The Overexpression of Hypomethylated miR-663 Induces Chemotherapy Resistance in Human Breast Cancer Cells by Targeting Heparin Sulfate Proteoglycan 2 (HSPG2)
The Overexpression of Hypomethylated miR-663 Induces Chemotherapy Resistance in Human Breast Cancer Cells by Targeting Heparin Sulfate Proteoglycan 2 (HSPG2)
复制标题
低甲基化 miR-663 的过度表达通过靶向硫酸肝素蛋白聚糖 2 (HSPG2) 诱导人乳腺癌细胞对化疗产生耐药性。
DOI：
10.1074/jbc.m112.434340
复制
发表时间：
2013-04-19
影响因子：
4.8
通讯作者：
Lin, Ling
中科院分区：
文献类型：
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作者：
Hu, Haiyan;Li, Shuqin;Lin, Ling
microRNA参与调节癌细胞的生物学，但它们在化学抗性中的作用尚未完全了解。我们发现miR-663在我们诱导的MDA-MB-231/ADM多药耐药细胞系中上调，并且这种上调与化疗敏感性密切相关。本研究旨在阐明miR-663在调节乳腺癌化疗耐药性中的作用。microRNA微阵列和定量RT-PCR分析用于鉴定差异表达的microRNA。通过膜联蛋白V/碘化丙啶染色、TUNEL和活性氧生成分析来评估细胞凋亡。采用原位杂交和免疫组化方法检测乳腺癌组织中miR-663和HSPG 2的表达。通过荧光素酶报告基因测定确定miR-663的潜在靶点。亚硫酸氢盐测序PCR分析甲基化状态。我们发现miR-663在MDA-MB-231/ADM细胞中显著升高，并且miR-663的下调使MDA-MB-231/ADM细胞对环磷酰胺和多西他赛都敏感。乳腺肿瘤组织中miR-663的过表达与化疗耐药性相关;在MDA-MB-231细胞中，这种化疗耐药性伴随着HSPG 2的下调，HSPG 2被鉴定为miR-663的靶点。MDA-MB-231/ADM细胞中甲基化CpG位点的数量少于其亲本细胞，并且5-aza-29-deoxycytidine处理后MDA-MB-231细胞中miR-663的表达被重新激活，表明DNA甲基化可能在miR-663的表达中发挥功能性作用。我们的研究结果表明，低甲基化miR-663的过度表达通过下调HSPG 2诱导乳腺癌细胞的化疗耐药性，从而为开发基于microRNA的乳腺癌治疗方法提供了潜在的靶点。
MicroRNAs are involved in regulating the biology of cancer cells, but their involvement in chemoresistance is not fully understood. We found that miR-663 was up-regulated in our induced multidrug-resistant MDA-MB-231/ADM cell line and that this up-regulation was closely related to chemosensitivity. In the present study, we aimed to clarify the role of miR-663 in regulating the chemoresistance of breast cancer. MicroRNA microarray and quantitative RT-PCR assays were used to identify differentially expressed microRNAs. Cell apoptosis was evaluated by annexin V/propidium iodide staining, TUNEL, and reactive oxygen species generation analysis. The expression of miR-663 and HSPG2 in breast cancer tissues was detected by in situ hybridization and immunohistochemistry. The potential targets of miR-663 were defined by a luciferase reporter assay. Bisulfite sequencing PCR was used to analyze the methylation status. We found that miR-663 was significantly elevated in MDA-MB-231/ADM cells, and the down-regulation of miR-663 sensitized MDA-MB-231/ADM cells to both cyclophosphamide and docetaxel. The overexpression of miR-663 in breast tumor tissues was associated with chemoresistance; in MDA-MB-231 cells, this chemoresistance was accompanied by the down-regulation of HSPG2, which was identified as a target of miR-663. MDA-MB-231/ADM contained fewer methylated CpG sites than its parental cell line, and miR-663 expression in MDA-MB-231 cells was reactivated by 5-aza-29-deoxycytidine treatment, indicating that DNA methylation may play a functional role in the expression of miR-663. Our findings suggest that the over-expression of hypomethylated miR-663 induced chemoresistance in breast cancer cells by down-regulating HSPG2, thus providing a potential target for the development of an microRNA-based approach for breast cancer therapy.