Xu Cheng-Bo, Liao Bin, Fu Hai-Ying, Qi Yan, Shen Jian-Zhen
Department of Hematology, The Affiliated People's Hospital to Fujian University of Traditional Chinese Medicine, Fuzhou 350004, Fujian Province, China.
Department of Hematology, Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fuzhou 350001, Fujian Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Jun;30(3):790-796. doi: 10.19746/j.cnki.issn.1009-2137.2022.03.021.
To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells.
The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein.
Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05)，while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05).
MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.
探讨DNA甲基化介导的miR-203/CREB1信号调控对多发性骨髓瘤（MM）细胞增殖、侵袭及凋亡的影响。
采用亚硫酸氢盐测序聚合酶链反应（BSP）检测RPMI 8226细胞中miR-203的甲基化水平。通过定量实时聚合酶链反应检测miR-203的mRNA表达。用DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理RPMI 8226细胞。将miR-203模拟物转染至MM细胞系RPMI 8226中，建立miR-203过表达细胞。分别采用CCK-8法、Transwell法及流式细胞术检测RPMI 8226细胞的增殖、侵袭能力及凋亡情况。通过双荧光素酶报告基因检测验证miR-203与CREB1的靶向关系。采用蛋白质免疫印迹法检测CREB1蛋白的表达。
RPMI 8226细胞中miR-203启动子区域存在高甲基化，且miR-203 mRNA表达水平较低，提示去甲基化可诱导miR-203表达。5-Aza-CdR处理后，RPMI 8226细胞的增殖和侵袭能力受到抑制，与空白对照组相比差异有统计学意义（均P<0.05），而凋亡率升高（P<0.05）。miR-203过表达组的增殖、侵袭能力及凋亡情况与去甲基化组相同。双荧光素酶报告基因检测证实CREB1是miR-203的直接靶点。去甲基化抑制了CREB1的蛋白水平，与对照组相比差异有统计学意义（P<0.05）。
DNA甲基化介导的miR-203靶向CREB1可维持MM细胞的恶性生物学行为。
