MiR-335表遗传学调控与胃癌临床病理特征和预后相关性研究  

Epigenetic regulation of microRNA-335 associated with poor clinicopathological characteristics and prognosis in gastric cancer tissues

作　　者：张家魁[1] 谢强[1] 刘继超[1] 张春东[1] 戴冬秋[1] 

机构地区：[1]中国医科大学附属第四医院胃肠外科,辽宁沈阳110032

出　　处：《中华肿瘤防治杂志》2015年第21期1680-1687,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：辽宁省自然科学基金(2013225021)

摘　　要：目的 miR-335在胃癌中的抑癌作用及表达下调的机制尚不明确,为此本研究探讨miR-335基因启动子区甲基化状态对胃癌miR-335表达水平、临床病理特征以及预后的影响。方法收集2009-07-01-2011-07-01中国医科大学附属第四医院就诊经病理诊断为原发性胃癌,并行根治性手术的231例新鲜胃癌组织及配对癌旁组织(距离癌组织>5cm);1株永生化的胃黏膜上皮细胞系(GES-1)和4株胃癌细胞系(SGC-7901、MKN-45、BGC-823和AGS)。实时荧光定量PCR(qRT-PCR)检测胃癌细胞株及231例胃癌组织中miR-335的表达水平。甲基化特异性PCR(MSP)和甲基化测序(BSP)方法检测胃癌细胞系及胃癌组织中miR-335的基因启动子区甲基化状态。综合分析miR-335表达水平,miR-335基因启动子区甲基化状态对胃癌患者临床病理特征及预后的影响。结果 qRT-PCR检测结果显示,miR-335在MKN-45细胞株表达水平为GES-1细胞株的0.154±0.016倍(P<0.01),在SGC-7901细胞株表达水平为GES-1细胞株的0.138±0.013倍(P<0.01),在BGC-823细胞株表达水平为GES-1细胞株的0.432±0.076倍(P<0.01),在AGS细胞株表达水平为GES-1细胞株的0.749±0.072倍(P=0.01),均显著低于正常人胃黏膜永生化细胞株GES-1。miR-335在231例胃癌组织中的表达水平较癌旁相对正常组织明显下降,P<0.001。甲基化测序及MSP的实验结果表明,MKN-45、SGC-7901和BGC-823细胞株均存在基因启动子区异常高甲基化状态,AGS细胞株亦呈部分高甲基化状态。miR-335低表达与肿瘤大小(P=0.030)、血管浸润(P=0.027)、腹膜播散(P=0.027)、淋巴结转移(P=0.033)、不良pT分期(P=0.040)、不良pN分期(P=0.038)、不良TNM分期(P=0.005)和淋巴管浸润(P=0.041)显著相关。miR-335基因启动子区的高甲基化状态与肿瘤大小(P=0.004)、淋巴结转移(P=0.046)、淋巴管浸润(P=0.001)和miR-335低表达(P<0.001)显著相关。Kaplan-Meier生存分析表明,高miR-335表达水平以及miR-335基因启动子区低甲基OBJECTIVE To investigate the role of miR-335 methylation and expression in gastric cancer cell lines and gastric tumor tissues, the clinicopathological and prognostic values of miR-335 methylation and expression in patients with gastric cancer （GC）. METHODS Two hundred and thirty-one pairs of fresh GC tissues and adjacent non-tumor gastric tissues were obtained from patients with a diagnosis of primary GC and then underwent elective surgery in the Fourth Affiliated Hospital of China Medical University （Shenyang, China） between June 2009 and June 2011. Relative expression of miR-335 in 4 gastric cancer cell lines and 231 gastric cancer tissues was detected by real-time quantitative reverse transcriptase-PCR （qRT-PCR） compared with controls. Methylation-specifc PCR （MSP） and bisulfte sequence-PCR （BSP） were used to evaluate the DNA methylation status in the CpG islands upstream of miR-335 in gastric cancer cell lines and tissues. RESULTS In 231 GC patients, the miR-335 levels were remarkably downregulated in GC tissues compared with paired adjacent nontumorous tissues （P〈0. 001）. miR-335 was downregulated in gastric cancer cell lines SGC-7901, MKN-45, BGC 823 and AGS compared with a normal gastric epithelial cell line GES-1 MKN 45, （0. 154＋- 0.016） fold （P〈0.01）; SGC-7901, （0.138＋-0.013） fold （P〈0.01）; BGC823, （0.432＋-0.076） fold （P〈0.01）; AGS, （0. 749＋-0. 072） fold （P=0.01）. Bisulfite sequencing of the promoter region of miR-335 in all 4 GC cell lines veri fied the marked methylation of the promoter region of miR-335. The MSP results showed that in the 231 gastric cancer tissues, there were 127 samples that showed hypermethylation （55%） and 104 samples that showed hypomethylation （45%）. Hypermethylation of the miR-335 gene promoter region was associated with tumor size （P=0. 004）, metastasis lymph node （P=0. 046）, miR-335 expression level （P〈0.001） and invasion into lymphatic vessels （P=0. 001）. Low expression of miR-

关 键 词：胃肿瘤 微小RNA miR-335 甲基化 预后 

分 类 号：R735.2[医药卫生—肿瘤]