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

doi:10.11958/20221753

Abstract

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

CLC Number:

R739.4

Figures/Tables 9

References 20

[1]	FENG Z, MAO Z, WANG Z, et al. Non-adenomatous pituitary tumours mimicking functioning pituitary adenomas[J]. Br J Neurosurg, 2020, 34(5):487-491. doi:10.1080/02688697.2018.1464121.
[2]	KOBALKA P J, HUNTOON K, BECKER A P. Neuropathology of pituitary adenomas and sellar lesions[J]. Neurosurgery, 2021, 88(5):900-918. doi:10.1093/neuros/nyaa548.
[3]	ZHOU D, TANG W, XU Y, et al. METTL3/YTHDF2 m6A axis accelerates colorectal carcinogenesis through epigenetically suppressing YPEL5[J]. Mol Oncol, 2021, 15(8):2172-2184. doi:10.1002/1878-0261.12898.
[4]	MA Z, LI Q, LIU P, et al. METTL3 regulates m6A in endometrioid epithelial ovarian cancer independently of METTl14 and WTAP[J]. Cell Biol Int, 2020, 44(12):2524-2531. doi:10.1002/cbin.11459.
[5]	王天工, 叶孟. m-6A甲基化与肿瘤研究进展[J]. 遗传, 2018, 40(12):1055-1065.
	WANG T G, YE M. Advances on the roles of m6A in tumorigenesis[J]. Hereditas, 2018, 40(12):1055-1065. doi:10.16288/j.yczz.18-098.
[6]	CHANG M, WANG Z, GAO J, et al. METTL3-mediated RNA m6A hypermethylation promotes tumorigenesis and GH secretion of pituitary somatotroph adenomas[J]. J Clin Endocrinol Metab, 2022, 107(1):136-149. doi:10.1210/clinem/dgab652.
[7]	YAN W, HUANG J, ZHANG Q, et al. Role of metastasis suppressor KAI1/CD82 in different cancers[J]. J Oncol, 2021, 2021:9924473. doi:10.1155/2021/9924473.
[8]	AL-KHATER K M, ALMOFTY S, RAVINAYAGAM V, et al. Role of a metastatic suppressor gene KAI1/CD82 in the diagnosis and prognosis of breast cancer[J]. Saudi J Biol Sci, 2021, 28(6):3391-3398. doi:10.1016/j.sjbs.2021.03.001.
[9]	HE X, MA X, WANG C, et al. The peptide mimicking small extracellular ring domain of CD82 inhibits tumor cell migration in vitro and metastasis in vivo[J]. J Cancer Res Clin Oncol, 2021, 147(7):1927-1934. doi:10.1007/s00432-021-03595-6.
[10]	WANNA-UDOM S, TERASHIMA M, LYU H, et al. The m6A methyltransferase METTL3 contributes to transforming growth factor-beta-induced epithelial-mesenchymal transition of lung cancer cells through the regulation of JUNB[J]. Biochem Biophys Res Commun, 2020, 524(1):150-155. doi:10.1016/j.bbrc.2020.01.042.
[11]	LIU Y, LIU Z, TANG H, et al. The N6-methyladenosine (m6A)-forming enzyme METTL3 facilitates M1 macrophage polarization through the methylation of STAT1 mRNA[J]. Am J Physiol Cell Physiol, 2019, 317(4):C762-C775. doi:10.1152/ajpcell.00212.2019.
[12]	LIU L, WU Y, LI Q, et al. METTL3 promotes tumorigenesis and metastasis through BMI1 m6A methylation in oral squamous cell carcinoma[J]. Mol Ther, 2020, 28(10):2177-2190. doi:10.1016/j.ymthe.2020.06.024.
[13]	PÉREZ-LÓPEZ C, ÁLVAREZ-ESCOLÁ C, ISLA GUERRERO A. Therapeutic approach to non-functioning pituitary adenomas[J]. Med Clin (Barc), 2021, 156(6):284-289. doi:10.1016/j.medcli.2020.08.019.
[14]	LAMBACK E B, GUTERRES A, BARBOSA M A, et al. Cyclin A in nonfunctioning pituitary adenomas[J]. Endocrine, 2020, 70(2):380-387. doi:10.1007/s12020-020-02402-5.
[15]	YANG N, WANG T, LI Q, et al. HBXIP drives metabolic reprogramming in hepatocellular carcinoma cells via METTL3-mediated m6A modification of HIF-1α[J]. J Cell Physiol, 2021, 236(5):3863-3880. doi:10.1002/jcp.30128.
[16]	CHEN H, GAO S, LIU W, et al. RNA N6-methyladenosine methyltransferase METTL3 facilitates colorectal cancer by activating the m6A-GLUT1-mTORC1 axis and is a therapeutic target[J]. Gastroenterology, 2021, 160(4):1284-1300.e16. doi:10.1053/j.gastro.2020.11.013.
[17]	YANG Z, JIANG X, LI D, et al. HBXIP promotes gastric cancer via METTL3-mediated MYC mRNA m6A modification[J]. Aging (Albany NY), 2020, 12(24):24967-24982. doi:10.18632/aging.103767.
[18]	MILLER J, DREYER T F, BÄCHER A S, et al. Differential tumor biological role of the tumor suppressor KAI1 and its splice variant in human breast cancer cells[J]. Oncotarget, 2018, 9(5):6369-6390. doi:10.18632/oncotarget.23968.
[19]	KRISHNA LATHA T, VERMA A, THAKUR G K, et al. Down regulation of KAI1/CD82 in lymph node positive and advanced T-stage group in breast cancer patients[J]. Asian Pac J Cancer Prev, 2019, 20(11):3321-3329. doi:10.31557/APJCP.2019.20.11.3321.
[20]	HUANG C, HAYS F A, TOMASEK J J, et al. Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle-mediated release of ezrin to inhibit tumour cell movement[J]. J Extracell Vesicles, 2020, 9(1):1692417. doi:10.1080/20013078.2019.1692417.

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

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

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^＊＊

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^＊＊

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^＊＊