m6a甲基转移酶METTL3调控KAI1/CD82表达介导垂体神经内分泌肿瘤细胞生物学行为的机制研究

doi:10.11958/20221753

天津医药 ›› 2023, Vol. 51 ›› Issue (8): 785-790.doi: 10.11958/20221753

• 细胞与分子生物学 • 下一篇

m6a甲基转移酶METTL3调控KAI1/CD82表达介导垂体神经内分泌肿瘤细胞生物学行为的机制研究

郑锴¹(), 罗秀玲², 张玮豪¹, 刘宇利¹, 李玉明¹, 廖尚高³

1 贵州医科大学附属医院神经外科（邮编550004）
2 贵州医科大学附属医院头颈肿瘤科（邮编550004）
3 贵州医科大学药学院

收稿日期:2022-10-27 修回日期:2022-12-02 出版日期:2023-08-15 发布日期:2023-08-10
作者简介:郑锴（1972），男，副主任医师，主要从事神经创伤与肿瘤方面研究。E-mail：ZK1972Z@163.com
基金资助:
贵州省高层次创新型人才资助项目(黔科合平台人才[2020]6011)

Mechanism of m6a methyltransferase METTL3 mediating the biological behavior of pituitary neuroendocrine tumor cells by regulating the expression of KAI1/CD82

ZHENG Kai¹(), LUO Xiuling², ZHANG Weihao¹, LIU Yuli¹, LI Yuming¹, LIAO Shanggao³

1 Department of Neurosurgery, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China
2 Department of Head and Neck Oncology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China
3 School of Pharmacy, Guizhou Medical University

Received:2022-10-27 Revised:2022-12-02 Published:2023-08-15 Online:2023-08-10

郑锴, 罗秀玲, 张玮豪, 刘宇利, 李玉明, 廖尚高. m6a甲基转移酶METTL3调控KAI1/CD82表达介导垂体神经内分泌肿瘤细胞生物学行为的机制研究[J]. 天津医药, 2023, 51(8): 785-790.

ZHENG Kai, LUO Xiuling, ZHANG Weihao, LIU Yuli, LI Yuming, LIAO Shanggao. Mechanism of m6a methyltransferase METTL3 mediating the biological behavior of pituitary neuroendocrine tumor cells by regulating the expression of KAI1/CD82[J]. Tianjin Medical Journal, 2023, 51(8): 785-790.

摘要/Abstract

摘要：

目的研究m6a甲基转移酶METTL3调控KAI1/CD82表达介导垂体神经内分泌肿瘤细胞增殖、迁移与侵袭的机制。方法通过实时荧光聚合酶链反应（qPCR）和蛋白免疫印迹测定大鼠垂体细胞、大鼠垂体神经内分泌肿瘤细胞系GH3和MMQ中的METTL3与KAI1/CD82表达水平。体外培养GH3细胞，将其随机分为对照组、METTL3过表达质粒组、METTL3空质粒组、METTL3 siRNA组、METTL3 siRNA阴性对照组，经分组转染后，通过qPCR和蛋白免疫印迹检测各组细胞METTL3与KAI1/CD82表达；通过CCK-8实验检测各组细胞活力；通过细胞划痕、Transwell侵袭实验检测各组细胞迁移侵袭情况；通过甲基化RNA免疫共沉淀（MeRIP）实验检测各组细胞KAI1/CD82 m6A甲基化修饰情况。结果相比大鼠垂体细胞，大鼠垂体神经内分泌肿瘤细胞系GH3及MMQ中的METTL3蛋白及mRNA表达水平明显升高，KAI1/CD82蛋白及mRNA表达水平明显降低（P<0.05）。与对照组相比，METTL3空质粒组、METTL3 siRNA阴性对照组细胞各指标差异无统计学意义；与对照组、METTL3空质粒组相比，METTL3过表达质粒组细胞活力、迁移距离、侵袭细胞数、METTL3蛋白及mRNA表达水平、KAI1/CD82 m6A甲基化水平升高（P<0.05），KAI1/CD82蛋白及mRNA表达水平降低（P<0.05）；与对照组、METTL3 siRNA阴性对照组相比，METTL3 siRNA组细胞活力、迁移距离、侵袭细胞数、METTL3蛋白及mRNA表达水平、KAI1/CD82 m6A甲基化水平降低（P<0.05），KAI1/CD82蛋白及mRNA表达水平升高（P<0.05）。结论下调METTL3表达可降低KAI1/CD82 m6A甲基化水平，促进KAI1/CD82 mRNA转录表达，抑制垂体神经内分泌肿瘤细胞增殖、侵袭及迁移。

关键词: 垂体肿瘤, 神经内分泌瘤, 甲基转移酶类, Kangai-1蛋白质, 甲基化, m6a甲基转移酶, 甲基转移酶样3

Abstract:

Objective To study the mechanism of m6a methyltransferase METL3 mediated proliferation, migration and invasion of pituitary neuroendocrine tumor cells by regulating the expression of KAI1/CD82. Methods The expression levels of METTL3 and KAI1/CD82 in rat pituitary cells, rat pituitary tumor cell lines GH3, and MMQ were determined by qPCR and Western blot assay. GH3 cells were cultured in vitro and randomly divided into the control group, the METL3 overexpression plasmid group, the METL3 empty plasmid group, the METL3 siRNA group and the METL3 siRNA negative control group. After grouping and transfection, the expression levels of METL3 and KAI1/CD82 of each cell group were detected by qPCR and Western blot assay. The cell viability of each group was detected by CCK-8 experiment. The cell migration and invasion in each group were detected by cell scratch and Transwell invasion experiment. The methylation modification of KAI1/CD82 m6A in each group was detected by methylated RNA immunoprecipitation (MeRIP) experiment. Results Compared with rat pituitary cells, the METL3 protein and mRNA expression level in rat pituitary tumor cell lines GH3 and MMQ were significantly increased (P<0.05). KAI1/CD82 protein and mRNA expression levels were significantly reduced (P<0.05). There were no significant differences in cell indicators between the control group, the METTL3 empty plasmid group and the METTL3 siRNA negative control group (P>0.05). Compared with the control group and the METTL3 empty plasmid group, the cell viability, migration distance, number of invaded cells, METL3 protein and mRNA expression level, KAI1/CD82 m6A methylation level increased in the METL3 overexpression plasmid group (P<0.05), and the KAI1/CD82 protein and mRNA expression levels decreased (P<0.05). Compared with the control group and the METTL3 siRNA negative control group, the cell viability, migration distance, number of invaded cells, METTL3 protein and mRNA expression level and KAI1/CD82 m6A methylation level decreased in the METTL3 siRNA group (P<0.05), and KAI1/CD82 protein and mRNA expression levels increased (P<0.05). Conclusion Down-regulating the expression of METTL3 can reduce the level of KAI1/CD82 m6A methylation, promote the transcriptional expression of KAI1/CD82 mRNA, reduce the proliferation, invasion and migration of pituitary tumor cells, and inhibit the occurrence and development of pituitary tumors.

Key words: pituitary neoplasms, neuroendocrine tumors, methyltransferases, Kangai-1 protein, methylation, m6a methyltransferase, methyltransferase-like 3

中图分类号:

R739.4

图/表 9

表1 qPCR引物序列

Tab.1 Primer sequence for qPCR

基因名称	引物序列(5'→3')	产物大小（bp）
METTL3	上游：CAAGCTGCACTTCAGACGAA 下游：GCTTGGCGTGTGGTCTTT	128
KAI1/CD82	上游：AGCCTGTATCAAAGTCACCAAAT 下游：TTGCAGGACAGAGATGAAACTG	92
β-actin	上游：CAAGGACCTCTACGCCAACAC 下游：TGGAGGCGCGATGATCTT	114

图1 蛋白免疫印迹检测大鼠垂体细胞与GH3、MMQ细胞METTL3、KAI1/CD82表达

Fig.1 Expression of METTL3 and KAI1/CD82 in rat pituitary cells, GH3 and MMQ cells detected by Western blot assay

表2 垂体神经内分泌肿瘤与正常垂体组织细胞中METTL3、KAI1/CD82蛋白及mRNA相对表达水平（n=6，$\bar{x}±s$）

Tab.2 Relative expression levels of METTL3, KAI1/CD82 protein and mRNA in pituitary neuroendocrine tumors and normal pituitary tissue cells

组别	METTL3 mRNA	KAI1/CD82 mRNA	METTL3 蛋白	KAI1/CD82 蛋白
大鼠垂体细胞	0.99±0.14	1.02±0.20	0.48±0.05	1.78±0.13
GH3细胞	2.75±0.56^a	0.39±0.05^a	1.21±0.16^a	0.54±0.06^a
MMQ细胞	2.34±0.41^a	0.42±0.11^a	1.15±0.12^a	0.60±0.09^a
F	30.450^＊＊	41.637^＊＊	69.558^＊＊	307.720^＊＊

图2 蛋白免疫印迹检测各组GH3细胞METTL3与KAI1/CD82蛋白表达 A：对照组；B：METTL3空质粒组；C：METTL3过表达质粒组；D：METTL3 siRNA阴性对照组；E：METTL3 siRNA组。

Fig.2 Western blot detection of METTL3 and KAI1/CD82 protein expression in GH3 cells of each group

表3 各组细胞METTL3与KAI1/CD82蛋白及mRNA水平（n=6，$\bar{x}±s$）

Tab.3 Protein and mRNA levels of METTL3 and KAI1/CD82 in cells of each group

组别	METTL3 mRNA	KAI1/CD82 mRNA	METTL3蛋白	KAI1/CD82蛋白
对照组	1.01±0.15	1.00±0.13	0.81±0.13	1.07±0.11
METTL3空质粒组	0.99±0.16	1.01±0.12	0.79±0.14	1.08±0.13
METTL3过表达质粒组	2.73±0.43^ab	0.41±0.08^ab	1.43±0.30^ab	0.24±0.03^ab
METTL3 siRNA阴性对照组	1.02±0.24	0.98±0.19	0.82±0.15	1.09±0.12
METTL3 siRNA组	0.42±0.11^ac	1.08±0.25^ac	0.15±0.01^ac	1.63±0.20^ac
F	75.722^＊＊	16.558^＊＊	41.248^＊＊	88.210^＊＊

表4 各组GH3细胞活力、迁移和侵袭能力以及KAI1/CD82 mRNA的m6A甲基化水平比较

Tab.4 Comparison of GH3 cell viability, migration and invasion ability and the level of m6A methylation of KAI1/CD82 mRNA between the five groups

组别	细胞活力/%	迁移距离/μm	侵袭细胞数/（个/视野）	m6A甲基化水平
对照组	100.00±0.00	149.87±12.56	89.37±13.12	0.61±0.13
METTL3空质粒组	99.75±23.21	152.95±14.01	92.16±19.31	0.63±0.16
METTL3过表达质粒组	178.96±36.58^ab	234.21±32.07^ab	197.48±30.27^ab	1.28±0.15^ab
METTL3 siRNA阴性对照组	100.12±25.04	150.19±15.04	91.05±21.36	0.58±0.14
METTL3 siRNA组	43.97±10.12^ac	83.43±10.12^ac	18.53±2.84^ac	0.22±0.03^ac
F	26.677^＊＊	50.170^＊＊	63.753^＊＊	50.481^＊＊

图3 细胞划痕实验检测各组GH3细胞迁移情况（比例尺：100 μm）

Fig.3 The migration of GH3 cells in each group detected by cell scratch assay (scale:100 μm）

图4 Transwell侵袭实验检测各组GH3细胞侵袭情况（×400）

Fig.4 The invasion of GH3 cells in each group detected by Transwell invasion test (×400)

图5 通过SRAMP数据库预测KAI1/CD82基因存在多个m6A甲基化修饰位点

Fig.5 Multiple m6A methylation modification sites in KAI1/CD82 gene inferred from SRAMP database

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m6a甲基转移酶METTL3调控KAI1/CD82表达介导垂体神经内分泌肿瘤细胞生物学行为的机制研究

Mechanism of m6a methyltransferase METTL3 mediating the biological behavior of pituitary neuroendocrine tumor cells by regulating the expression of KAI1/CD82

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