摘要
目的探讨miR-101-3p靶向E2F转录因子2(E2F2)调控黑色素瘤细胞增殖的作用机制。方法使用GEPIA、OSskcm等数据库联合分析E2F2在黑色素瘤中的表达,并评估转录因子E2F2在黑色素瘤预后中的价值;利用短发夹RNA(shRNA)抑制黑色素瘤细胞MV3中E2F2表达后,分别采用PI染色和流式细胞术检测细胞周期的改变;Western blot实验检测细胞周期蛋白E(CyclinE)和细胞周期蛋白依赖性激酶2(CDK2)的表达水平;免疫荧光实验检测细胞中自噬相关蛋白LC3B的表达水平;裸鼠荷瘤实验观察黑色素瘤在体内生长的变化。经miR-101-3p mimics处理后,采用实时荧光定量PCR(qPCR)检测MV3细胞中miR-101-3p的表达水平,同时利用qPCR和Western blot实验检测E2F2的表达水平;通过MTT、BrdU法检测MV3细胞增殖水平。结果E2F2表达升高的患者预后更差(P<0.05);与shGFP组相比,shE2F2组中处于G0/G1期的细胞比例显著增多(P<0.05),CyclinE和CDK2的表达水平显著降低,自噬相关蛋白LC3B荧光信号明显减少(P<0.05),体内黑色素瘤形成质量显著降低(P<0.05);采用miR-101-3p mimics处理后,MV3细胞中miR-101-3p的表达水平明显升高(P<0.05),E2F2的mRNA和蛋白表达均显著低于NC mimics处理组(P<0.05),同时MTT结果表明miR-101-3p mimics组MV3细胞的活力显著降低(P<0.05),BrdU标记试验结果显示miR-101-3p mimics组细胞中BrdU阳性信号明显减少(P<0.05)。结论miR-101-3p可靶向E2F2并抑制其表达,进而降低黑色素瘤细胞增殖水平。
Objective To explore effect and mechanism of miR-101-3p targeting E2F transcription factor 2(E2F2)in regulating the melanoma cell proliferation.Methods GEPIA,OSskcm databases were used to analyze the expression of E2F2 in melanoma and evaluate the value of transcription factor E2F2 in the prognosis of melanoma.The expression of E2F2 in melanoma cells MV3 was inhibited by shRNA,and the cell cycle changes were detected by PI staining flow cytometry.The expression levels of CyclinE and Cyclin-dependent kinase 2(CDK2)were detected by Western blot assay.The expression level of autophagy-associated protein LC3B was detected by immunofluorescence,and the growth changes of melanoma in vivo were observed by tumor-bearing experiments in nude mice.The expression level of miR-101-3p in MV3 cells was detected by real-time fluorescence quantitative PCR,the expression level of E2F2 was detected by qPCR and Western blot assay,and the proliferation level of MV3 cells was detected by MTT and BrdU methods.Results Melanoma patients with increased E2F2 expression had worse prognosis(P<0.05).Compared with the shGFP group,the proportion of cells in G0/G1 phase increased significantly in the shE2F2 group(P<0.05),the expression levels of CyclinE and CDK2 decreased significantly,the fluorescence signal of autophagy-associated protein LC3B decreased significantly(P<0.05),and the weight of melanoma formation in vivo decreased significantly(P<0.05).The expression level of miR-101-3p in MV3 cells increased significantly after treated with miR-101-3p mimics(P<0.05),and the mRNA and protein expression of E2F2 were significantly lower than those in the NC mimics treatment group(P<0.05).Meanwhile,MTT results showed that the vitality of MV3 cells decreased significantly in the miR-101-3p mimics group(P<0.05).BrdU labeling test results showed that the BrdU positive signal decreased significantly in the miR-101-3p mimics group(P<0.05).Conclusion miR-101-3p targets to E2F2 and inhibits its expression,and then reduces the proliferation level of melanoma cells.
作者
李吻吻
朱智鑫
韩利民
焦艳林
翁宗琴
赵海龙
LI Wenwen;ZHU Zhixin;HAN Limin;JIAO Yanlin;WENG Zongqin;ZHAO Hailong(Department of Pathophysiology,School of Preclinical Medicine,Zunyi Medical University,Zunyi 563000,China)
出处
《天津医药》
CAS
北大核心
2022年第11期1121-1127,共7页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(82060503)
贵州省科技计划项目(黔科合基础[2019]1334号)
贵州省卫健委科学技术基金资助项目(gzwjkj2019-1-033)。
作者简介
李吻吻(1993),女,硕士在读,主要从事肿瘤靶向治疗方面研究。E-mail:lw20200701@163.com;通信作者:赵海龙,E-mail:Hailongzhao@zmu.edu.cn。
