miR-32通过抑制靶基因KLF4促进胶质瘤的增殖和侵袭

Q4 Medicine

中华神经外科杂志 Pub Date : 2019-11-28 DOI:10.3760/CMA.J.ISSN.1001-2346.2019.11.020

Zhang Liang, Li Peidong, Jie Yang, Tong Luqing, Yi Li, Liu Jiabo, Zhang Jinhao, Zhang Yiming, Wang Xuya, Yang Xuejun

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引用次数: 0

摘要

目的探讨miR-32靶向KLF4影响胶质瘤细胞增殖和侵袭的分子机制。方法采用实时荧光定量PCR (RT-qPCR)检测miR-32在胶质细胞系(HA)和胶质瘤细胞系(U87、U373MG、U251)中的表达。通过细胞增殖(MTT)、细胞侵袭和划痕实验检测miR-32对U87胶质瘤细胞系的影响。随后，通过target Scan Human 7.1在线检测系统发现miR-32的靶基因，并通过双荧光素酶报告基因实验进行验证。验证成功后，采用RT-qPCR和Western blot (WB)检测miR-32对靶基因KLF4 mRNA的调控作用，并采用MTT、细胞侵袭和划痕实验检测靶基因KLF4对胶质瘤细胞株的影响。最后通过挽救实验探讨miR-32如何调控靶基因KLF4从而影响胶质瘤的增殖和侵袭。结果RT-qPCR结果显示，miR-32在U87、U373MG和U251细胞中的表达水平均显著高于HA细胞(P<0.01)。与转染miRNA-NC组比较，转染miR-32 mimic组U87细胞的miR-32表达水平、增殖、侵袭和迁移能力均显著上调(均P<0.05)。转染miR-32 inhibitor组miR-32的表达水平、增殖、侵袭和迁移水平均显著下调(P<0.05)。生物信息学和双荧光素酶报告结果显示，KLF4是miR-32的靶基因，当miR-32过表达时，细胞荧光素酶活性显著降低，KLF4表达下调。miR-32沉默后，荧光素酶活性显著升高，KLF4表达上调。KLF4在U87、U373MG和U251细胞中的表达均显著低于HA细胞(均P<0.01)。高表达KLF4可抑制胶质瘤细胞系的增殖、侵袭和迁移。拯救实验表明KLF4沉默逆转了miR-32沉默诱导的胶质瘤细胞增殖和侵袭的下调。KLF4过表达逆转了miR-32过表达诱导的胶质瘤细胞增殖和侵袭的促进作用。结论miR-32通过抑制其靶基因KLF4的表达促进胶质瘤细胞的增殖和侵袭。关键词:胶质瘤;细胞增殖;肿瘤侵袭性;miR-32;KLF4

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miR-32 promotes proliferation and invasion of glioma by inhibiting the target gene KLF4

Objective To explore the molecular mechanism through which miR-32 affects the proliferation and invasion of glioma cells by targeting KLF4. Methods The expression of miR-32 in glial cell lines (HA) and glioma cell lines (U87, U373MG, U251) was detected by real-time quantitative PCR (RT-qPCR). The effects of miR-32 on U87 glioma cell lines were detected by cell proliferation (MTT), cell invasion and scratch assay. Afterwards, the target gene of miR-32 was found by the Target Scan Human 7.1 online detection system and verified by the dual luciferase reporter assay. After successful verification, the RT-qPCR and Western blot (WB) assay were used to determine the regulation of target gene KLF4 mRNA by miR-32, and the effects of target gene KLF4 on glioma cell lines were detected by MTT, cell invasion and scratch assay. Finally, rescue experiments were used to explore how miR-32 regulated the target gene KLF4 and thus affected the proliferation and invasion of glioma. Results The results of RT-qPCR showed that the expression levels of miR-32 in U87, U373MG and U251 cells were significantly higher than those in HA cells (all P<0.01). Compared with the group transfected with miRNA-NC, the miR-32 expression level, proliferation, invasion and migration ability of U87 cells were significantly up-regulated in the group transfected with miR-32 mimic (all P<0.05). The expression levels of miR-32, proliferation, invasion and migration were significantly down-regulated in the group transfected with miR-32 inhibitor (all P<0.05). The bioinformatics and dual luciferase reporter results showed that KLF4 was a target gene of miR-32, and when miR-32 was overexpressed, the cell luciferase activity was significantly decreased, and the expression of KLF4 was down-regulated. When miR-32 was silenced, the luciferase activity was significantly increased, and the expression of KLF4 was up-regulated. The expression of KLF4 in U87, U373MG and U251 cells was significantly lower than that in HA cell lines (all P<0.01). The proliferation, invasion and migration of glioma cell lines were inhibited when KLF4 was highly expressed. The rescue experiments showed that KLF4 silencing reversed the down-regulation of proliferation and invasion of glioma cell induced by miR-32 silencing. Overexpression of KLF4 reversed the promotion of proliferation and invasion of glioma cells induced by miR-32 overexpression. Conclusion miR-32 promoted proliferation and invasion of glioma cells by inhibiting the expression of its target gene KLF4. Key words: Glioma; Cell proliferation; Neoplasm invasiveness; miR-32; KLF4

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来源期刊

中华神经外科杂志 Medicine-Surgery

CiteScore

0.10

自引率

0.00%

发文量

10706

期刊介绍： Chinese Journal of Neurosurgery is one of the series of journals organized by the Chinese Medical Association under the supervision of the China Association for Science and Technology. The journal is aimed at neurosurgeons and related researchers, and reports on the leading scientific research results and clinical experience in the field of neurosurgery, as well as the basic theoretical research closely related to neurosurgery.Chinese Journal of Neurosurgery has been included in many famous domestic search organizations, such as China Knowledge Resources Database, China Biomedical Journal Citation Database, Chinese Biomedical Journal Literature Database, China Science Citation Database, China Biomedical Literature Database, China Science and Technology Paper Citation Statistical Analysis Database, and China Science and Technology Journal Full Text Database, Wanfang Data Database of Medical Journals, etc.