摘要
目的探讨miR-125在鼻咽癌组织中的表达及其调控肿瘤细胞生物学特性的可能机制。方法收集2018年6月至2019年6月上海交通大学附属第六人民医院南院收治的鼻咽癌患者癌组织及癌旁组织各30例,通过荧光定量PCR法检测miR-125和成纤维细胞生长因子2(FGF-2)mRNA的表达。鼻咽癌细胞CNE-2转染miR-125 mimic(miR-125 mimic组),并设阴性对照组(NC组)。Transwell小室实验检测细胞侵袭能力;划痕愈合实验检测细胞迁移能力;WST-1用于评估细胞的增殖活力;流式细胞术和电镜观察分别用于检测细胞的凋亡和自噬;双荧光素酶报告分析miR-125靶标;Western blotting检测相关蛋白的表达情况。结果鼻咽癌组织中miR-125 mRNA的相对表达量为0.692±0.316,明显低于癌旁组织的1.501±0.748(t=5.242,P<0.001);鼻咽癌组织中FGF-2 mRNA的相对表达量为1.317±0.552,明显高于癌旁组织的0.783±0.241(t=7.360,P<0.001)。miR-125 mimic组CNE-2细胞中miR-125 mRNA的表达量为4.091±0.145,显著高于NC组的0.993±0.137(t=85.062,P<0.001)。miR-125 mimic组完成转染48、96 h后,CNE-2细胞的增殖活性均显著低于NC组(0.891±0.214 vs.1.295±0.245,t=6.802,P<0.001;0.934±0.208 vs.1.488±0.269,t=8.924,P<0.001)。miR-125 mimic组的穿膜细胞数和细胞的迁移率分别为(36000±3820)个和(39.4±6.5)%,均显著低于NC组的(74000±7500)个和(102.7±10.6)%(t=24.728,P<0.001;t=27.883,P<0.001)。miR-125 mimic组CNE-2细胞凋亡率为(22.5±1.4)%,显著高于NC组的(1.4±0.5)%(t=77.740,P<0.001),且miR-125 mimic组细胞凋亡蛋白Bax的相对表达量为0.983±0.158,显著高于NC组的0.418±0.122(t=15.503,P<0.001),Bcl-2的相对表达量为0.688±0.174,显著低于NC组的1.013±0.109(t=8.670,P<0.001)。电镜观察到miR-125过表达的CNE-2细胞出现自噬现象,miR-125 mimic组自噬蛋白LC3Ⅱ/LC3Ⅰ相对表达量比值为2.517±0.209,显著高于NC组的1.238±0.135(t=28.156,P<0.001)。miR-125 mimic组FGF-2蛋白的表达量为0.504±0.118,显著低于NC组的1.228±0.134(t=22.210,P<0.001)。双荧光素酶报告证实FGF-2是miR-125的靶基因。FGF-2基因质粒和miR-125 mimic共转染的CNE-2细胞完成转染12、24、48及96 h细胞增殖活性均显著高于miR-125 mimic转染细胞(均P<0.05),细胞凋亡率显著低于miR-125 mimic转染细胞[(6.2±1.5)%vs.(17.6±2.4)%,t=22.062,P<0.001]。结论miR-125在鼻咽癌组织中表达下降,过表达miR-125可能通过下调FGF-2抑制鼻咽癌细胞CNE-2的增殖、迁移和侵袭,促进CNE-2的凋亡与自噬。
Objective To explore the expression of miR-125 in nasopharyngeal carcinoma tissues and the possible regulatory mechanism of biological characteristics of tumor cells.Methods Thirty cases of carcinoma and paracancerous tissues from patients with nasopharyngeal carcinoma admitted to Southern Hospital of Sixth People's Hospital Affiliated to Shanghai Jiaotong University from June 2018 to June 2019 were collected.The expressions of miR-125 and fibroblast growth factor 2(FGF-2)mRNA were detected by fluorescent quantitative PCR.Nasopharyngeal carcinoma CNE-2 cells were transfected by miR-125 mimic(miR-125 mimic group),and negative control group was set(NC group).Transwell chamber assay was used to determine cell invasion ability,scratch healing assay was used to determine cell migration ability,WST-1 assay was used to assess cell viability,flow cytometry and electron microscopy were used respectively to detect apoptosis and autophagy,dual luciferase reporter assay was used to analyze the target of miR-125,and Western blotting was used to detect the expressions of related proteins.Results The relative expression of miR-125 mRNA in nasopharyngeal carcinoma tissues was 0.692±0.316,which was significantly lower than 1.501±0.748 in the adjacent tissues(t=5.242,P<0.001).The relative expression of FGF-2 mRNA in nasopharyngeal carcinoma tissues was 1.317±0.552,which was significantly higher than 0.783±0.241 in the adjacent tissues(t=7.360,P<0.001).The miR-125 mRNA expression of CNE-2 cells in the miR-125 mimic group was 4.091±0.145,which was significantly higher than 0.993±0.137 in the NC group(t=85.062,P<0.001).The proliferative activities of CNE-2 cells in the miR-125 mimic group at 48 and 96 h after transfection were significantly lower than those in the NC group(0.891±0.214 vs.1.295±0.245,t=6.802,P<0.001;0.934±0.208 vs.1.488±0.269,t=8.924,P<0.001).The number of transmembrane cells and cell migration rate of the miR-125 mimic group were 36000±3820 and(39.4±6.5)%,which were significantly lower than 74000±7500 and(102.7±10.6)%of the NC group(t=24.728,P<0.001;t=27.883,P<0.001).The apoptosis rate of CNE-2 cells in the miR-125 mimic group was(22.5±1.4)%,which was significantly higher than that in the NC group(1.4±0.5)%(t=77.740,P<0.001).The relative expression of the apoptotic protein Bax in the miR-125 mimic group was 0.983±0.158,which was significantly higher than that in the NC group(0.418±0.122;t=15.503,P<0.001),and the relative expression of Bcl-2 was 0.688±0.174,which was significantly lower than that of the NC group(1.013±0.109;t=8.670,P<0.001).Autophagy was observed in miR-125 overexpressing CNE-2 cells by electron microscopy,and the relative expression ratio of autophagy protein LC3Ⅱ/LC3Ⅰin the miR-125 mimic group was 2.517±0.209,which was significantly higher than 1.238±0.135 in the NC group(t=28.156,P<0.001).The expression of FGF-2 protein in the miR-125 mimic group was 0.504±0.118,which was significantly lower than 1.228±0.134 in the NC group(t=22.210,P<0.001).The double luciferase report confirmed FGF-2 as the target gene of miR-125.At 12,24,48 and 96 h after the transfection,the cell proliferative activities of CNE-2 cells co-transfected by FGF-2 gene plasmid and miR-125 mimic were significantly higher than those of CNE-2 cells transfected by miR-125 mimic(all P<0.05),and the apoptosis rate was significantly lower than that of CNE-2 cells transfected by miR-125 mimic[(6.2±1.5)%vs.(17.6±2.4)%,t=22.062,P<0.001].Conclusion The expression of miR-125 is down-regulated in nasopharyngeal carcinoma tissues.Overexpression of miR-125 may inhibit the proliferation,migration and invasion of nasopharyngeal carcinoma CNE-2 cells and promote the apoptosis and autophagy of CNE-2 cells by down-regulating FGF-2 expression.
作者
杨超
王建波
张菊红
王佳龙
Yang Chao;Wang Jianbo;Zhang Juhong;Wang Jialong(Department of Otolaryngology,Southern Hospital of Sixth People's Hospital Affiliated to Shanghai Jiaotong University,Shanghai 201400,China;Central Laboratory,Southern Hospital of Sixth People's Hospital Affiliated to Shanghai Jiaotong University,Shanghai 201400,China)
出处
《国际肿瘤学杂志》
CAS
2020年第5期257-263,共7页
Journal of International Oncology
