受甲基化调控的lncRNA在乳腺癌中的表达谱及意义

目的：研究乳腺癌细胞去甲基化处理株和亲本株的lncRNA的差异表达情况，并从差异表达谱中筛选lncRNA，探讨受DNA启动子甲基化调控的lncRNA是否参与乳腺癌发生与发展。方法：取乳腺癌BT474细胞株去甲基化处理后，采用高通量lncRNA芯片技术分别检测去甲基化组和亲本组中lncRNA的表达谱，并在BT474、MB231和BT549三株乳腺癌细胞株及乳腺癌组织中应用qRT-PCR技术检测lncRNA的表达。同时寻找lncRNA的基因甲基化位点，并以重亚硫酸盐测序法（BSP）确定甲基化情况。结果：去甲基化株和亲本株lncRNA表达谱发生了显著的变化。qRT-PCR检测结果与芯片检测结果基本一致。qRT-PCR检测发现乳腺癌细胞株中lncRNA uc003lxs及uc002btf相比正常乳腺上皮细胞株表达升高（P＜0.01），乳腺癌组织中lncRNA uc003lxs及uc002btf相比癌旁组织表达升高（P＜0.01）。BSP方法显示相比正常乳腺上皮细胞株，乳腺癌细胞株中lncRNA uc003lxs及uc002btf DNA甲基化水平显著降低（P＜0.01）；相比癌旁组织，乳腺癌组织中lncRNA uc003lxs及uc002btf DNA甲基化水平显著降低（P＜0.01）。结论：乳腺癌细胞株去甲基化处理后lncRN表达谱发生显著变化，并在差异表达谱中筛选出lncRNA uc003lxs及uc002btf，为研究受甲基化调节的lncRNA调控乳腺癌发生发展奠定前期的研究基础。

Expression profile of methylation regulated lncRNA and its significance in breast cancer

Objective: To study the differential expression of lncRNA between demethylated breast cancer cells and their parental lines, and to screen lncRNA from differential expression profile, so as to explore the role of lncRNA regulated by DNA promoter methylation in the occurrence and development of breast cancer. Methods: After demethylation of breast cancer BT474 cell line, the expression profiles of lncRNA in demethylation group and parental group were detected by high-throughput lncRNA microarray technology, and the expression of lncRNA in breast cancer cell line BT474, MB231, BT549 and cancer tissue were detected by qRT-PCR technology. Meanwhile, the gene methylation sites were searched by bisulfite sequencing PCR. Results: LncRNA expression profiles of demethylated and parental cells have significantly changed. The results of quantitative RT-PCR and microarray were basically consistent. Quantitative RT-PCR showed that the expression of lncRNA uc003lxs and uc002btf in breast cancer tissues and breast cancer cell lines were higher than that in normal breast epithelial cell lines (P<0.01), and the expression of lncRNA uc003lxs and uc002btf in breast cancer tissues was higher than that in adjacent tissues (P<0.01). Compared with normal breast epithelial cell lines, the DNA methylation levels of lncRNA uc003lxs and uc002btf in breast cancer tissues and breast cancer cell lines were significantly decreased by BSP method (P<0.01); compared with adjacent tissues, the DNA methylation levels of lncRNA uc003lxs and uc002btf in breast cancer tissues were significantly decreased (P<0.01). Conclusion: The expression of lncRNA in breast cancer cell lines after demethylation treatment has been found to be significantly changed, and lncRNA uc003lxs and uc002btf have been screened out, which lays a preliminary research foundation for the study of methylation regulated lncRNA in the regulation of the occurrence and development of breast cancer.