Wnt5a 基因启动子区域异常甲基化在人上皮性卵巢癌中的作用：一项临床和实验研究。

The Role of Abnormal Methylation of Wnt5a Gene Promoter Regions in Human Epithelial Ovarian Cancer: A Clinical and Experimental Study.

机构信息

The Shenzhen Maternity & Child Healthcare Hospital Affiliated to Southern Medical University, ShenZhen 518028, China.

The First Clinical Medical College of Lanzhou University, Lanzhou, China.

出版信息

Anal Cell Pathol (Amst). 2018 Jul 16;2018:6567081. doi: 10.1155/2018/6567081. eCollection 2018.

DOI:10.1155/2018/6567081

PMID:30079293

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6069701/

Abstract

OBJECTIVE

In the current study, the role of abnormal methylation of Wnt5a gene promoter regions in human epithelial ovarian cancer was investigated.

METHODS

Wnt5a expressions were examined by immunohistochemistry in epithelial ovarian tissues (30 normal and 79 human EOC tissues). SKOV3 cells were treated with different concentrations of 5-Aza-CdR (0.5, 5, and 50 mol/L). The methylation status of the Wnt5a promoter was analyzed using a methylation-specific polymerase chain reaction (MSP), and the expression level of Wnt5a mRNA was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was measured by MTT assay, and apoptosis was analyzed using flow cytometry.

RESULTS

(1) Compared with normal tissues, Wnt5a expressions were reduced or lost in EOC ( < 0.05). Wnt5a expression had a close relationship with histological grade, FIGO stage, and lymph node metastasis ( = 0.005, = 0.022, and = 0.037, resp.). (2) Wnt5a abnormal methylation status existed in ovarian cancer tissues and was higher than that of normal ovarian tissue ( < 0.01). (3) Before treatment with 5-Aza-CdR, the promoter of the Wnt5a gene was methylated in SKOV3 cells; accordingly, Wnt5a mRNA levels were low to absent in SKOV3 cells. (4) Following 5-Aza-CdR treatment, MSP analysis revealed complete demethylation of the Wnt5a promoter in the SKOV3 cell line, particularly at 5 mol/L 5-Aza-CdR. Wnt5a expression increased in SKOV3 cells following treatment with a demethylating agent ( ≤ 0.001). (5) The growth rate of the cells was inhibited in a dose-dependent manner by treatment with 5-Aza-CdR. (6) The cell apoptosis rate increased gradually after treatment with 0.5, 5, and 50 mol/L 5-Aza-CdR. The apoptosis rate exists in a dose-dependent relationship with 5-Aza-CdR concentration ( = 779.73, < 0.01).

CONCLUSIONS

Wnt5a gene region promoter aberrant methylation existed in epithelial ovarian cancer, and abnormal methylation of Wnt5a gene promoter regions may be a new target for the treatment of epithelial ovarian cancer.

摘要

目的

本研究旨在探讨 Wnt5a 基因启动子区异常甲基化在人上皮性卵巢癌中的作用。

方法

采用免疫组织化学法检测上皮性卵巢组织（30 例正常和 79 例人卵巢上皮性癌组织）中 Wnt5a 的表达。用不同浓度的 5-氮杂-2′-脱氧胞苷（5-Aza-CdR，0.5、5 和 50 μmol/L）处理 SKOV3 细胞。采用甲基化特异性聚合酶链反应（MSP）分析 Wnt5a 启动子的甲基化状态，并用实时定量聚合酶链反应（qRT-PCR）检测 Wnt5a mRNA 的表达水平。用 MTT 法检测细胞增殖，用流式细胞术分析细胞凋亡。

结果

（1）与正常组织相比，EOC 组织中 Wnt5a 的表达降低或缺失（<0.05）。Wnt5a 的表达与组织学分级、FIGO 分期和淋巴结转移密切相关（=0.005、=0.022 和=0.037）。（2）卵巢癌组织中存在 Wnt5a 异常甲基化状态，且高于正常卵巢组织（<0.01）。（3）5-Aza-CdR 处理前，Wnt5a 基因启动子呈甲基化状态，因此 SKOV3 细胞中 Wnt5a mRNA 水平低或缺失。（4）经 5-Aza-CdR 处理后，MSP 分析显示 SKOV3 细胞中 Wnt5a 启动子完全去甲基化，尤其是在 5 μmol/L 5-Aza-CdR 时。用去甲基化剂处理后，SKOV3 细胞中 Wnt5a 的表达增加（≤0.001）。（5）Wnt5a 基因启动子区异常甲基化存在于上皮性卵巢癌中，Wnt5a 基因启动子区异常甲基化可能成为上皮性卵巢癌治疗的新靶点。