LAMA3 DNA甲基化对卵巢上皮性癌铂耐药及预后影响的生物信息学和临床分析

Clinical and bioinformatics analysis of the relationship between LAMA3 DNA methylation expression and platinum resistance and prognosis in epithelial ovarian cancer

目的:探讨层粘连蛋白α3(LAMA3)DNA甲基化对卵巢上皮性癌(卵巢癌)铂耐药及预后的影响及可能的机制。方法:(1)通过数据库中生物信息学数据分析LAMA3 DNA甲基化水平与卵巢癌铂耐药的关系。(2)选取2000年1月至2012年12月在广西医科大学附属肿瘤医院诊治的卵巢癌患者共67例(包括铂耐药组35例和铂敏感组32例),采用焦磷酸测序技术检测两组卵巢癌组织中LAMA3 DNA甲基化水平,探讨其对卵巢癌患者铂耐药及预后的诊断效能,并进一步分析其对卵巢癌铂耐药患者化疗效果及预后的影响。结果:(1)从基因互作检索平台(STRING)数据库中筛选出与LAMA3高度互作的10个蛋白,包括层粘连蛋白β(LAMB)3、层粘连蛋白γ(LAMC)3、整合素α(ITGA)6、整联蛋白β4(ITGB4)、ITGA3、LAMC1、LAMB2、肌营养不良蛋白关联糖蛋白1(DAG1)、LAMB1、17型胶原蛋白α1链(COL17A1)蛋白;京都基因与基因组百科全书(KEGG)富集分析显示,LAMA3及其相关互作蛋白通过细胞凋亡、细胞周期、DNA损伤反应、上皮间质转化(EMT)、雄激素受体(AR)、雌激素受体(ER)、磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)、大鼠肉瘤癌基因(RAS)/有丝分裂原活化蛋白激酶(MAPK)、受体酪氨酸激酶(RTK)、结节性硬化症蛋白复合体(TSC)/雷帕霉素靶蛋白(mTOR)等信号通路,参与恶性肿瘤发生、发展过程的调控,其表达水平与卵巢癌顺铂等化疗药物的敏感性相关。(2)本院临床数据分析发现,铂耐药组、铂敏感组卵巢癌组织中LAMA3 DNA甲基化水平分别为71%(25/35)、69%(22/32),两组比较,差异无统计学意义(χ^(2)=0.057,P=0.811);LAMA3 DNA甲基化水平判断卵巢癌铂耐药的曲线下面积(AUC)为0.601,预测患者预后的AUC值为0.686。LAMA3 DNA高甲基化铂耐药卵巢癌患者(24例)的化疗效果较低甲基化铂敏感患者(15例)更差[有效率分别为50%(12/24)和15/15;χ^(2)=10.833,P=0.001],且LAMA3 DNA甲基化水平对卵巢癌患者的无进展生存时间和总生存时间均有显著影响(P均<0.05)。结论:LAMA3 DNA甲基化水平对卵巢癌患者的铂耐药及预后具有一定的诊断和预测价值,可能与卵巢癌的调控机制、铂耐药及预后存在密切联系。

Objective To investigate the effect of DNA methylation of lamininα3(LAMA3)on the prognosis of platinum-resistant epithelial ovarian cancer(EOC)and its possible mechanism.Methods(1)The relationship between DNA methylation of LAMA3 and platinum resistance in EOC was evaluated by bioinformatics.(2)A total of 67 EOC patients treated at Guangxi Medical University Cancer Hospital from January 2000 to December 2012 were selected to detect the levels of LAMA3 DNA methylation in EOC tissues using pyrophosphate sequencing technology to explore its diagnostic efficacy for platinum resistance and prognosis in EOC patients.Furthermore,its impact on chemotherapy efficacy and prognosis of platinum resistant EOC patients were also analyzed.Results(1)Ten proteins highly interacting with LAMA3 were screened from the Gene Interaction Retrieval Platform(STRING)database,including lamininβ(LAMB)3,lamininγ(LAMC)3,integrinα(ITGA)6,intestine proteinβ4(ITGB4),ITGA3,LAMC1,LAMB2,dystrophin associated glycoprotein 1(DAG1),LAMB1 and cytochrome P450c17α(COL17A1)protein;kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis showed that LAMA3 and its related interacting proteins participate in the regulation of malignant tumor occurrence and development through signaling pathways such as apoptosis,cell cycle,DNA damage response,epithelial mesenchymal transition(EMT),androgen receptor(AR),estrogen receptor(ER),phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt),RAS/mitogen activated protein kinase(MAPK),receptor tyrosine kinase(RTK),tuberous sclerosis protein complex(TSC)/mammalian target of rapamycin(mTOR),and their expression levels were related to the sensitivity of chemotherapy drugs such as cisplatin in EOC.(2)Our clinical data analysis found that the LAMA3 DNA methylation level in EOC tissue of the platinum-sensitive group(35 cases)was 71%(25/35),which was higher than 69%(22/32)in the platinum-resistant group(32 cases),with statistically insignificant difference(χ^(2)=0.057,P=0.811).The area under the curve(AUC)of LAMA3 DNA methylation level for assessing platinum resistance in EOC was 0.601,and the AUC for predicting EOC patient prognosis was 0.686.The chemotherapy efficacy of EOC patients with high methylation of LAMA3 DNA was worse than that of patients with low methylation,50%(12/24)vs 15/15,with statistically significant difference(χ^(2)=10.833,P=0.001).The level of LAMA3 DNA methylation had a significant impact on the progression free survival and overall survival of EOC patients(both P<0.05).Conclusion The level of LAMA3 DNA methylation has certain diagnostic and predictive value for platinum resistance and prognosis in EOC patients,which may be closely related to the regulatory mechanism,platinum resistance and prognosis of EOC.