miR-10a-5p基因甲基化对卵巢癌铂类耐药的影响

Effects of miR-10a-5p genomic methylation on the response to platinum drugs in ovarian cancer

目的卵巢癌是死亡率最高的妇科肿瘤,较强的化疗耐药性是其预后差的主要原因之一,为了阐明卵巢癌对铂类药物的耐药机制,本研究探讨miRNA基因的甲基化水平对卵巢癌铂类耐药的影响。方法将卵巢癌组织分为敏感组和耐药组,每组各3例;采用基因芯片技术,对比分析了两组微RNA(microRNA,miRNA)的表达差异;采用实时荧光定量PCR,分别在6例敏感和3例耐药组织、铂类药物敏感(CoC1)和耐药的卵巢癌细胞系(CoC1/DDP),检测了候选miRNA的表达差异;应用Massarray技术,检测敏感组织(15例)与耐药组织(6例)中miRNA基因启动子的甲基化差异;应用生物信息学分析,鉴定目标miRNA的潜在靶基因。结果以铂类药物敏感的卵巢癌组织样本为对照,利用基因芯片筛选,鉴定了6条在耐药组织样本中出现表达上调的miRNA(miR-493-3p、miR-10a-5p、miR-16-2-3p、miR-1248、miR-451a、miR-628-3p)和6条表达下调的miRNA(miR-509-3p、miR-1197、miR-376a-3p、miR-1273a、miR-550a-3p、miR-19b-3p)。组织验证发现,miR-509-3p、miR-493-3p、miR-10a-5p、miR-16-2-3p和miR-451a,与芯片结果一致;培养细胞研究发现,4条miRNA的表达调控方式与组织芯片结果一致,miR-10a-5p、miR-16-2-3p、miR-1248和miR-628-3p在耐药细胞系中高表达。进一步研究发现,与敏感肿瘤组织相比,耐药组织中miR-10a-5p基因启动子的甲基化水平出现显著降低,P=0.04。结合生物信息学预测HOXA1和USF2为miR-10a-5p与耐药相关的靶基因。结论与敏感组相比,耐药组miR-10a-5p基因启动子甲基化水平显著降低,miR-10a-5p表达升高,通过抑制HOXA1和USF2,抑制细胞凋亡,导致铂类化疗药物耐受。

OBJECTIVE Ovarian cancer is the highest mortality rate of gynecological tumors and drug resistance is one of the main reasons for poor prognosis. In order to elucidate the resistance of ovarian cancer to platinum drugs, this paper systematically studied the effect of miRNA gene methylation on drug resistance. METHODS According to the rela-tionship between no disease progression and 6-month after application of platinum-based combination of chemotherapy, the ovarian cancer tissue is divided into sensitive group and drug-resistant group; Gene chip technique was used to evaluate the expression levels of miRNAs in 3 sensitive tissues and 3 drug-resistant ovarian cancer tissues ; The real-time quantitative PCR （RT-PCR） was used to evaluate the expression levels of miRNAs in 6 sensitive tissues, 3 drug-resistant tissues and human ovarian cancer cell lines （CoC1 and CoC1/DDP） ; The methylation of the miRNAs promoter region was detected by the Massarray; Bioinformatics programs were used to identify potential target genes for miRNAs. RESULTS Compared with sensitive group the miRNA microarray profiling revealed 6 up-regulated （miR-493-3p, miR-10a-5p, miR-16-2-3p, miR-1248, miR-451a, miR-628-3p） and 6 up-regulated （miR-509-3p, miR-1197, miR-376a-3p, miR-1273a, miR-550a-3p, miR-19b-3p） miRNA in resistant group. Among the miRNAs, miR-509-3p, miR-493-3p, miR-10a-5p, miR-16-2-3p and miR-451a were expressed consistently with chip in tissues. 4 were expressed consistently in CoC1 and CoC1/DDP ovarian cancer cell lines, miR-10a-Sp, miR-16-2-3p, miR-1248, and miR-628-3p were express higher in resistant group. The genomie methylation was lower in resistant group （P= 0.04）. Combined with bioinformatics prediction and litera- ture, HOXA1 and USF2 were found as the target gene of miR-10a-5p. CONCLUSIONS Compared with the sensitive tumor tissue, the promoter methylation level of the miR-10a-Sp gene in the drug-resistant tumor was significantly decreased, the expression level of the miR-10a-Sp was increased, and the biological activity of the HOXA1 and USF2 was inhibited, leading to platinum-based chemotherapy drug tolerance.