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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。
The purpose of ovarian cancer is the highest mortality of gynecologic tumors, strong resistance to chemotherapy is one of the main reasons for its poor prognosis. In order to elucidate the mechanism of ovarian cancer resistance to platinum drugs, the study of miRNA gene methylation Level on Platinum Resistance in Ovarian Cancer. Methods The ovarian cancer tissues were divided into sensitive group and drug-resistant group, 3 cases in each group. Gene microarray was used to analyze the difference of microRNA (miRNA) expression between the two groups. Real-time fluorescence quantitative PCR Sensitive and 3 drug-resistant tissues, CoC1 / resistant CoC1 / DDP cell line were detected, and the expression of candidate miRNAs was detected. Massarray technique was used to detect the expression of The methylation of miRNA promoter in drug-resistant tissues (6 cases) was analyzed. Bioinformatics analysis was used to identify the potential target genes of miRNAs. Results The samples of ovarian cancer sensitive to platinum-based drugs were used as controls. Six miRNAs (miR-493-3p, miR-10a-5p and miR-16) that were up-regulated in drug- -2-3p, miR-1248, miR-451a, miR-628-3p) and six downregulated miRNAs (miR-509-3p, miR- 1197, miR- 376a-3p, miR- 1273a, miR- 550a -3p, miR-19b-3p). Tissue verification found that miR-509-3p, miR-493-3p, miR-10a-5p, miR-16-2-3p and miR-451a, consistent with the chip results; cultured cells found that four miRNA expression and regulation In a manner consistent with the results of tissue microarrays, miR-10a-5p, miR-16-2-3p, miR-1248 and miR-628-3p were highly expressed in drug-resistant cell lines. Further study found that, compared with sensitive tumor tissue, the methylation level of miR-10a-5p gene promoter was significantly decreased in drug-resistant tissues, P = 0.04. Bioinformatics prediction of HOXA1 and USF2 miR-10a-5p resistance-related target genes. Conclusions Compared with the susceptible group, the methylation level of miR-10a-5p gene promoter is significantly decreased and the expression of miR-10a-5p is increased in drug-resistant group. By inhibiting HOXA1 and USF2, it inhibits cell apoptosis, Tolerance.