Department of Neurosurgery, The Second Affiliated Hospital of XinJiang Medical University, Urumqi, China.
Eur Rev Med Pharmacol Sci. 2020 Feb;24(4):1922-1931. doi: 10.26355/eurrev_202002_20370.
This study aimed to explore the effects of microRNA-29b (miR-29b) on chemoresistance of glioma and to examine the underlying mechanisms.
MiR-29b expression in glioma tissues and cell lines was analyzed by quantitative real time-polymerase chain reaction (qRT-PCR). The cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was analyzed by Annexin V-Fluorescein isothiocyanate (FITC) assay. The relationship between miR-29b and signal transducer and activator of transcription 3 (STAT3) was examined by the Dual-Luciferase reporter gene assay. The levels of cleaved caspase-3, Bax, Bcl-2, and STAT3 were detected by Western blotting assay.
The expression of miR-29b was downregulated in glioma tissues compared to normal brain tissue. In addition, the expression level of miR-29b was lower in glioma tissues from patients at late stages (III and IV) compared with early stages (I and II). Besides, miR-29b expression was significantly lower in LN229, U87MG, and U251 cells compared to normal human astrocytes (NHA) cells. Moreover, our results showed that miR-29b expression in Temozolomide (TMZ)-resistance cell lines U251/TMZ and U87MG/TMZ was markedly lower than that of TMZ-sensitivity cell lines U251 and U87MG. The protein levels of STAT3 and the phosphorylation of STAT3 were increased in U251/TMZ and U87MG/TMZ compared to U251 and U87MG. When the expression of miR-29b was repressed, cell viability was increased. Meanwhile, cell apoptosis was reduced, the protein levels of cleaved caspase-3 and (Bcl-2 Associated X Protein) Bax were decreased, whereas the protein level of B-cell lymphoma 2 (Bcl-2) was increased. Moreover, the effects of miR-29b knockdown on the cell growth and apoptosis in U251 and U87MG cells were markedly attenuated by knockdown of STAT3. In TMZ-resistant U251/TMZ and U87MG/TMZ cells, transfection with miR-29b decreased cell growth, promoted apoptotic cell death, elevated the protein levels of cleaved caspase-3, and Bax protein, while downregulated Bcl-2 protein. As expected, the effect of miR-29b upregulation on cell growth and apoptosis of TMZ-resistant glioma cells was reversed by STAT3 overexpression. The results from the Luciferase assay demonstrated miR-29b modulated STAT3 expression by directly bound with 3'-Untranslated Region (3'-UTR).
MiR-29b enhances the cell sensitivity to TMZ by inhibiting STAT3 in glioma. Our study might provide a novel target for treating TMZ-resistant glioma.
本研究旨在探讨 microRNA-29b (miR-29b) 对神经胶质瘤化疗耐药性的影响，并探讨其潜在机制。
通过实时定量聚合酶链反应 (qRT-PCR) 分析神经胶质瘤组织和细胞系中 miR-29b 的表达。通过细胞计数试剂盒-8 (CCK-8) 测定细胞活力。通过 Annexin V-Fluorescein isothiocyanate (FITC) 测定细胞凋亡。通过双荧光素酶报告基因检测 miR-29b 与信号转导和转录激活因子 3 (STAT3) 之间的关系。通过 Western blot 检测裂解的 caspase-3、Bax、Bcl-2 和 STAT3 的水平。
与正常脑组织相比，miR-29b 在神经胶质瘤组织中的表达下调。此外，晚期 (III 和 IV 期) 神经胶质瘤组织中 miR-29b 的表达水平低于早期 (I 和 II 期)。此外，与正常星形胶质细胞 (NHA) 细胞相比，LN229、U87MG 和 U251 细胞中 miR-29b 的表达明显降低。此外，我们的研究结果表明，在 Temozolomide (TMZ) 耐药细胞系 U251/TMZ 和 U87MG/TMZ 中，miR-29b 的表达明显低于 TMZ 敏感细胞系 U251 和 U87MG。与 U251 和 U87MG 相比，U251/TMZ 和 U87MG/TMZ 中 STAT3 的蛋白水平和磷酸化 STAT3 增加。当抑制 miR-29b 的表达时，细胞活力增加。同时，细胞凋亡减少，裂解的 caspase-3 和 (Bcl-2 相关 X 蛋白) Bax 的蛋白水平降低，而 B 细胞淋巴瘤 2 (Bcl-2) 的蛋白水平升高。此外，STAT3 敲低明显减弱了 miR-29b 敲低对 U251 和 U87MG 细胞生长和凋亡的影响。在 TMZ 耐药的 U251/TMZ 和 U87MG/TMZ 细胞中，miR-29b 的转染降低了细胞生长，促进了凋亡性细胞死亡，上调了裂解的 caspase-3 和 Bax 蛋白水平，而下调了 Bcl-2 蛋白水平。预期，STAT3 过表达逆转了 miR-29b 上调对 TMZ 耐药神经胶质瘤细胞生长和凋亡的影响。荧光素酶检测结果表明，miR-29b 通过直接与 3'-非翻译区 (3'-UTR) 结合来调节 STAT3 的表达。
miR-29b 通过抑制 STAT3 增强神经胶质瘤对 TMZ 的细胞敏感性。我们的研究为治疗 TMZ 耐药性神经胶质瘤提供了一个新的靶点。
