PRMT7通过调控Notch信号转导通路抑制膀胱癌细胞增殖和迁移

doi:10.19401/j.cnki.1007-3639.2023.05.003

摘要/Abstract

摘要：

背景与目的：膀胱癌是常见的泌尿系统恶性肿瘤之一，蛋白质精氨酸甲基转移酶7（protein arginine methyltransferase 7，PRMT7）在消化道肿瘤中的作用已有研究报道，但在膀胱癌中的生物学作用及机制尚未明确，本文旨在探究PRMT7对人膀胱癌细胞增殖和迁移能力的影响及其可能的作用机制。方法：体外培养人膀胱癌细胞系5637、T24、RT112、UM-UC-3和输尿管上皮永生化细胞系SV-HUC-1，采用蛋白质印迹法（Western blot）检测PRMT7的表达情况。采用RNA干扰技术沉默PRMT7基因的表达，设立阴性对照组（si-NC）及实验组（siPRMT7#1、siPRMT7#2），采用质粒转染法过表达PRMT7基因，设立阴性对照组（p-PCMV3）及实验组（p-PRMT7）。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）及Western blot验证PRMT7的转染效率，采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）和集落形成实验检测细胞增殖能力，采用transwell实验检测细胞迁移能力，采用Western blot检测增殖和迁移相关靶蛋白Cyclin D1、CDK4和MMP9以及Notch信号转导通路关键蛋白Notch1、HEY1和Hes1的表达情况。使用转染siPRMT7#2的细胞加入Notch信号特异性抑制剂γ-分泌酶抑制剂DAPT，进一步验证PRMT7在膀胱癌中的表达情况。结果：Western blot结果显示，PRMT7在膀胱癌细胞中低表达（P<0.05）。在5637细胞中沉默PTMT7后，细胞增殖和迁移能力显著增强（P<0.05），增殖和迁移相关靶蛋白Cyclin D1、CDK4和MMP9以及Notch信号通路关键蛋白Notch1、HEY1和Hes1的表达明显增加（P <0.05）；而在T24细胞中过表达PRMT7后，呈相反的趋势。使用DAPT后，明显逆转了PRMT7在膀胱癌细胞中的表达。结论：PRMT7抑制膀胱癌细胞增殖和迁移，其作用机制与Notch信号转导通路有关。

关键词: 膀胱癌, 蛋白质精氨酸甲基转移酶7, 增殖, 迁移, Notch信号通路

Abstract:

Background and purpose: Bladder cancer is one of the common tumor of the urinary system. The protein arginine methyltransferase 7 (PRMT7) has been reported in gastrointestinal tumors, but its biological role and mechanism in bladder cancer are still unknown. In this study, the effect of PRMT7 on the proliferation and migration of human bladder cancer cells and its possible mechanism were investigated. Methods: Human bladder cancer cell lines 5637, T24, RT112, UM-UC-3 and ureteral epithelial immortalized cells SV-HUC-1 were cultured in vitro, and the expression of PRMT7 was detected by Western blot. The expression of PRMT7 gene was silenced by RNA interference, and the negative control group (si-NC) and experimental group (siPRMT7#1 and siPRMT7#2) were established. PRMT7 gene were overexpressed by plasmid transfection, and the negative control group (p-PCMV3) and experimental group (p-PRMT7) were established. The transfection efficiency of PRMT7 was verified by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot, cell proliferation was detected by cell counting kit-8 (CCK-8) assay and colony formation assay, and cell migration was detected by Transwell assay. Western blot was used to detect the expressions of proliferation and migration related target proteins including Cyclin D1, CDK4 and MMP9, as well as the key proteins Notch1, HEY1 and Hes1 in Notch signaling pathway. Notch signaling specific inhibitors γ-secretase inhibitor DAPT was added to siPRMT7#2 transfected cells to further verify PRMT7 expression in bladder cancer. Results: Western blot results showed that PRMT7 expression was low in bladder cancer cells (P<0.05). After silencing PTMT7 in 5637 cells, cell proliferation and migration were significantly enhanced (P<0.05), and the expressions of proliferation and migration related target proteins including Cyclin D1, CDK4 and MMP9, and the key proteins of Notch signaling pathway Notch1, HEY1 and Hes1 were significantly increased (P<0.05). The overexpression of PRMT7 in T24 cells showed an opposite trend. DAPT significantly reversed the expression of PRMT7 in bladder cancer cells. Conclusion: PRMT7 inhibits the proliferation and migration of bladder cancer cells, and its mechanism is related to Notch signaling pathway.

Key words: Bladder Cancer, Protein arginine methyltransferase 7, Proliferation, Migration, Notch signaling pathway

中图分类号:

R737.14

王雪梅, 程玉, 齐洁敏. PRMT7通过调控Notch信号转导通路抑制膀胱癌细胞增殖和迁移[J]. 中国癌症杂志, 2023, 33(5): 437-444.

WANG Xuemei, CHENG Yu, QI Jiemin. PRMT7 inhibits proliferation and migration of bladder cancer cells by regulating Notch signaling pathway[J]. China Oncology, 2023, 33(5): 437-444.

图/表 5

参考文献 19

[1]	SUNG H, FERLAY J, SIEGEL R L, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249. doi: 10.3322/caac.v71.3
[2]	钟键, 金正贤, 卞卫星, 等. FGFR3和PIK3CA基因突变影响膀胱癌的预后[J]. 中国癌症杂志, 2019, 29(11): 880-886.
	ZHONG J, JIN Z X, BIAN W X, et al. Effects of FGFR3 and PIK3CA mutations on prognosis of bladder carcinoma[J]. China Oncol, 2019, 29(11): 880-886.
[3]	LENIS A T, LEC P M, CHAMIE K, et al. Bladder cancer: a review[J]. JAMA, 2020, 324(19): 1980-1991. doi: 10.1001/jama.2020.17598 pmid: 33201207
[4]	CHEN X, XIE R H, GU P, et al. Long noncoding RNA LBCS inhibits self-renewal and chemoresistance of bladder cancer stem cells through epigenetic silencing of SOX2[J]. Clin Cancer Res, 2019, 25(4): 1389-1403. doi: 10.1158/1078-0432.CCR-18-1656
[5]	XU J, RICHARD S. Cellular pathways influenced by protein arginine methylation: implications for cancer[J]. Mol Cell, 2021, 81(21): 4357-4368. doi: 10.1016/j.molcel.2021.09.011 pmid: 34619091
[6]	HE H C, CHEN J L, ZHAO J, et al. PRMT7 targets of Foxm1 controls alveolar myofibroblast proliferation and differentiation during alveologenesis[J]. Cell Death Dis, 2021, 12(9): 841. doi: 10.1038/s41419-021-04129-1 pmid: 34497269
[7]	YAO R S, JIANG H, MA Y H, et al. PRMT7 induces epithelial-to-mesenchymal transition and promotes metastasis in breast cancer[J]. Cancer Res, 2014, 74(19): 5656-5667. doi: 10.1158/0008-5472.CAN-14-0800 pmid: 25136067
[8]	ASTER J C, PEAR W S, BLACKLOW S C. The varied roles of Notch in cancer[J]. Annu Rev Pathol, 2017, 12: 245-275. doi: 10.1146/annurev-pathol-052016-100127 pmid: 27959635
[9]	LI W J, HE Y H, YANG J J, et al. Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth[J]. Nat Commun, 2021, 12(1): 1946. doi: 10.1038/s41467-021-21963-1
[10]	JEONG H J, LEE S J, LEE H J, et al. PRMT7 promotes myoblast differentiation via methylation of p38MAPK on arginine residue 70[J]. Cell Death Differ, 2020, 27(2): 573-586. doi: 10.1038/s41418-019-0373-y
[11]	CHEN M, WANG Y B, LIN L M, et al. PRMT7 is involved in regulation of germ cell proliferation during embryonic stage[J]. Biochem Biophys Res Commun, 2020, 533(4): 938-944. doi: 10.1016/j.bbrc.2020.09.099
[12]	LIU L X, ZHANG X L, DING H Y, et al. Arginine and lysine methylation of MRPS23 promotes breast cancer metastasis through regulating OXPHOS[J]. Oncogene, 2021, 40(20): 3548-3563. doi: 10.1038/s41388-021-01785-7 pmid: 33927350
[13]	SHEN T, NI T, CHEN J X, et al. An enhancer variant at 16q22.1 predisposes to hepatocellular carcinoma via regulating PRMT7 expression[J]. Nat Commun, 2022, 13(1): 1232. doi: 10.1038/s41467-022-28861-0 pmid: 35264579
[14]	ROPER N, VELEZ M J, CHIAPPORI A, et al. Notch signaling and efficacy of PD-1/PD-L1 blockade in relapsed small cell lung cancer[J]. Nat Commun, 2021, 12(1): 3880. doi: 10.1038/s41467-021-24164-y pmid: 34162872
[15]	GENG Y W, FAN J, CHEN L Y, et al. A Notch-dependent inflammatory feedback circuit between macrophages and cancer cells regulates pancreatic cancer metastasis[J]. Cancer Res, 2021, 81(1): 64-76. doi: 10.1158/0008-5472.CAN-20-0256 pmid: 33172931
[16]	HE H D, SHAO X N, LI Y N, et al. Targeting signaling pathway networks in several malignant tumors: progresses and challenges[J]. Front Pharmacol, 2021, 12: 675675. doi: 10.3389/fphar.2021.675675
[17]	ZHOU B H, LIN W L, LONG Y L, et al. Notch signaling pathway: architecture, disease, and therapeutics[J]. Signal Transduct Target Ther, 2022, 7(1): 95.
[18]	HARADA Y, YAMADA M, IMAYOSHI I, et al. Cell cycle arrest determines adult neural stem cell ontogeny by an embryonic Notch-nonoscillatory Hey1 module[J]. Nat Commun, 2021, 12(1): 6562. doi: 10.1038/s41467-021-26605-0 pmid: 34772946
[19]	ZHU J J, LI X, CAI X L, et al. Arginine monomethylation by PRMT7 controls MAVS-mediated antiviral innate immunity[J]. Mol Cell, 2021, 81(15): 3171-3186.e8. doi: 10.1016/j.molcel.2021.06.004