Department of Image Diagnostics, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China.
Department of Dermatology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China.
Neoplasma. 2019 Jul 23;66(4):543-554. doi: 10.4149/neo_2018_180830N659. Epub 2019 Mar 7.
In the present study, a series of studies were conducted to explore the function of miR-374b in glioma and the regulatory relationship among miR-374b, GATA3 and SEMA3B. In the present study, miR-374b mimics and inhibitors were employed to regulate miR-374b expression. Besides, qRT-PCR assay was used for detecting the expression level of miR-374b, GATA3 and SEMAB mRNAs. To verify the targeting relationship between miR-374b and GATA3, dual luciferase analysis was utilized. Moreover, chromatin immunoprecipitationn (ChIP) assay was performed for identify the correlation of GATA3 with SEMA3B. Furthermore, si1-GATA3, si2-GATA3 and pc-GATA3 were used to regulate GATA3 expression, and pc-SEMA3B was taken advantage for dysregulating SEMA3B. For assessing the significance of miR374b alone or co-operated with GATA3 or SEMA3B in cell viability, migration and apoptosis, CCK-8, transwell and FCM assay were performed, respectively. We found that overexpression of miR-374b, which was identified in glioma tissues and cell lines, U251, LN-299 and GOS-3, promoting cell migration and enhancing cell viability but inhibiting cell apoptosis were suggested in this research. Besides, GATA3 contributed to increase in cell viability and migration and decrease in cell apoptosis targeted by miR-374b as evidenced by dual luciferase assay. Moreover, GATA3 binding to the promoter of SEMA3B involved in regulating SEMA3B was revealed. Further, a series of studies demonstrated that miR-374b targeting GATA3 regulating SMEA3B resulted in elevation in cell viability and migration but suppression in cell apoptosis. But the promotion effects of miR-374 in glioma process were reversed by co-transfecting pc-GATA3 or pc-SEMA3B. In conclusion, miR-374b promotes glioma process in vitro through suppressing SEMA3B via targeting GATA3. The result of this study provides an important clue to the optimal treatment schedule for glioblastoma.
在本研究中，进行了一系列研究以探讨 miR-374b 在神经胶质瘤中的功能以及 miR-374b、GATA3 和 SEMA3B 之间的调控关系。在本研究中，使用 miR-374b 模拟物和抑制剂来调节 miR-374b 的表达。此外，qRT-PCR 测定用于检测 miR-374b、GATA3 和 SEMAB mRNA 的表达水平。为了验证 miR-374b 与 GATA3 之间的靶向关系，使用了双荧光素酶分析。此外，进行染色质免疫沉淀（ChIP）测定以鉴定 GATA3 与 SEMA3B 的相关性。进一步，使用 si1-GATA3、si2-GATA3 和 pc-GATA3 来调节 GATA3 的表达，并利用 pc-SEMA3B 来失调 SEMA3B。为了评估 miR374b 单独或与 GATA3 或 SEMA3B 共同作用在细胞活力、迁移和凋亡中的意义，分别进行了 CCK-8、Transwell 和 FCM 测定。我们发现，在神经胶质瘤组织和细胞系 U251、LN-299 和 GOS-3 中，miR-374b 的过表达促进细胞迁移，增强细胞活力，但抑制细胞凋亡。此外，双荧光素酶测定表明，GATA3 通过靶向 miR-374b 增加细胞活力和迁移，减少细胞凋亡。此外，揭示了 GATA3 与 SEMA3B 启动子结合参与调节 SEMA3B。进一步的一系列研究表明，miR-374b 通过靶向 GATA3 调节 SMEA3B 导致细胞活力和迁移增加，细胞凋亡减少。但是，共转染 pc-GATA3 或 pc-SEMA3B 逆转了 miR-374 在神经胶质瘤过程中的促进作用。总之，miR-374b 通过靶向 GATA3 抑制 SEMA3B 促进体外神经胶质瘤的发生。本研究的结果为胶质母细胞瘤的最佳治疗方案提供了重要线索。
