长链非编码RNA 068在黑素瘤细胞迁移中的功能研究

Role of long non-coding RNA 068 in the migration of melanoma cells

目的探索长链非编码RNA(lncRNA)068对黑素瘤细胞系A375迁移的影响及潜在机制。方法2015年12月至2020年11月在南通大学附属医院皮肤科门诊收集经病理确诊的皮肤黑素瘤患者21例,采用定量PCR(qPCR)检测其黑素瘤组织与相邻癌旁组织中lncRNA 068的表达。通过慢病毒转染在A375细胞中过表达或敲降lncRNA 068,过表达实验分为LV-GFP组和LV-lncRNA 068组,低表达实验分为LV-LacZ shRNA组和LV-lncRNA 068 shRNA组,其中LV-GFP组和LV-LacZ shRNA组均作为对照;Transwell实验及划痕实验检测过表达或敲降lncRNA 068对A375细胞迁移的影响,EdU细胞增殖实验和CCK8法分别检测各组增殖细胞比例及细胞活力,细胞免疫荧光染色检测过表达或敲降lncRNA 068对上皮-间质转化的影响。将各组慢病毒转染的A375细胞接种于BALB/c裸鼠腋窝,每3天测量并计算肿瘤体积,30 d后处死所有裸鼠,切除肿瘤组织并测量肿瘤体积和质量。将培养的B16F10细胞接种于BALB/c裸鼠背部和足部皮下构建B16F10移植瘤模型,2周后处死裸鼠,qPCR和Western印迹法分别检测B16F10移植瘤及癌旁组织中炎症因子mRNA的表达及IKK/P65信号通路相关蛋白的表达。两组间比较采用t检验,各组间不同时间点肿瘤体积及质量的比较采用重复测量方差分析。结果qPCR显示,人黑素瘤组织及裸鼠B16F10移植瘤组织中lncRNA 068相对表达量(0.414±0.109、0.717±0.041)均明显低于相应的癌旁组织(1.050±0.103、1.011±0.023,t=19.48、10.83,均P<0.001)。Transwell实验及划痕实验均显示,LV-lncRNA 068组细胞迁移能力显著低于LV-GFP组(均P<0.01),而LV-lncRNA 068 shRNA组明显强于LV-LacZ shRNA组(均P<0.05)。细胞免疫荧光实验显示,与LV-GFP组相比,LV-lncRNA 068组E钙黏蛋白荧光强度增强,而N钙黏蛋白荧光强度减弱(均P<0.001);与LV-LacZ shRNA组相比,LV-lncRNA 068 shRNA组E钙黏蛋白荧光强度减弱,而N钙黏蛋白荧光强度增强(均P<0.05)。体内裸鼠成瘤实验显示,过表达组与敲降组黑素瘤的体积、质量差异均无统计学意义(P>0.05)。qPCR和Western印迹实验显示,LV-lncRNA 068组A375细胞中白细胞介素(IL)-10、紧密连接蛋白1 mRNA及蛋白的表达均显著高于LV-GFP组(均P<0.05),而LV-lncRNA 068 shRNA组IL-10、紧密连接蛋白1 mRNA及蛋白表达均显著低于LV-LacZ shRNA组(均P<0.05)。B16F10黑素瘤组织中IL-10 mRNA的表达量显著低于癌旁组织(P<0.01),而IL-6、TNF-αmRNA表达及p-P65蛋白表达均显著高于癌旁组织(均P<0.001),p-IKK蛋白表达量显著高于癌旁组织(P<0.01)。结论LncRNA 068过表达可抑制黑素瘤细胞A375的迁移,并可能通过抑制IKK/P65信号通路影响炎症的发生,抑制上皮-间质转化过程。

Objective To investigate the effect of long non-coding RNA 068(lncRNA 068)on the migration of a melanoma cell line A375,and to explore its mechanism of action.Methods From December 2015 to November 2020,21 patients with pathologically confirmed cutaneous melanoma were collected from Department of Dermatology,Affiliated Hospital of Nantong University,and quantitative PCR(qPCR)was performed to determine the expression of lncRNA 068 in melanoma and paracancerous tissues.LncRNA 068 was overexpressed or knocked down via lentiviral transfection in A375 human melanoma cells in the following experiments.Specifically,A375 cells were divided into lentiviral vector(LV)-green fluorescent protein(GFP)group and LV-lncRNA 068 group to be transfected with a GFP-expressing LV and a LV containing lncRNA 068 respectively in the overexpression experiment,and were divided into LV-LacZ short hairpin RNA(shRNA)group and LV-lncRNA 068 shRNA group to be transfected with a LV containing the reporter gene LacZ-specific shRNA and a LV containing the lncRNA 068-targeting shRNA respectively in the low-expression experiment,with the LV-GFP group and LV-LacZ shRNA group serving as the control groups.Transwell and scratch assays were performed to evaluate cell migration,EdU cell proliferation assay and cell counting kit-8(CCK8)assay to determine the proportion of proliferative cells and cell viability respectively,and immunofluorescence staining was conducted to evaluate epithelial-mesenchymal transformation in the above groups.Lentivirus-transfected A375 cells from the above groups were inoculated into the axillae of BALB/c nude mice,and tumor volume was measured and calculated every 3 days.After 30 days,all mice were sacrificed,and tumor tissues were resected to measure the tumor volume and weight.Cultured B16F10 cells were subcutaneously inoculated into the back and foot of BALB/c nude mice to construct mouse models of subcutaneously transplanted B16F10 melanoma.After 2 weeks,the mice were sacrificed,and qPCR and Western blot analysis were performed to determine the mRNA expression of inflammatory factors in transplanted B16F10 melanoma and paracancerous tissues,and expression of IκB kinase(IKK)/P65 signaling pathway-related proteins,respectively.Comparisons between 2 groups were done by t test,and comparisons of tumor volume and weight at different time points among groups were done by repeated measures analysis of variance.Results qPCR showed that the relative expression of lncRNA 068 was significantly lower in human melanoma tissues and transplanted B16F10 murine melanoma tissues(0.414±0.109,0.717±0.041,respectively)than in the corresponding paracancerous tissues(1.050±0.103,1.011±0.023,t=19.48,10.83,respectively,both P<0.001).Transwell and scratch assays both showed that the cellular migratory ability was significantly lower in the LV-lncRNA 068 group than in the LV-GFP group(both P<0.01),and significantly higher in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group(both P<0.05).Immunofluorescence assay showed significantly increased fluorescence intensity of E-cadherin and decreased fluorescence intensity of N-cadherin in the LV-lncRNA 068 group compared with the LV-GFP group(both P<0.001),but significantly decreased fluorescence intensity of E-cadherin and increased fluorescence intensity of N-cadherin in the LV-lncRNA 068 shRNA group compared with the LV-LacZ shRNA group(both P<0.05).In vivo tumor formation experiment in nude mice showed that there were no significant differences in the volume or weight of melanoma between the LV-lncRNA 068 group and LV-GFP group(both P>0.05),as well as between the LV-lncRNA 068 shRNA group and LV-LacZ shRNA group(both P>0.05).As qPCR and Western blot analysis showed,the mRNA and protein expression of interleukin-10(IL-10)and claudin-1 in A375 cells were significantly higher in the LV-lncRNA 068 group than in the LV-GFP group(both P<0.05),but significantly lower in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group(both P<0.05).Compared with the paracancerous tissues,B16F10 melanoma tissues showed significantly decreased mRNA expression of IL-10(P<0.01),but significantly increased mRNA expression of IL-6 and tumor necrosis factor-α,as well as protein expression of phosphorylated P65 and phosphorylated IKK(P<0.01).Conclusion Overexpression of lncRNA 068 can inhibit the migration of A375 melanoma cells,and may affect the development of inflammation and inhibit the epithelial-mesenchymal transformation by inhibiting the IKK/P65 signaling pathway.