miR-127-3p对口腔鳞状细胞癌细胞增殖、迁移及Zwint-1基因表达的影响

doi:10.13591/j.cnki.kqyx.2022.02.004

摘要/Abstract

摘要： 目的探讨miR-127-3p调控Zeste white 10(ZW10)相互作用着丝粒蛋白1(Zwint-1)对口腔鳞状细胞癌(OSCC)细胞增殖、迁移及侵袭的影响。方法体外培养OSCC细胞系PE/CA-PJ15、CAL27,对其转染并分为空白对照组(NG组)、阴性转染组(NC组)、过表达miR-127-3p组(miR-127-3p-mimics组)。倒置荧光显微镜下观察转染效果;采用实时荧光定量PCR法检测miR-127-3p、Zwint-1 mRNA水平;MTT法检测各组细胞增殖情况;划痕实验检测PE/CA-PJ15、CAL27细胞迁移能力;Transwell实验检测PE/CA-PJ15、CAL27细胞侵袭能力;免疫印迹法检测人增殖细胞核抗原(Ki-67),凋亡相关蛋白Bax、Bcl-2,侵袭及迁移相关蛋白E-钙粘附蛋白(E-cadherin)、N-钙粘附蛋白(N-cadherin)、波形蛋白(Vimentin)及Zwint-1蛋白表达情况;应用TargetScan数据库预测miR-127-3p与Zwint-1靶向关系并用双荧光素酶报告基因实验验证。结果细胞转染成功;与NG组、NC组比较,miR-127-3p-mimics组PE/CA-PJ15、CAL27细胞miR-127-3p、增殖抑制率、E-cadherin、Bax蛋白表达显著升高(P<0.05),划痕治愈率、细胞侵袭数、Zwint-1 mRNA及其蛋白、Ki-67、N-cadherin、Vimentin、Bcl-2蛋白表达显著减少或降低(P<0.05)。TargetScan数据库预测显示Zwint-1是miR-127-3p的潜在靶基因,双荧光素酶报告基因实验证实二者存在靶向关系,且miR-127-3p可负向调控Zwint-1表达。结论上调miR-127-3p表达可靶向下调Zwint-1表达,并抑制OSCC细胞增殖、侵袭及迁移。

关键词: miR-127-3p, Zeste white 10(ZW10)相互作用着丝粒蛋白1, 口腔鳞状细胞癌细胞, 增殖, 侵袭迁移

Abstract: Objective To investigate the effect of miR-127-3p on proliferation, migration and invasion of oral squamous cell carcinoma (OSCC) cells by regulating Zeste white 10 (ZW10)-interacting kinetochore protein 1 (Zwint-1). Methods PE/CA-PJ15, CAL27 cells were cultured in vitro, transfected and divided into blank control group (NG group), negative transfection group (NC group), miR-127-3p overexpression group (miR-127-3p-mimics group). The transfection effect was observed under inverted fluorescence microscope; levels of miR-127-3p and Zwint-1 mRNA were detected by real-time PCR; cell proliferation was detected by MTT assay; the migration ability and invasion ability of PE/CA-PJ15 and CAL27 cells were detected by scratch test and Transwell assay respectively; expressions of human proliferating cell nuclear antigen (Ki-67), Apoptosis-associated protein Bax, Bcl-2, E-cadherin, N-cadherin, Vimentin and Zwint-1 were detected by Western blotting; the targeting relationship between miR-127-3p and Zwint-1 was predicted by TargetScan database and verified by double luciferase reporter gene experiment. Results Cells were successfully transfected; compared with those in NG group and NC group, the expression of miR-127-3p, proliferation inhibition rate and E-cadherin, Bax in PE/CA-PJ15 and CAL27 cells in miR-127-3p-mimics group were significantly increased (P<0.05); the wound healing rate, the number of cell invasion, the expression of Zwint-1 mRNA and protein, Ki-67, Bcl-2, N-cadherin and Vimentin protein were significantly decreased (P<0.05). TargetScan database prediction showed that Zwint-1 was a potential target gene of miR-127-3p; double luciferase reporter gene experiment confirmed that there was a targeting relationship between Zwint-1 and miR-127-3p, and miR-127-3p could negatively regulate the expression of Zwint-1. Conclusion Up-regulating the expression of miR-127-3p can target and down-regulate the expression of Zwint-1, and inhibit the proliferation, invasion and migration of OSCC cells.

Key words: miR-127-3p, Zeste white 10 (ZW10)-interacting kinetochore protein 1, oral squamous cell carcinoma cell, proliferation, invasion and migration

中图分类号:

R739.85

钱伟祥, 武艳飞, 杨卫平, 邵莉. miR-127-3p对口腔鳞状细胞癌细胞增殖、迁移及Zwint-1基因表达的影响[J]. 口腔医学, 2022, 42(2): 117-124.

QIAN Weixiang, WU Yanfei, YANG Weiping, SHAO Li. Effects of miR-127-3p on proliferation, migration and Zwint-1 gene expression of oral squamous cell carcinoma cells[J]. Stomatology, 2022, 42(2): 117-124.

参考文献

[1] Feng X, Luo Q, Wang H, et al. MicroRNA-22 suppresses cell proliferation, migration and invasion in oral squamous cell carcinoma by targeting NLRP3[J]. J Cell Physiol, 2018, 233(9):6705-6713.
[2] Gallenkamp J, Spanier G, Wörle E, et al. A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma[J]. Oncotarget, 2018, 9(3):3001-3013.
[3] Huang TH, Li KY, Choi WS. Lymph node ratio as prognostic variable in oral squamous cell carcinomas:Systematic review and meta-analysis[J]. Oral Oncol, 2019, 89:133-143.
[4] Roat R, Hossain MM, Christopherson J, et al. Circulating miRNA-150-5p is associated with immune-mediated early β-cell loss in a humanized mouse model[J]. Xenotransplantation, 2019, 26(2):e12474.
[5] Ji L, Zhu ZN, He CJ, et al. MiR-127-3p targets KIF₃B to inhibit the development of oral squamous cell carcinoma[J]. Eur Rev Med Pharmacol Sci, 2019, 23(2):630-640.
[6] 王波, 郭小军, 丁韬, 等. 三阴性乳腺癌组织ZWINT表达及临床意义[J]. 中华肿瘤防治杂志, 2020, 27(16):1292-1297.
[7] Musio A, Mariani T, Montagna C, et al. Recapitulation of the Roberts syndrome cellular phenotype by inhibition of INCENP, ZWINT-1 and ZW10 genes[J]. Gene,2004, 28(331):33-40.
[8] 周洁, 潘芳名, 王晶, 等. Zwint-1蛋白在头颈部鳞状细胞癌中表达上调及其与临床特征的相关性[J]. 中国耳鼻咽喉颅底外科杂志, 2019, 25(5):508-512.
[9] Zhou GR, Shen MY, Zhang ZY. ZW10 binding factor (ZWINT), a direct target of mir-204, predicts poor survival and promotes proliferation in breast cancer[J]. Med Sci Monit, 2020, 26:e921659.
[10] Yakop F, Abd Ghafar SA, Yong YK, et al. Silver nanoparticles Clinacanthus Nutans leaves extract induced apoptosis towards oral squamous cell carcinoma cell lines[J]. Artif Cells Nanomed Biotechnol, 2018, 46(sup2):131-139.
[11] Shao TR,Zheng ZN, Chen YC, et al. LncRNA AC007271.3 promotes cell proliferation, invasion, migration and inhibits cell apoptosis of OSCC via the Wnt/β-catenin signaling pathway[J]. Life Sci, 2019, 239:117087.
[12] Nakashima C, Yamamoto K, Fujiwara-Tani R, et al. Expression of cytosolic malic enzyme (ME1) is associated with disease progression in human oral squamous cell carcinoma[J]. Cancer Sci, 2018, 109(6):2036-2045.
[13] 杨珊珊, 朱慧勇. microRNA与舌鳞状细胞癌相关性的研究进展[J]. 口腔医学, 2021, 41(3):269-273.
[14] 谢丹, 文丹宁, 罗丹. miR-127-5p靶向IRAK4对肺炎链球菌诱导的肺泡上皮细胞凋亡及炎症因子表达的影响[J]. 临床肺科杂志, 2020, 25(2):261-266,274.
[15] 周联明, 张学利, 杨治, 等. 血清miR-34 c和miR-127-3p表达在结肠癌诊治中的临床意义[J]. 现代中西医结合杂志, 2018, 27(27):3061-3064.
[16] Gastal GDA, Aguiar FLN, Rodrigues APR, et al. Cryopreservation and in vitro culture of white-tailed deer ovarian tissue[J]. Theriogenology, 2018, 113:253-260.
[17] Zhang HG, Pan YW,Feng J, et al. TRIM66 promotes malignant progression of hepatocellular carcinoma by inhibiting E-cadherin expression through the EMT pathway[J]. Eur Rev Med Pharmacol Sci, 2019, 23(5):2003-2012.
[18] Kim JH, Youn Y, Lee JC, et al. Involvement of the NF-κB signaling pathway in proliferation and invasion inhibited by Zwint-1 deficiency in pancreatic cancer cells[J]. J Cancer, 2020, 11(19):5601-5611.
[19] WooSeo D, Yeop You S, Chung WJ, et al. Zwint-1 is required for spindle assembly checkpoint function and kinetochore-microtubule attachment during oocyte meiosis[J]. Sci Rep, 2015, 5:15431.
[20] Jiang HW, Jin CM, Liu J,et al. Next generation sequencing analysis of miRNAs:MiR-127-3p inhibits glioblastoma proliferation and activates TGF-β signaling by targeting SKI[J]. OMICS, 2014, 18(3):196-206.