miR-374靶向下调TRIM35表达可促进乳腺癌细胞增殖和侵袭

doi:10.3969/j.issn.1673-8640.2023.09.002

摘要/Abstract

摘要：

目的探讨miR-374在乳腺癌中的作用及其与三基序蛋白35（TRIM35）的关系。方法选取乳腺癌患者42例，收集癌组织和癌旁组织（距肿瘤切缘>2 cm）。收集所有患者的临床病理资料。检测癌组织和癌旁组织miR-374和TRIM35的表达。根据不同处理方法将乳腺癌细胞系MCF-7分为3组：对照组（未作任何处理）、干扰组（加入100 nmol/L miR-374 抑制剂）和空载体组（加入100 nmol/L随机序列）。采用细胞增殖试验和Transwell试验评估miR-374对乳腺癌细胞系MCF-7增殖活性和侵袭能力的影响。采用双荧光素酶报告基因实验分析miR-374和TRIM35之间的相互作用。结果与癌旁组织比较，癌组织miR-374相对表达量显著升高（P<0.001），TRIM35相对表达量显著降低（P<0.001）。不同肿瘤大小和有无淋巴转移、远隔转移的乳腺癌患者之间miR-374相对表达量差异均有统计学意义（P<0.05）。不同年龄和TNM分期的乳腺癌患者之间miR-374相对表达量差异均无统计学意义（P>0.05）。干扰组miR-374相对表达量显著低于对照组和空载体组（P<0.001）。干扰组培养48、72 h后的细胞增殖率和侵袭细胞数均显著低于空载体组和对照组（P<0.001），TRIM35相对表达量显著高于空载体组和对照组（P<0.001）。双荧光素酶报告基因实验结果显示，miR-374可直接抑制靶基因TRIM35的转录表达。结论 miR-374与TRIM35存在相互作用，miR-374通过靶向下调TRIM35表达促进乳腺癌细胞的增殖和侵袭。

关键词: 微小RNA-374, 三基序蛋白35, 乳腺癌, 细胞增殖, 细胞转移

Abstract:

Objective To investigate the role of miR-374 in breast cancer and the relationship between miR-374 and tripartite motif-containing protein 35（TRIM35）. Methods A total of 42 patients with breast cancer were enrolled，and cancer tissues and adjacent tissues（>2 cm away from tumor resection margin） were collected，and the data of patients were collected as well. The expression of miR-374 and TRIM35 were determined in cancer tissues and adjacent tissues. Breast cancer cell line MCF-7 was classified into 3 groups according to different treatment methods，including control group（without any treatment），interference group（adding 100 nmol/L miR-374 inhibitor） and mock-vehicle group（adding 100 nmol/L negative control sequence）. The effects of miR-374 on the proliferative activity and invasiveness of breast cancer cell line MCF-7 were evaluated by cell proliferation assay and Transwell assay. Dual luciferase reporter gene assay was used to determine the interaction between miR-374 and TRIM35. Results The expression of miR-374 in cancer tissues was higher than that in adjacent tissues（P<0.001）. TRIM35 relative expression in cancer tissues was lower than that in adjacent tissues（P<0.001）. The miR-374 relative expression had statistical significance with different breast tumor sizes，distant metastasis and lymphatic metastasis（P<0.05）. There was no statistical significance for miR-374 relative expression with different ages and TNM stages（P>0.05）. The relative expression of miR-374 in interference group was lower than those in control group and mock-vehicle group（P<0.001）. The cell proliferation rate and the number of invasive cells in interference group were lower than those in mock-vehicle group and control group（P<0.001）. TRIM35 relative expressions were higher than those in mock-vehicle group and control group（P<0.001）. Dual luciferase reporter gene assay confirmed that miR-374 directly inhibited the transcriptional expression of target gene TRIM35. Conclusions MiR-374 interacts with TRIM35，and miR-374 promotes the proliferation and invasion of breast cancer cells by targeting and down-regulating the expression of TRIM35.

Key words: MicroR-374, Tripartite motif-containing protein 35, Breast cancer, Cell proliferation, Cell metastasis

中图分类号:

R393

王蓉, 邢连翔, 黄克亮, 李欣. miR-374靶向下调TRIM35表达可促进乳腺癌细胞增殖和侵袭[J]. 检验医学, 2023, 38(9): 812-817.

WANG Rong, XING Lianxiang, HUANG Keliang, LI Xin. MiR-374 promoting proliferation and invasion of breast cancer cells by targeting and down-regulating TRIM35[J]. Laboratory Medicine, 2023, 38(9): 812-817.

图/表 8

表1 引物序列

基因名称	引物序列（5'~3'）	产物长度/bp
TRIM35	F：GAGGTGCTGGCACATGAGAT	112
	R：GCTCCATGGTGCAGAAGAGT
GAPDH	F：CACCCACTCCTCCACCTTTG	110
	R：CCACCACCCTGTTGCTGTAG
miR-374	F：ATATAATACAACCTGCTAAGTG	134
	R：GCAGGGTCCGAGGTATTC
U6	F：CTCGCTTCGGCAGCACA	94
	R：AACGCTTCACGAATTTGCGT

图1 癌组织和癌旁组织miR-374、TRIM35相对表达量比较和两者之间的相关性注：（a）miR-374；（b）TRIM35；（c）miR-374与TRIM35的相关性（r=-0.114，P<0.01）。

表2 不同临床病理特征乳腺癌患者miR-374相对表达量比较

临床病理特征	例数	miR-374
年龄
<60岁	16	3.52±1.68
≥60岁	26	3.30±1.64
t值		0.389 1
P值		0.678 4
肿瘤大小
<3 cm	18	2.64±1.20
≥3 cm	24	3.81±1.67
t值		2.148 3
P值		0.023 2
TNM分期
T1~T2	26	3.40±1.51
T3~T4	16	3.29±1.63
t值		0.532 6
P值		0.551 4
淋巴转移
无	14	3.80±1.55
有	28	2.32±1.07
t值		7.724 2
P值		0.002 1
远隔转移
无	31	2.49±1.54
有	11	3.59±1.49
t值		1.934 5
P值		0.034 9

图2 干扰组、对照组和空载体组miR-374相对表达量比较注：与干扰组比较，*P<0.001。

图3 miR-374干扰对MCF-7细胞增殖的影响注：对照组；空载体组；干扰组；与干扰组比较，*P<0.001。

图4 miR-374干扰对MCF-7细胞侵袭性的影响（×100）注：（a）对照组；（b）空载体组；（c）干扰组；（d）对照组、空载体组和干扰组MCF-7细胞侵袭数比较；与干扰组比较，*P<0.001。

图5 miR-374干扰对TRIM35相对表达量的影响注：与干扰组比较，*P<0.001。

图6 miR-374对TRIM35转录调控的影响

参考文献 24

[1]	JOKAR N, VELIKYAN I, AHMADZADEHFAR H, et al. Theranostic approach in breast cancer:a treasured tailor for future oncology[J]. Clin Nucl Med, 2021, 46(8):e410-e420. DOI URL
[2]	LI C L, YANG D, CAO X, et al. Fibronectin induces epithelial-mesenchymal transition in human breast cancer MCF-7 cells via activation of calpain[J]. Oncol Lett, 2017, 13(5):3889-3895. DOI URL
[3]	LAGES E, IPAS H, GUTTIN A, et al. MicroRNAs:molecular features and role in cancer[J]. Front Biosci(Landmark Ed), 2012, 17(7):2508-2540.
[4]	LUO J, ZHAO Q, ZHANG W, et al. A novel panel of microRNAs provides a sensitive and specific tool for the diagnosis of breast cancer[J]. Mol Med Rep, 2014, 10(2):785-791. DOI PMID
[5]	KIMURA F, SUZU S, NAKAMURA Y, et al. Cloning and characterization of a novel RING-B-box-coiled-coil protein with apoptotic function[J]. J Biol Chem, 2003, 278(27):25046-25054. DOI PMID
[6]	WANG Y, YAN S, YANG B, et al. TRIM35 negatively regulates TLR7- and TLR9-mediated type Ⅰ interferon production by targeting IRF7[J]. FEBS Lett, 2015, 589(12):1322-1330. DOI URL
[7]	中国抗癌协会乳腺癌专业委员会. 中国抗癌协会乳腺癌诊治指南与规范(2021年版)[J]. 中国癌症杂志, 2021, 31(10):954-1040.
[8]	GIAQUINTO A N, SUNG H, MILLER K D, et al. Breast cancer statistics,2022[J]. CA Cancer J Clin, 2022, 72(6):524-541. DOI URL
[9]	JIANG Y, JIANG Z, WANG M, et al. Current understandings and clinical translation of nanomedicines for breast cancer therapy[J]. Adv Drug Deliv Rev, 2022, 180:114034. DOI URL
[10]	NATHANSON S D, DETMAR M, PADERA T P, et al. Mechanisms of breast cancer metastasis[J]. Clin Exp Metastasis, 2022, 39(1):117-137. DOI
[11]	IACONA J R, LUTZ C S. MiR-146a-5p:expression,regulation,and functions in cancer[J]. Wiley Interdiscip Rev RNA, 2019, 10(4):e1533. DOI URL
[12]	WU J, WANG Y, SHANG L, et al. Five common functional polymorphisms in microRNAs and susceptibility to breast cancer:an updated meta-analysis[J]. Genet Test Mol Biomarkers, 2018, 22(6):350-358. DOI URL
[13]	SANDIFORD O A, MOORE C A, DU J, et al. Human aging and cancer:role of miRNA in tumor microenvironment[J]. Adv Exp Med Biol, 2018, 1056:137-152.
[14]	ZHANG K, WANG Y Y, XU Y, et al. A two-miRNA signature of upregulated miR-185-5p and miR-362-5p as a blood biomarker for breast cancer[J]. Pathol Res Pract, 2021, 222:153458. DOI URL
[15]	BIAN H, ZHOU Y, ZHOU D, et al. The latest progress on miR-374 and its functional implications in physiological and pathological processes[J]. J Cell Mol Med, 2019, 23(5):3063-3076. DOI PMID
[16]	HE H C, HAN Z D, DAI Q S, et al. Global analysis of the differentially expressed miRNAs of prostate cancer in Chinese patients[J]. BMC Genomics, 2013, 14:757. DOI
[17]	RAHIMI Z, GHORBANI Z, MOTAMED H, et al. Aberrant expression profile of miR-32,miR-98 and miR-374 in chronic lymphocytic leukemia[J]. Leuk Res, 2021, 111:106691. DOI URL
[18]	GAO J, BAI S, WANG Y, et al. MiR-374b targets GATA3 to promote progression and development of glioblastoma via regulating SEMA3B[J]. Neoplasma, 2019, 66(4):543-554. DOI PMID
[19]	HATAKEYAMA S. TRIM family proteins:roles in autophagy,immunity,and carcinogenesis[J]. Trends Biochem Sci, 2017, 42(4):297-311. DOI URL
[20]	EBERHARDT W, HAEUSSLER K, NASRULLAH U, et al. Multifaceted roles of TRIM proteins in colorectal carcinoma[J]. Int J Mol Sci, 2020, 21(20):7532. DOI URL
[21]	CHEN Z, WANG Z, GUO W, et al. TRIM35 interacts with pyruvate kinase isoform M2 to suppress the Warburg effect and tumorigenicity in hepatocellular carcinoma[J]. Oncogene, 2015, 34(30):3946-3956. DOI PMID
[22]	WANG R, HUANG K L, XING L X. TRIM35 functions as a novel tumor suppressor in breast cancer by inducing cell apoptosis through ubiquitination of PDK1[J]. Neoplasma, 2022, 69(2):370-382. DOI URL
[23]	LIANG A L, ZHANG T T, ZHOU N, et al. MiRNA-10b sponge:an anti-breast cancer study in vitro[J]. Oncol Rep, 2016, 35(4):1950-1958. DOI URL
[24]	WU H, GUO X, JIAO Y, et al. TRIM35 ubiquitination regulates the expression of PKM2 tetramer and dimer and affects the malignant behaviour of breast cancer by regulating the Warburg effect[J]. Int J Oncol, 2022, 61(6):144. DOI URL