Lv Lv, He Liang, Chen Shaohua, Yu Yaqun, Che Guosong, Tao Xuan, Wang Shengtao, Jian Zhiyuan, Zhang Xuemei
Department of Emergency and Trauma Surgery, Affiliated Hospital of Guilin Medical University, Guilin, China.
Department of Anesthesiology, Affiliated Hospital of Guilin Medical University, Guilin, China.
Front Oncol. 2019 Dec 17;9:1383. doi: 10.3389/fonc.2019.01383. eCollection 2019.
This study aimed to identify the roles of the long non-coding RNA in colorectal cancer (CRC) development. The expression levels of and miR-133b in CRC were determined by reverse transcription (RT)-polymerase chain reaction (PCR) and the functions of in CRC were evaluated and . Methylation-specific PCR assay was performed to detect the miR-133b promoter methylation in CRC cells. Bioinformatics analysis, RNA immunoprecipitation, dual luciferase assay, RNA pull-down, co-immunoprecipitation (IP), and chromatin IP (ChIP) assays were used to elucidate whether could recruit EZH2/DNMT1 and bind to the miR-133b promoter region, leading to dysregulated methylation and the depression of miR-133b. The expression levels of DNA methyltransferases (DNMTs), EZH2, and nucleoporin 214(NUP214) were analyzed by western blotting. Data showed that was highly expressed, whereas miR-133b was downregulated in the CRC tissues and cells. , silencing inhibited cell proliferation and impeded cell cycle at the G1/S phase by upregulating miR-133b. knockdown reduced tumor growth. Further analysis showed that the methylation in miR-133b promoter region was increased in the CRC and silencing increased miR-133b expression through depressing methylation of its promoter region. ChIP-PCR experiments demonstrated that EZH2 and DNMT1 could bind to the miR-133b promoter region and it was abolished by knockdown. sh-EZH2 reversed the overexpression of DNMTs and CRC cell cycle progression induced by the upregulation. LINC00114 could regulate the NUP214 protein expression by sponging miR-133b. These results demonstrated that suppressed miR-133b expression via EZH2/DNMT1-mediated methylation of its promoter region, indicating that might be a potential novel target for CRC diagnosis and treatment.
本研究旨在确定长链非编码RNA在结直肠癌（CRC）发生发展中的作用。通过逆转录（RT）-聚合酶链反应（PCR）测定CRC中[未提及的某个基因]和miR-133b的表达水平，并通过[未提及的某些实验]评估[未提及的某个基因]在CRC中的功能。采用甲基化特异性PCR检测CRC细胞中miR-133b启动子甲基化情况。运用生物信息学分析、RNA免疫沉淀、双荧光素酶测定、RNA下拉、免疫共沉淀（IP）和染色质免疫沉淀（ChIP）实验，以阐明[未提及的某个基因]是否能招募EZH2/DNMT1并与miR-133b启动子区域结合，导致甲基化失调和miR-133b表达受抑。通过蛋白质免疫印迹法分析DNA甲基转移酶（DNMTs）、EZH2和核孔蛋白214（NUP214）的表达水平。数据显示，[未提及的某个基因]在CRC组织和细胞中高表达，而miR-133b表达下调。[未提及的某个基因]沉默后，通过上调miR-133b抑制细胞增殖并使细胞周期阻滞在G1/S期。[未提及的某个基因]敲低可减少肿瘤生长。进一步分析表明，CRC中miR-133b启动子区域甲基化增加，[未提及的某个基因]沉默通过抑制其启动子区域甲基化增加miR-133b表达。ChIP-PCR实验表明，EZH2和DNMT1可与miR-133b启动子区域结合，而[未提及的某个基因]敲低可消除这种结合。sh-EZH2可逆转由[未提及的某个基因]上调诱导的DNMTs过表达和CRC细胞周期进程。LINC00114可通过吸附miR-133b调节NUP214蛋白表达。这些结果表明，[未提及的某个基因]通过EZH2/DNMT1介导的miR-133b启动子区域甲基化抑制其表达，提示[未提及的某个基因]可能是CRC诊断和治疗的潜在新靶点。
