蛋白精氨酸甲基转移酶5在肺癌中的表达及其促进肺癌的作用机制

doi:10.3969/j.issn.1674-8115.2021.08.003

摘要/Abstract

摘要：

目的·探讨蛋白精氨酸甲基转移酶5（protein arginine methyltransferase 5，PRMT5）在肺癌中的表达及其对肺癌细胞增殖、迁移、侵袭能力的作用。方法·采用免疫组织化学法检测73对肺癌及癌旁组织中PRMT5的表达，并与患者的临床特征行相关性分析。在肺癌细胞NCI-H1299中下调PRMT5后，通过CCK8法和Transwell小室观察PRMT5对细胞增殖、迁移和侵袭功能的影响，并采用蛋白质印迹法检测上皮细胞-间充质转化相关蛋白（E钙黏蛋白、波形蛋白和转录因子SNAI1）的表达。利用公共数据库GEO（Gene Expression Omnibus，基因表达综合）和TCGA（The Cancer Genome Atlas，癌症基因组图谱）分别分析PRMT5在肿瘤细胞系、肺癌组织中的表达，并进一步分析组织中PRMT5表达和肺癌患者预后的关系。结果·免疫组织化学法检测结果显示，PRMT5在肺癌组织中的表达显著高于癌旁组织（P=0.000），且在肿瘤Ⅲ~Ⅳ期的表达高于Ⅰ~Ⅱ期（P=0.033）。下调PRMT5后，NCI-H1299细胞的增殖、迁移和侵袭能力下降，E钙黏蛋白表达增加、波形蛋白及转录因子SNAI1表达减少。TCGA数据库的肺癌测序数据显示PRMT5在肺癌组织中表达显著高于正常肺组织；且PRMT5高表达的肺腺癌患者总生存率较低（P=0.005），PRMT5高表达的肺鳞癌患者无复发生存率较低（P=0.028）。结论·PRMT5在肺癌组织中表达较高，可促进肺癌细胞的增殖、迁移和侵袭，PRMT5高表达和肺癌的不良预后有关。

关键词: 蛋白精氨酸甲基转移酶5, 肺癌, 增殖, 迁移, 上皮细胞-间充质转化

Abstract:

Objective·To investigate the expression of protein arginine methyltransferase 5 (PRMT5) in lung cancer and its effect on proliferation, migration and invasion of lung cancer cells.

Methods· Immunohistochemistry was used to detect the expression of PRMT5 in 73 pairs of lung cancer tissues and adjacent tissues, and its correlation with clinical characteristics of patients was analyzed. After PRMT5 was down-regulated in lung cancer cell line NCI-H1299, the effects of PRMT5 on cell proliferation, migration and invasion were observed by CCK8 assay and Transwell chamber, and the expressions of E-cadherin, vimentin and transcription factor SNAI1 were detected by Western blotting. The expression of PRMT5 in tumor cell lines and lung cancer tissues was analyzed by using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database, respectively, and the relationship between the expression of PRMT5 in tissues and the prognosis of lung cancer patients was further analyzed.

Results·Immunohistochemistry showed that the expression of PRMT5 in lung cancer tissues was significantly higher than that in adjacent tissues (P=0.000), and the expression of PRMT5 in stage Ⅲ?Ⅳ was higher than that in stage Ⅰ?Ⅱ (P=0.033). After PRMT5 down-regulation, the proliferation, migration and invasion ability of NCI-H1299 cells decreased, the expression of E-cadherin increased and the expression of vimentin and SNAI1 decreased. The lung cancer sequencing data of TCGA database showed that the expression of PRMT5 in lung cancer tissues was significantly higher than that in normal lung tissues, and the lung adenocarcinoma patients and the lung squamous cell carcinoma patients with higher expression of PRMT5 had lower overall survival rate (P=0.005) and relapse-free survival rate (P=0.028), respectively.

Conclusion· PRMT5 is highly expressed in lung cancer tissues, which can promote the proliferation, migration and invasion of lung cancer cells. High expression of PRMT5 is associated with poor prognosis of lung cancer.

Key words: protein arginine methyltransferase 5 (PRMT5), lung cancer, proliferation, migration, epithelial-mesenchymal transition (EMT)

中图分类号:

R734.2

周冰倩, 韩丽, 陈哲逸, 陈诗宇, 郑英霞. 蛋白精氨酸甲基转移酶5在肺癌中的表达及其促进肺癌的作用机制[J]. 上海交通大学学报(医学版), 2021, 41(8): 1009-1016.

Bing-qian ZHOU, Li HAN, Zhe-yi CHEN, Shi-yu CHEN, Ying-xia ZHENG. Expression of protein arginine methyltransferase 5 in lung cancer and its mechanism of promoting lung cancer[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(8): 1009-1016.

图/表 9

表 1 PRMT5表达评分标准

Tab 1 PRMT5 expression scoring criteria

Intensity of PRMT5 expression	Proportion of PRMT5-positive cells	Score
Negative	0	0
Weakly positive	0‒25%	1
Moderately positive	25%‒50%	2
Strongly positive	50%‒75%	3
No stronger intensity	75%‒100%	4

表 2 shRNA寡核苷酸序列

Tab 2 Oligonucleotide sequence of shRNA

shRNA	Sequence （5′→3′）
NC-shRNA	F： GATCTGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTC
	R： AATTCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAACA
PRMT5-shRNA1	F： GATCCGCCCAGTTTGAGATGCCTTATTTCAAGAGAATAAGGCATCTCAAACTGGGCTTTTTTG
	R： AATTCAAAAAAGCCCAGTTTGAGATGCCTTATTCTCTTGAAATAAGGCATCTCAAACTGGGCG
PRMT5-shRNA2	F： GATCCGGCTGACCTCCCATCTAATCATTCAAGAGATGATTAGATGGGAGGTCAGCCTTTTTTG
	R： AATTCAAAAAAGGCTGACCTCCCATCTAATCATCTCTTGAATGATTAGATGGGAGGTCAGCCG
PRMT5-shRNA3	F： GATCCCCCATCCTCTTCCCTATTAAGCTTCCTGTCAGACTTAATAGGGAAGAGGATGGGTTTTTG
	R： AATTCAAAAACCCATCCTCTTCCCTATTAAGTCTGACAGGAAGCTTAATAGGGAAGAGGATGGGG

表 3 qPCR引物序列

Tab 3 Primer sequences for qPCR

Primer	Forward （5’→3’）	Reverse （5’→3’）
PRMT5	CGATCAGACCTACTGCTGTCA	CTCGGAGTTCCTGCGAATCT
β-actin	CATGTACGTTGCTATCCAGGC	CTCCTTAATGTCACGCACGAT
VIM	GACGCCATCAACACCGAGTT	CTTTGTCGTTGGTTAGCTGGT
SNAI1	TCGGAAGCCTAACTACAGCGA	AGATGAGCATTGGCAGCGAG
CDH1	ATTTTTCCCTCGACACCCGAT	TCCCAGGCGTAGACCAAGA

图 1 免疫组织化学检测PRMT5在肺癌及癌旁组织中的表达Note：A. Staining results of PRMT5 expression (×40). B. Statistical analysis of PRMT5 expression. ①P=0.000, compared with adjacent tissues.

Fig 1 Expression of PRMT5 in lung cancer tissues and adjacent tissues by immunohistochemistry

表 4 PRMT5的表达水平与肺癌患者临床特征间的关系

Tab 4 Relationship between the expression of PRMT5 and the clinical features of lung cancer patients

Clinical feature	Relative expression of PRMT5/score	t value	P value
Gender		0.191	0.849
Male （n=39）	6.21±3.00
Female （n=34）	6.08±2.84
Age		0.490	0.626
≥60 year （n=36）	5.93±2.84
<60 year （n=37）	6.32±2.99
TNM stage		2.178	0.033
Ⅰ‒Ⅱ （n=51）	5.65±2.75
Ⅲ‒Ⅳ （n=22）	7.24±3.01
Pathological grading		0.370	0.712
T1‒T2 （n=61）	6.21±3.01
T3‒T4 （n=12）	5.86±2.45
Lymph node metastasis metastasis		0.884	0.379
Yes （n=35）	5.83±2.54
No （n=38）	6.43±3.21

图 2 PRMT5在多种癌细胞中的表达Note：A. Expression of PRMT5 mRNAin the different primary cancer cell lines in GSE36139. B. Expression of PRMT5 mRNA in different pathological types of lung cancer cell lines in GSE36139. ①P=0.034, ②P=0.028, compared with the squamous cell carcinoma. C. Expression of PRMT5 in human embryonic lung fibroblasts and lung cancer cells detected by Western blotting.

Fig 2 Expression of PRMT5 in various cancer cells

图 3 沉默PRMT5对肺癌细胞NCI-H1299的增殖、迁移和侵袭能力的影响Note：A/B. Transfection efficiency of PRMT5-shRNAs in NCI-H1299 cells verified by qPCR (A) and Western blotting (B). ①P=0.000, compared with NC-shRNA. C. Effect of PRMT5-shRNA on the proliferation of NCI-H1299 cells detected by CCK8 assay. ② P=0.000, compared with NC-shRNA. D/E. Effects of PRMT5-shPRMT5 on the migration (D) and invasion (E) ability of NCI-H1299 cells (crystal violet staining, ×100). ③P=0.000, compared with NC-shRNA.

Fig 3 Effects of silencing PRMT5 on proliferation, migration and invasion abilities of lung cancer cell NCI-H1299

图 4 敲除PRMT5后EMT相关蛋白及其基因的表达Note：A. Expression of EMT-associated proteins detected by Western blotting. B. Expression of EMT-associated genes detected by qPCR.①P=0.000, compared with NC-shRNA.

Fig 4 Expression of EMT-associated proteins and the genes after PRMT5 knockout

图 5 TCGA数据库中PRMT5 mRNA在肺癌及正常肺组织中的表达及其表达与患者预后的关系Note：A/B. Expression of PRMT5 mRNAin the LUAD tissues (A), LUSC tissues (B) and normal tissues in TCGA database. ①P=0.000, compared with normal tissues. C. Correlation between PRMT5 mRNA expression and OS in patients with LUAD. D. Correlation between PRMT5 mRNA expression and RFS in patients with LUSC.

Fig 5 Expression of PRMT5 mRNA in lung cancer tissues and normal lung tissues from TCGA database and the correlation with prognosis

参考文献 17

1	Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020[J]. CA Cancer J Clin, 2020, 70(1): 7-30.
2	Feng RM, Zong YN, Cao SM, et al. Current cancer situation in China: good or bad news from the 2018 Global Cancer Statistics?[J]. Cancer Commun, 2019, 39(1): 22.
3	Woodard GA, Jones KD, Jablons DM. Lung cancer staging and prognosis[J]. Cancer Treat Res, 2016, 170: 47-75.
4	Mehlen P, Puisieux A. Metastasis: a question of life or death[J]. Nat Rev Cancer, 2006, 6(6): 449-458.
5	Stopa N, Krebs JE, Shechter D. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond[J]. Cell Mol Life Sci, 2015, 72(11): 2041-2059.
6	Pal S, Baiocchi RA, Byrd JC, et al. Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma[J]. EMBO J, 2007, 26(15): 3558-3569.
7	Migliori V, Müller J, Phalke S, et al. Symmetric dimethylation of H3R2 is a newly identified histone mark that supports euchromatin maintenance[J]. Nat Struct Mol Biol, 2012, 19(2): 136-144.
8	Jing PY, Zhao N, Ye MX, et al. Protein arginine methyltransferase 5 promotes lung cancer metastasis via the epigenetic regulation of miR-99 family/FGFR3 signaling[J]. Cancer Lett, 2018, 427: 38-48.
9	Chen H, Lorton B, Gupta V, et al. A TGFβ-PRMT5-MEP50 axis regulates cancer cell invasion through histone H3 and H4 arginine methylation coupled transcriptional activation and repression[J]. Oncogene, 2017, 36(3): 373-386.
10	Jiang H, Zhu Y, Zhou ZY, et al. PRMT5 promotes cell proliferation by inhibiting BTG2 expression via the ERK signaling pathway in hepatocellular carcinoma[J]. Cancer Med, 2018, 7(3): 869-882.
11	Chiang K, Zielinska AE, Shaaban AM, et al. PRMT5 is a critical regulator of breast cancer stem cell function via histone methylation and FOXP1 expression[J]. Cell Rep, 2017, 21(12): 3498-3513.
12	Nicholas C, Yang J, Peters SB, et al. PRMT5 is upregulated in malignant and metastatic melanoma and regulates expression of MITF and p27(Kip1.)[J]. PLoS One, 2013, 8(9): e74710.
13	Brabletz T, Kalluri R, Nieto MA, et al. EMT in cancer[J]. Nat Rev Cancer, 2018, 18(2): 128-134.
14	Ge L, Wang H, Xu X, et al. PRMT5 promotes epithelial-mesenchymal transition via EGFR-β-catenin axis in pancreatic cancer cells[J]. J Cell Mol Med, 2020, 24(2): 1969-1979.
15	Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6): 394-424.
16	Shailesh H, Zakaria ZZ, Baiocchi R, et al. Protein arginine methyltransferase 5 (PRMT5) dysregulation in cancer[J]. Oncotarget, 2018, 9(94): 36705-36718.
17	Yuan YY, Nie H. Protein arginine methyltransferase 5: a potential cancer therapeutic target[J]. Cell Oncol (Dordr), 2021, 44(1): 33-44.

[1]	凌徐心仪, 张瑶, 钟华. 非小细胞肺癌免疫治疗获益人群筛选的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(8): 1114-1119.
[2]	贺艳琪, 迟锐, 陈梦萍, 陈思, 刘春良, 刘云霞, 孙海鹏. 支链氨基酸分解代谢在肺癌细胞中的功能[J]. 上海交通大学学报(医学版), 2021, 41(7): 858-864.
[3]	童徐, 舒林径. miR-877-3p对骨质疏松症小鼠骨髓间充质干细胞增殖能力的影响[J]. 上海交通大学学报(医学版), 2021, 41(7): 884-890.
[4]	鲁昱晟, 杨文艺, 马海龙, 胡镜宙. Ras相关结构域家族成员5对头颈鳞癌细胞迁移和侵袭能力的影响[J]. 上海交通大学学报(医学版), 2021, 41(6): 717-723.
[5]	马韵芳, 潘丽娜, 李圳, 高蓓莉, 胡家安, 徐志红. 司美替尼下调KRAS G12V突变型非小细胞肺癌细胞PD-L1水平的探索性研究[J]. 上海交通大学学报(医学版), 2021, 41(6): 741-748.
[6]	徐建华, 江萍, 邓炯. ATP结合盒蛋白G超家族成员2在肺癌中的表达及意义[J]. 上海交通大学学报(医学版), 2021, 41(6): 830-833.
[7]	程龙1，徐五琴2，张鹏1，黄建军1，李小宁1. 下调miR-27a表达对三阴乳腺癌细胞增殖、迁移和侵袭的影响[J]. 上海交通大学学报（医学版）, 2020, 40(2): 188-.
[8]	许书琳，吴京，娄加陶. 肺癌和贫血的相关性分析[J]. 上海交通大学学报(医学版), 2020, 40(11): 1500-1504.
[9]	何春明，尹航，唐健，丁一宗，傅于捷，赵晓菁. 基于SEER数据库的老年肺癌术后患者预后模型构建与内部验证[J]. 上海交通大学学报(医学版), 2020, 40(11): 1554-1561.
[10]	周箴1，徐云华1，张菲菲2，赵艺1，郭林2，付国龙2，沈盛萍1，冷雪娇1. 人源化免疫肺癌小鼠模型的建立及其在程序性死亡受体1抑制剂疗效评估中的作用[J]. 上海交通大学学报（医学版）, 2020, 40(1): 37-.
[11]	严宇青，沈超琴，陈豪燕，洪洁#，王震华#. lnc-MTBP-5在结直肠癌中的表达及其对细胞侵袭能力的影响[J]. 上海交通大学学报(医学版), 2020, 40(09): 1193-1201.
[12]	邹琛1, 2，徐润灏1, 3，张泓2，马展2，陈黎2，张洁3，李敏3，张舒林1. 小分子代谢物在肺癌和肺炎鉴别诊断中的潜在作用[J]. 上海交通大学学报(医学版), 2020, 40(08): 1041-1047.
[13]	陈巍，韩峥，邹艳丽，黄莎莎，田霞. 溃疡性结肠炎小鼠血清miR-23a-3p和miR-27a-3p的表达水平及其作用的研究[J]. 上海交通大学学报(医学版), 2020, 40(08): 1069-1074.
[14]	何春明，尹航，郑佳杰，唐健，傅于捷，赵晓菁. 肺癌免疫治疗：免疫抑制细胞和肺内免疫[J]. 上海交通大学学报(医学版), 2020, 40(08): 1137-1142.
[15]	公绪华1，朱樑2，陈超3，钱黎俊1. miR-214通过激活肝星状细胞促进肝纤维化进程[J]. 上海交通大学学报(医学版), 2020, 40(06): 776-784.