lnc⁃NEAT1通过miR⁃29c⁃3p/CSPG4信号轴调节黑色素瘤B16细胞的增殖、迁移和侵袭  

Lnc⁃NEAT1 regulates the proliferation,migration and invasion of melanoma B16 cells through the miR⁃29c⁃3p/CSPG4 signaling axis

作　　者：谢奇 樊鑫梅 韩永红 陶娟 刘旭 刘家秀 XIE Qi;FAN Xinmei;HANG Yonghong;TAO Juan;LIU Xu;LIU Jiaxiu(College of Pharmacy and Traditional Chinese Medicine,Jiangsu College of Nursing,Huai’an 223005,China)

机构地区：[1]江苏护理职业学院药学与中药学院,江苏淮安223005

出　　处：《南京医科大学学报（自然科学版）》2023年第4期492-501,共10页Journal of Nanjing Medical University(Natural Sciences)

基　　金：淮安市自然科学研究计划(联合专项)项目(HABL202112)。

摘　　要：目的:探讨长链非编码RNA⁃核富集转录本1(long non⁃coding RNA⁃nuclear enriched abundant transcript 1,lnc⁃NEAT1)对黑色素瘤(melanoma,MM)细胞增殖、迁移和侵袭的调控机制。方法:体外培养MM细胞系(A375、A875、M14、B16)和人表皮黑色素细胞(HEMa⁃LP),采用实时荧光定量PCR(real⁃time quantitative PCR,RT⁃qPCR)检测细胞中lnc⁃NEAT1、microRNA⁃29c⁃3p(miR⁃29c⁃3p)、硫酸软骨素蛋白多糖4(chondroitin sulfate proteoglycan 4,CSPG4)mRNA表达。取对数生长期B16细胞,分为对照组(control组)、si⁃NC组、si⁃NEAT1组、si⁃NEAT1+inhibitor⁃NC组、si⁃NEAT1+miR⁃29c⁃3p inhibitor组,用Lipofectamine 3000将相应质粒转染到细胞中。RT⁃qPCR检测转染效率,MTT法检测细胞增殖活力,Transwell小室检测细胞迁移和侵袭能力,蛋白质印迹法检测细胞CSPG4及增殖、迁移与侵袭相关蛋白表达。双荧光素酶报告基因检测miR⁃29c⁃3p与lnc⁃NEAT1、CSPG4的靶向关系。裸鼠移植瘤实验探究lnc⁃NEAT1敲低对MM细胞体内生长的影响。结果:MM细胞系中lnc⁃NEAT1和CSPG4 mRNA水平显著高于HEMa⁃LP细胞,miR⁃29c⁃3p水平显著低于HEMa⁃LP细胞(P均<0.05)。敲低lnc⁃NEAT1可显著升高miR⁃29c⁃3p表达,降低CSPG4 mRNA和蛋白水平,抑制细胞增殖、迁移和侵袭,并降低Ki⁃67、N⁃cadherin与Vimentin蛋白水平,升高E⁃cadherin蛋白水平(P均<0.05);下调miR⁃29c⁃3p表达可显著升高CSPG4 mRNA和蛋白水平,减弱lnc⁃NEAT1敲低对MM细胞增殖、迁移和侵袭的抑制作用(P<0.05)。双荧光素酶报告基因检测结果显示,转染miR⁃29c⁃3p mimic后,细胞中NEAT1⁃WT和CSPG4⁃WT的荧光素酶活性显著降低(P<0.05)。裸鼠移植瘤实验结果显示,敲低lnc⁃NEAT1可显著抑制体内移植瘤生长,而抑制miR⁃29c⁃3p可显著减弱lnc⁃NEAT1敲低对移植瘤生长的抑制作用(P均<0.05)。结论:lnc⁃NEAT1可能通过调节miR⁃29c⁃3p/CSPG4轴促进MM细胞增殖、迁移和侵袭。Objective:To investigate the regulatory mechanism of long non⁃coding RNA⁃nuclear enriched transcript 1(lnc⁃NEAT1)on the proliferation,migration and invasion of melanoma(MM)cells.Methods:MM cell lines(A375,A875,M14,B16)and human epidermal melanocytes(HEMa⁃LP)were cultured in vitro,real⁃time quantitative PCR(RT⁃qPCR)was performed to determine the mRNA expression of lnc⁃NEAT1,microRNA⁃29c⁃3p(miR⁃29c⁃3p)and chondroitin sulfate proteoglycan 4(CSPG4)in cells.B16 cells at logarithmic growth phase were taken and separated into control group,si⁃NC group,si⁃NEAT1 group,si⁃NEAT1+inhibitor⁃NC group,and si⁃NEAT1+miR⁃29c⁃3p inhibitor group.Lipofectamine 3000 was applied to transfect the corresponding plasmids into cells.RT⁃qPCR was performed to determine the transfection efficiency.MTT method was used to determine cell proliferation.Transwell assay was performed to determine cell migration and invasion abilities.Western blotting was performed to determine the expression of CSPG4 and proteins related to the proliferation,migration and invasion.Dual⁃luciferase reporter gene assay was performed to determine miR⁃29c⁃3p targeting relationship with lnc⁃NEAT1 and CSPG4.Nude mice xenograft experiment was performed to explore the effect of lnc⁃NEAT1 knockdown on the growth of MM cells in vivo.Results:The mRNA levels of lnc⁃NEAT1 and CSPG4 in MM cell lines were obviously higher than those in HEMa⁃LP cells,and the levels of miR⁃29c⁃3p were obviously lower than those in HEMa⁃LP cells(all P<0.05).Knockdown of lnc⁃NEAT1 obviously increased miR⁃29c⁃3p expression,decreased CSPG4 mRNA and protein levels,inhibited cell proliferation,migration and invasion,and decreased Ki⁃67,N⁃cadherin and Vimentin protein levels as well as increased E⁃cadherin protein level(all P<0.05).Down⁃regulation of miR⁃29c⁃3p expression obviously increased CSPG4 mRNA and protein levels,and attenuated the inhibitory effects of lnc⁃NEAT1 knockdown on MM cell proliferation,migration and invasion(P

关 键 词：黑色素瘤 长链非编码RNA核富集转录本1 microRNA⁃29c⁃3p 硫酸软骨素蛋白多糖4 增殖 迁移 侵袭 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]