Expression And Methylation Of GPC5and Its Clinical Significance In Lung Adenocarcinoma

Background and objectives:Lung cancer remians the leading cause of death for malignant tumors in China.Adenocarcinoma is the most frequent type of lung cancer. The occurrence and developmentof lung cancer is a complex process with multiple stages affected by multiple factors andinvolved changes of many genes. Some oncogenes and tumor suppressor genes have beenidentified, and progressions have been made in prevention and treatment of lung cancer.However, the majority of lung cancer patients are diagnosed at advanced stages, and theoverall treatment effectis still poor; the five-year overall survival rate is only about15%.The main reason is the mechanism of lung cance remains unclear. Thus, further research onthe molecular mechanisms of occurrence and development of lung cancer, and looking fornew targets of diagnosis and treatment have become the urgent priority for improving theeffects of prevention and treatment.A genomic wide association study found that the genetic polymorphism of GPC5wassignificantly associated with lung adenocarcinoma. To further explore the mechanism ofGPC5in lung cancer, we will detect and analyze the different expression and methylationof GPC5between lung adenocarcinoma and paired normal tissue, and explore theregulatory role of the methylation in the GPC5gene expression and their potential clinicalfeatures. The results of our study will provide the oretical basis to reveal the molecularmechanisms of lung cancer and targeted prevention and treatment for lung adenocarcinoma.Methods:1. We used real-time quantitative PCR to detect expression level of GPC5in39lungadenocarcinoma and paired normal tissues, and analyzed the differences of expression andits correlation with clinical features. We further validated our results using two datasets from GEO database.2. Bisulfite sequencing PCR (BSP) was used to detect the methylation of tumor andpaired normal tissues from30lung adenocarcinoma patients. We also analyzed thedifferences of methylation between tumor and normal tissue and its correlation with clinicalfeatures.3. We analyzed the association of GPC5gene expression and overall survival (OS) oflung adenocarcinoma by using online survival analysis tool. Logrank test and Kaplan-Meiercurve were used to analyze the survival difference. Multivariate COX model was used toanalysis the relationship of GPC5gene expression with OS by adjusting potentialconfounders.Results:1. GPC5expression in tumor tissue (0.07±0.08) is significantly lower than in normaltissue（(0.19±0.10, P <0.0001）. In subgroup analysis by gender, age, smoking history, grade,stage, tumor size,and lymph node metastasis, GPC5expression in tumor tissues is alsosignificantly lower than that in normal tissues (P <0.05) in each subgroup. The results oftwo datasets from GEO database are consistent with ours. Furthermore, we found that theGPC5expression level in tumor tissues of stage I was significantly higher than that ofstageⅡ+Ⅲa (P<0.05). In lung adenocarcinoma tissues, there was no significant differenceof GPC5expression between subgroups by gender, age, smoking history, grade, tumor size,and lymph node metastasis (P>0.05).2. There were2CpG islands in GPC5, CpG island1was located in promoter, andisland2in gene body. The methylation levels of15CpG sites of CpG island2in tumortissues were significant higher than in normal tissue (P＜0.05); there was no significantdifference in CpG island1. Gene expression level of GPC5was positively correlated withmethylation level of2CpG sites of island2in tumor tissue (r=0.42and0.38,P＜0.05).Methylation of GPC5in tumor tissue was significantly associated with clinical stage;methylation levels in9CpG sites of island2in stage II was significant lower than stage I(P<0.05). Methylation of GPC5in tumor tissues was also significantly related with grade;methylation levels of1CpG sites of island2in poor differentiation grade was significantlower than middle and high differentiation grades. 3. The OS of patients with GPC5high expression was higher than low expression inlung adenocarcinoma. Subgroup analysis by stageⅠ, stage Ⅱ, male and female revealedthe consistent results. Multivariate COX model analysis found that GPC5gene expressionmight be an independent prognostic factor (HR=0.45;95%CI,0.19-1.02; P=0.057）afteradjusting stage, gender and smoking history.Conclusions:1. GPC5expression in tumor tissues is significantly down-regulated compared tonormal tissues. In lung adenocarcinoma tissues, significant higher level of GPC5expressionwas foundin stage I than stageⅡ+Ⅲa. These results indicated that GPC5might be a tumorsuppressor gene for lung adenocarcinoma.2. Methylation of CpG island in gene body was positively correlated with GPC5geneexpression, and was significantly associated with stage and grade in tumor tissues. Theseresults suggested that methylation in GPC5gene body might regulate gene expression toinfluence development of lung adenocarcinoma.3. Gene expresssion of GPC5may be an independent prognostic factor for lungadenocarcinoma.