miR-212-3p靶向调控NAP1L1抑制胶质瘤细胞增殖、迁移和上皮-间充质转化

郭小兵; 李晓文; 李恒希; 曹艳; 李坪

doi:10.12259/j.issn.2095-610X.S20241104

miR-212-3p靶向调控NAP1L1抑制胶质瘤细胞增殖、迁移和上皮-间充质转化

doi: 10.12259/j.issn.2095-610X.S20241104

昆明医科大学基础医学院人体解剖学与组织胚胎学系，云南昆明　650500

基金项目: 云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目（202101AY070001-037）

详细信息

作者简介:
郭小兵（1982～），男，湖南邵东人，医学硕士，讲师，主要从事神经系统疾病与损伤研究工作

通讯作者:
李坪，E-mail：liping@kmmu.edu.cn

中图分类号: R735.7
计量
- 文章访问数: 104
- HTML全文浏览量: 137
- PDF下载量: 6
- 被引次数: 0
出版历程
- 收稿日期: 2024-06-24
- 网络出版日期: 2024-11-09
- 刊出日期: 2024-11-25

miR-212-3p Targeted Regulation of NAP1L1 Inhibits Glioma Cell Proliferation，Migration and EMT

Dept. of Human Anatomy and Histoembryology，School of Basic Medical Sciences，Kunming Medical University，Kunming Yunnan 650500，China

摘要

摘要: 目的探究miR-212-3p在胶质瘤细胞增殖、侵袭和上皮-间充质转化（EMT）中的分子机制。方法 RT-qPCR用于衡量胶质瘤细胞中miR-212-3p和NAP1L1表达，构建NC mimic、miR-212-3p mimic、oe-NC和oe-NAP1L1转染至细胞中，利用CCK-8 、Transwell和伤口实验评估细胞生物学行为。通过Western blot分析EMT相关标志物的蛋白表达。miR-212-3p与NAP1L1的关系通过双荧光素酶报告基因和AgO2-RIP实验进行证实。结果 miR-212-3p的表达水平在胶质瘤细胞中显著下调（P < 0.0001），过表达miR-212-3p 可以显著降低胶质瘤细胞增殖（P < 0.0001）、侵袭（P = 0.0011）和迁移能力（P < 0.0001），并且抑制了EMT标志物N-钙黏蛋白（N-cadherin）（P = 0.000861）和波形蛋白（Vimentin）（P = 0.007430）的表达，而上调了E-钙黏蛋白（E-cadherin）（P < 0.0001）的表达。miR-212-3p靶向负调控NAP1L1的表达。过表达NAP1L1逆转了miR-212-3p对胶质瘤细胞增殖（P < 0.0001）、迁移（P < 0.0001）和EMT（P < 0.0001）的抑制作用。结论 miR-212-3p通过靶向负调节NAP1L1的表达抑制胶质瘤细胞增殖、迁移和EMT。
- 胶质瘤 /
- miR-212-3p /
- NAP1L1 /
- 上皮-间充质转化 /
- 细胞增殖
Abstract: Objective To explore the molecular mechanism of miR-212-3p in glioma cell proliferation, invasion and epithelial-mesenchymal transition （EMT）. Methods The expression of miR-212-3p and NAP1L1 were detected by RT-qPCR in glioma cells. NC mimic, miR-212-3p mimic, oe-NC and oe-NAP1L1 were built and transfected in cells. CCK-8, Transwell and wound healing assay were used to evaluate the cell biological behaviour. Western blot was used to detect the expression of EMT-related biomarkers. The relationship between miR-212-3p and NAP1L1 was confirmed by the dual-luciferase reporter gene and AgO2-RIP assay. Results miR-212-3p was lowly expressed in glioma cells （P < 0.0001）. miR-212-3p mimic significantly inhibited the glioma cell proliferation （P < 0.0001）, invasion （P = 0.0011） and migration （P < 0.0001）, and reduced the expression of EMT-related biomarkers N-cadherin （P = 0.000861） and Vimentin （P = 0.007430）, while upregulating the expression of E-cadherin （P < 0.0001）. miR-212-3p targeted and negatively regulated the NAP1L1 expression. Overexpression of NAP1L1 reversed the inhibitory effects of miR-212-3p on glioma cell proliferation （P < 0.0001）, migration （P < 0.0001）, and EMT （P < 0.0001）. Conclusion miR-212-3p inhibits the glioma cell proliferation, migration and EMT by targeting the negative regulation of NAP1L1 expression.
- Glioma /
- miR-212-3p /
- NAP1L1 /
- Epithelial-mesenchymal transition /
- Cell proliferation

HTML全文

图 1 miR-212-3p在胶质瘤细胞中低表达

与NHA组比较，^***P < 0.001。

Figure 1. Low expression of miR-212-3p in glioma cells

下载: 全尺寸图片幻灯片

图 2 miR-212-3p抑制胶质瘤细胞增殖、迁移和EMT

A：RT- qPCR检测miR-212-3p转染效率；B：CCK-8分析；C：Transwell评估细胞侵袭；D：伤口愈合实验评估细胞迁移；E：Western blot分析EMT相关标志物的表达（N-cadherin、E-cadherin和Vimentin）的蛋白水平。与NC组比较，^**P < 0.01，^***P < 0.001。

Figure 2. miR-212-3p inhibits glioma cell proliferation，migration and EMT

下载: 全尺寸图片幻灯片

图 3 miR-212-3p靶向调控NAP1L1的表达

A：miR-212-3p与NAP1L1的结合序列；B：双荧光素酶基因报告验证miR-212-3p与NAP1L1的靶向关系；C：AgO2-RIP检测miR-212-3p与NAP1L1的关系；D：生存率预测，high-risk样本33例，low-risk样本41例，P-value=0.023500；E：RT-qPCR检测细胞中NAP1L1的表达；与NC-mimic组比较，^***P < 0.001。

Figure 3. miR-212-3p targets and regulates NAP1L1 expression

下载: 全尺寸图片幻灯片

图 4 miR-212-3p通过下调NAP1L1抑制胶质瘤细胞增殖、迁移和EMT

A：RT- qPCR检测NAP1L1转染效率；B：RT- qPCR检测NAP1L1的表达；C：CCK-8分析；D：Transwell评估细胞侵袭；E：伤口愈合实验评估细胞迁移；F：Western blot检测N-cadherin、E-cadherin和Vimentin的蛋白水平。与NC组比较，^**P < 0.01，^***P < 0.001；与miR-212-3p mimic组比较，^#P < 0.05，^##P < 0.01，^###P < 0.001。

Figure 4. miR-212-3p inhibits glioma cell proliferation，migration and EMT through down-regulation of NAP1L1

下载: 全尺寸图片幻灯片

表 1 PCR引物序列

Table 1. PCR primer sequences

基因	引物	序列（5'-3'）
miR-212-3p	forward	5'-GCGCGTAACAGTCTCCAGTC -3'
	reverse	5'-AGTGCAGGGTCCGAGGTATT -3'
U6	forward	5'-CAAATTCGTGAAGCGTTCCA-3'
	reverse	5'-AGTGCAGGGTCCGAGGTATT-3'
NAP1L1	forward	5'-GGCAGACATTGACAACAA-3'
	reverse	5'-TTCCTCCGAAATCTCATC-3'
GAPDH	forward	5'-TGACCACAGTCCATGCCATCAC-3'
	reverse	5'-CGCCTGCTTCACCACCTTCTT-3'

下载: 导出CSV

参考文献(31)

[1]	Tan A C,Ashley D M,López G Y,et al. Management of glioblastoma: State of the art and future directions[J]. CA Cancer J Clin,2020,70(4):299-312. doi: 10.3322/caac.21613
[2]	程铃,陈蓉,李朝今,等. 山茶花提取物抑制胶质瘤细胞SW1088增殖、迁移和侵袭的机制研究[J]. 西部医学,2023,35(12):1735-1739. doi: 10.3969/j.issn.1672-3511.2023.12.004
[3]	Smolle M A,Leithner A,Posch F,et al. MicroRNAs in different histologies of soft tissue sarcoma: A comprehensive review[J]. Int J Mol Sci,2017,18(9):1960. doi: 10.3390/ijms18091960
[4]	张绍卿,杜敏,刘兰,等. 微RNA-219通过ERK 1/2通路改善炎症所致新生SD大鼠脑内少突胶质细胞成熟障碍[J]. 海军军医大学学报,2023,44(12):1429-1434.
[5]	向发,吴红艳. MicroRNA在糖尿病诊治中的应用前景[J]. 中国现代医学杂志,2024,34(2):66-75. doi: 10.3969/j.issn.1005-8982.2024.02.011
[6]	赵强,赵瑞平. 血浆MicroRNA-296-5P、MicroRNA-107表达水平对急性心肌梗死诊断的预测价值[J]. 包头医学院学报,2023,39(12):36-39,54.
[7]	Liu Z,Su D,Qi X,et al. MiR‑500a‑5p promotes glioblastoma cell proliferation,migration and invasion by targeting chromodomain helicase DNA binding protein 5[J]. Mol Med Rep,2018,18(3):2689-2696.
[8]	任同,杨智君,陈思源,等. miR-141靶向ZEB2对神经胶质瘤细胞增殖、迁移及侵袭的影响[J]. 蚌埠医学院学报,2023,48(7):862-867.
[9]	林亦海,吴漳益,郭亮. miR-106a对胶质瘤增殖和凋亡的影响及机制研究[J]. 浙江医学,2023,45(4):342-349. doi: 10.12056/j.issn.1006-2785.2023.4.2022-1424
[10]	Wu X,Chen H,Zhang G,et al. MiR-212-3p inhibits cell proliferation and promotes apoptosis by targeting nuclear factor IA in bladder cancer[J]. J Biosci,2019,44(4):80. doi: 10.1007/s12038-019-9903-5
[11]	Zhang L,Zhang Y,Wang S,et al. MiR-212-3p suppresses high-grade serous ovarian cancer progression by directly targeting MAP3K3[J]. Am J Transl Res,2020,12(3):875-888.
[12]	谯钰琪,惠盼,南岩东. NAP1L1与肿瘤的关系及其研究进展[J]. 中华肺部疾病杂志(电子版),2021,14(6):827-829.
[13]	Liang X,Tang Z,Zhang Y,et al. NAP1L1 promotes the growth of colon cancer by activating HDGF/DDX5[J]. Acta Biochim Biophys Sin (Shanghai),2022,54(9):1234-1243.
[14]	Chen Z,Gao W,Pu L,et al. PRDM8 exhibits antitumor activities toward hepatocellular carcinoma by targeting NAP1L1[J]. Hepatology,2018,68(3):994-1009. doi: 10.1002/hep.29890
[15]	Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method[J]. Methods,2001,25(4):402-408. doi: 10.1006/meth.2001.1262
[16]	Li L,Lv G,Wang B,et al. The role of lncRNA XIST/miR-211 axis in modulating the proliferation and apoptosis of osteoarthritis chondrocytes through CXCR4 and MAPK signaling[J]. Biochem Biophys Res Commun,2018,503(4):2555-2562. doi: 10.1016/j.bbrc.2018.07.015
[17]	Delgado-Martín B,Medina M. Advances in the knowledge of the molecular biology of glioblastoma and its impact in patient diagnosis,stratification,and treatment[J]. Adv Sci (Weinh),2020,7(9):1902971. doi: 10.1002/advs.201902971
[18]	Stupp R,Mason W P,Van Den Bent M J,et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[J]. N Engl J Med,2005,352(10):987-996. doi: 10.1056/NEJMoa043330
[19]	Śledzińska P,Bebyn M G,Furtak J,et al. Prognostic and predictive biomarkers in gliomas[J]. Int J Mol Sci,2021,22(19):10373. doi: 10.3390/ijms221910373
[20]	Pang H,Zheng Y,Zhao Y,et al. miR-590-3p suppresses cancer cell migration,invasion and epithelial-mesenchymal transition in glioblastoma multiforme by targeting ZEB1 and ZEB2[J]. Biochem Biophys Res Commun,2015,468(4):739-745. doi: 10.1016/j.bbrc.2015.11.025
[21]	Liu Y,Duan N,Duan S. MiR-29a inhibits glioma tumorigenesis through a negative feedback loop of TRAF4/Akt signaling[J]. Biomed Res Int,2018,2018(1):1-8.
[22]	Qin Y,Chen W,Liu B,et al. MiR-200c inhibits the tumor progression of glioma via targeting moesin[J]. Theranostics,2017,7(6):1663-1673. doi: 10.7150/thno.17886
[23]	Wu Z,Yu B,Jiang L. MiR-212-3p mediates apoptosis and invasion of esophageal squamous cell carcinoma through inhibition of the Wnt/β-catenin signaling pathway by targeting SOX4[J]. J Thorac Dis,2020,12(8):4357-4367. doi: 10.21037/jtd-20-2558
[24]	Tripathi S,Levine H,Jolly M K. The physics of cellular decision making during epithelial-mesenchymal transition[J]. Annu Rev Biophys,2020,49(1):1-18. doi: 10.1146/annurev-biophys-121219-081557
[25]	Zhang J,Cai H,Sun L,et al. LGR5,a novel functional glioma stem cell marker,promotes EMT by activating the Wnt/β-catenin pathway and predicts poor survival of glioma patients[J]. J Exp Clin Cancer Res,2018,37(1):225. doi: 10.1186/s13046-018-0864-6
[26]	Chen J Q,Ou Y L,Huang Z P,et al. MicroRNA-212-3p inhibits the proliferation and invasion of human hepatocellular carcinoma cells by suppressing CTGF expression[J]. Sci Rep,2019,9(1):9820. doi: 10.1038/s41598-019-46088-w
[27]	Li T,Niu Z,Yu T,et al. Nucleosome assembly protein 1 like 1 (NAP1L1) promotes cardiac fibrosis by inhibiting YAP1 ubiquitination and degradation[J]. MedComm (2020),2023,4(5): e348.
[28]	Le Y,Kan A,Li Q J,et al. NAP1L1 is a prognostic biomarker and contribute to doxorubicin chemotherapy resistance in human hepatocellular carcinoma[J]. Cancer Cell Int,2019,19(1):228. doi: 10.1186/s12935-019-0949-0
[29]	Zhai W,Ma J,Zhu R,et al. MiR-532-5p suppresses renal cancer cell proliferation by disrupting the ETS1-mediated positive feedback loop with the KRAS-NAP1L1/P-ERK axis[J]. Br J Cancer,2018,119(5):591-604. doi: 10.1038/s41416-018-0196-5
[30]	Chen Z,Yan X,Miao C,et al. Targeting MYH9 represses USP14-mediated NAP1L1 deubiquitination and cell proliferation in glioma[J]. Cancer Cell Int,2023,23(1):220. doi: 10.1186/s12935-023-03050-1
[31]	Chen Z,Xie Y,Luo H,et al. NAP1L1 promotes proliferation and chemoresistance in glioma by inducing CCND1/CDK4/CDK6 expression through its interaction with HDGF and activation of c-Jun[J]. Aging (Albany NY),2021,13(24):26180-26200. doi: 10.18632/aging.203805

相关文章(20)

[1]	邓勇军, 陈倩, 邹建彬, 宫政, 刘焕鹏. ZIC1基因过表达激活P53信号通路抑制胸膜间皮瘤细胞增殖, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20240405
[2]	姜右川, 余妍, 赵国, 李世存, 丁鹏. 过表达三结构域蛋白48调控p-ERK1/2抑制胶质瘤生长的作用机制, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20240505
[3]	李鹏, 邹仁超, 代龙翱, 傅嵩洲, 魏东, 唐波. HIF-1α通过调控上皮-间充质转化对胆囊癌侵袭转移的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20240507
[4]	王惠, 曾文珺, 张海萍, 郭瑞威. 钙库操纵型钙通道Orai3分子对冠状动脉血管平滑肌细胞增殖的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230423
[5]	梁彩红, 孟明耀, 李欣欣, 熊晶晶, 李檬, 刘梅, 侯宗柳, 黄永坤. 肠道菌群代谢物脱氧胆酸对人脐带间充质干细胞hUC-MSCs增殖及细胞周期的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230402
[6]	冯毅, 王小峰, 白西民, 姚胜, 党俊涛, 赵云洁, 蔡冰. miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230823
[7]	钱石兵, 孟明耀, 于鸿滨, 段开文, 李昌全, 夏志刚. 三七总皂苷对人根尖牙乳头干细胞增殖的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20221018
[8]	马振桓, 李震, 周香林, 李国剑, 杨国凯, 万嘉, 杜玲娟, 杨镛. 碘-125粒子调控微小RNA-193b-5p抑制胃癌的增殖和侵袭, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220120
[9]	王晓寒, 牟善茂, 郝翠芳, 任琳琳, 王敏, 赵彤. 富血小板血浆促进人子宫内膜间充质干细胞（EnMSCs）增殖的机制, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220126
[10]	袁伟, 孟明耀, 李欣欣, 熊晶晶, 李檬, 曹佳, 刘梅, 侯宗柳, 黄永坤. 内毒素的量效和时效对人脐带间充质干细胞增殖的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220928
[11]	毕菲, 郭维华. 赫特维希上皮根鞘细胞的研究进展, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220628
[12]	殷顺会, 周建忠, 李自良. Tiger17促进口腔黏膜成纤维细胞的增殖和迁移, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20210823
[13]	王宏宇, 韦焘, 王进昆, 杨智勇, 王伟民, 丁鹏. MCP-1、VEGF在人脑胶质瘤中的表达及其相关性, 昆明医科大学学报.
[14]	陈希, 许智星, 刘旭杰, 田锦涛, 牛小群, 蒲军. 洛哌丁胺体外对胶质瘤干细胞的杀伤作用, 昆明医科大学学报.
[15]	徐波, 杨锐, 陆斌, 曾锐, 孙珂, 丁鹏. 趋化因子CCL2在胶质瘤细胞株U251、U373中的表达, 昆明医科大学学报.
[16]	高红强, 刘静, 李志强, 王海雷, 陈刚, 李立. MiR-199a-3p对大鼠肝细胞增殖的抑制作用, 昆明医科大学学报.
[17]	胡正雄. TGF-β2和geneX对BrdU标记骨髓间充质干细胞增殖与成骨分化的作用, 昆明医科大学学报.
[18]	袁勇. 适度低氧微环境对体外培养脑胶质瘤干细胞生长的影响, 昆明医科大学学报.
[19]	. SDF-1与VEGF在胶质瘤表达中的关系及意义, 昆明医科大学学报.
[20]	. TRB3在同型半胱氨酸抑制内皮细胞增殖中的作用研究, 昆明医科大学学报.