称为：临床样本）SHOX2及RASSF1A甲基化状态,以任一基因启动子区甲基化阳性为两基因联合甲基化阳性。以病理诊断为金标准,绘制受试者工作特征（ROC）曲线,分析基因甲基化阳性诊断早期肺腺癌的效能。筛选出石蜡样本中肿瘤细胞比例>80%的患者,对其肿瘤组织及正常肺组织进行mRNA高通量测序。分析两基因甲基化阳性与患者临床病理特征的关系,采用Spearman法分析临床样本和TCGA数据库样本中两基因启动子区甲基化水平与mRNA表达水平的相关性。采用基因集变异分析（GSVA）法分析两基因甲基化阳性临床肺腺癌样本和对应的甲基化阴性肺腺癌样本间京都基因与基因组百科全书（KEGG）富集通路的差异。 结果 在TCGA数据库中,SHOX2启动子区甲基化岛包含6个被测序的甲基化位点,其中位点cg04532033、cg01557547甲基化水平在肺腺癌组织中均高于正常肺组织（均P<0.05）;RASSF1A基因启动子区甲基化岛包含11个被测序的甲基化位点,其中6个位点的甲基化水平在肺腺癌组织中均高于正常肺组织（均P<0.05）;与正常肺组织比较,SHOX2启动子区甲基化水平在Ⅰ、Ⅱ期肺腺癌组织中升高（均P<0.05）;RASSF1A启动子区甲基化在肺腺癌各期中均处于较高水平（均P<0.001）。在临床54例早期肺腺癌患者中,癌组织SHOX2启动子区甲基化阳性28例,RASSF1A启动子区甲基化阳性21例,两基因联合甲基化阳性40例;31例良性肺结节SHOX2、RASSF1A甲基化均为阴性。ROC曲线分析示,SHOX2启动子区甲基化阳性诊断早期肺腺癌的灵敏度高于RASSF1A启动子区甲基化阳性诊断（51.8%比38.9%）,两基因联合甲基化阳性诊断的曲线下面积（AUC）均高于单独以SHOX2或RASSF1A甲基化阳性诊断（0.870比0.759、0.694）。Ⅰ、Ⅱ期肺腺癌SHOX2及RASSF1A甲基化实时荧光定量聚合酶链反应（qRT-PCR）检测的循环阈值（Ct）均低于正常肺组织（均P<0.05）;与两基因甲基化均阴性患者相比,两基因联合甲基化阳性患者具有年龄更高、肿瘤长径更长、病理分期更晚的特点（均P<0.05）。在临床Ⅰ~Ⅱ期肺腺癌样本中,SHOX2及RASSF1A启动子区甲基化qRT-PCR检测的Ct与其mRNA相对表达水平均呈负相关（r=-0.43,P=0.003;r=-0.48,P=0.001）;TCGA数据库Ⅰ~Ⅱ期肺腺癌样本中,SHOX2启动子区甲基化水平与其mRNA相对表达量呈负相关（r=-0.23,P<0.001）,RASSF1A启动子区甲基化水平与其mRNA相对表达量也有负相关趋势,但无统计学意义（r=-0.05,P=0.310）。两基因启动子区甲基化阳性肺腺癌样本中与叶酸代谢及DNA稳定性相关的通路上调,与血管收缩、细胞生长分化等相关的通路的下调。 结论 SHOX2及RASSF1A启动子区甲基化联合检测可作为早期肺腺癌筛查、诊断的指标。两基因启动子区甲基化异常可能影响多个肿瘤相关通路,促进早期肺腺癌的发生、发展。
Objective To investigate the value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma. Methods The mRNA sequencing data of 471 lung adenocarcinoma patients and corresponding methylation data of 413 cases were downloaded from The Cancer Genome Atlas (TCGA) database, the methylation levels of SHOX2 and RASSF1A gene promoter regions were calculated, and the difference in methy lation level between normal lung tissues and tumor tissues was analyzed. The clinical data of 54 patients with early-stage lung adenocarcinoma and 31 patients with benign lung tumors who underwent surgery at Drum Tower Hospital Affiliated to Nanjing University Medical School from January 2018 to January 2019 were retrospectively analyzed. The methylation status of SHOX2 and RASSF1A in tumor tissues and normal lung tissues (>5 cm from the edge of the tumor foci) (called clinical samples) was detect, and a positive methylation in the promoter region of either gene was considered as a combination of two genes methylation positivity. Using pathological diagnosis as the gold standard, the efficacy of gene methylation positivity in diagnosing early-stage lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve. Patients with >80% of tumor cells in paraffin samples were screened, and mRNA high-throughput sequencing was performed in their tumor tissues and normal lung tissues. The relationship between positive methylation of the two genes and clinicopathological features was analyzed, and the correlation between the promoter region methylation level of the two genes and mRNA expression levels in clinical samples and TCGA database samples was analyzed by Spearman method. Gene set variance analysis (GSVA) method was used to analyze the differences in Kyoto Encyclopedia of Genes and Genomes enrichment pathways between two-gene methylation-positive clinical lung adenocarcinoma samples and corresponding methylation-negative lung adenocarcinoma. Results In TCGA database, the SHOX2 promoter region methylation island contained 6 sequenced methylation sites, of which sites cg04532033 and cg01557547 methylation levels were higher in lung adenocarcinoma tissues than in normal lung tissues (both P < 0.05); the RASSF1A gene promoter region methylation island contained 11 sequenced methylation sites, and the methylation levels of 6 of these sites in lung adenocarcinoma tissues were higher than those in normal lung tissues (all P < 0.05). Compared with normal lung tissues, the methylation level of SHOX2 promoter region was higher in stage Ⅰ and Ⅱ lung adenocarcinoma tissues (both P < 0.05); the methylation level of RASSF1A promoter region was higher in all stages of lung adenocarcinoma ( P < 0.001). Among 54 patients with early-stage lung adenocarcinoma, 28 were positive for SHOX2 promoter region methylation in tumor tissues, 21 were positive for RASSF1A promoter region methylation, and 40 were positive for combined methylation of both genes; 31 benign lung nodules were negative for SHOX2 and RASSF1A methylation. ROC curve analysis showed that the sensitivity of positive SHOX2 promoter region methylation for diagnosing early-stage lung adenocarcinoma was higher than that of RASSF1A promoter region methylation positivity (51.8% vs. 38.9%), and the area under the curve (AUC) for diagnosis by two-gene methylation positivity was larger than that for diagnosis by SHOX2 or RASSF1A gene methylation positivity alone (0.870 vs. 0.759 and 0.694). The circulating thresholds (Ct) of SHOX2 and RASSF1A methylation tested by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in stage Ⅰ and Ⅱ lung adenocarcinoma were lower than those in normal lung tissues (all P < 0.05); patients with two-gene methylation positivity were characterized by older age, longer tumor longest diameter and more advanced pathological stage compared with patients with two-gene methylation negativity (all P < 0.05). In clinical stage Ⅰ-Ⅱ lung adenocarcinoma samples, the Ct of SHOX2 and RASSF1A promoter region methylation tested by qRT-PCR was negatively correlated with their mRNA relative expression levels ( r=-0.43, P = 0.003; r = -0.48, P = 0.001); in TCGA database stage Ⅰ-Ⅱ lung adenocarcinoma samples, the level of SHOX2 promoter region methylation was negatively correlated with its mRNA relative expression level (r = -0.23, P < 0.001), and the level of RASSF1A promoter region methylation was also negatively correlated with its mRNA relative expression level, but without statistical difference ( r = -0.05, P = 0.310). In two-gene promoter methylation-positive lung adenocarcinoma samples, the pathways related to folate metabolism and DNA stability were upregulated, and the pathways related to vasoconstriction and cell growth and differentiation were downregulated. Conclusions The combined detection of SHOX2 and RASSF1A promoter region methylation can be used as an indicator for screening and diagnosis of early-stage lung adenocarcinoma. Abnormal promoter region methylation of the two genes may affect multiple tumor-related pathways and promote the occurrence and progression of early-stage lung adenocarcinoma.
