甲基莲心碱通过影响微小RNA抑制鼻咽癌细胞侵袭转移的机制研究

Mechanisms of inhibition of invasion and metastasis of nasopharyngeal carcinoma cells by neferine through its influence on microRNAs

本文以鼻咽癌CNE-1和5-8F细胞为研究对象,检测甲基莲心碱(neferine, Nef)抑制鼻咽癌侵袭转移的作用,探讨其抑制侵袭转移的机制。CCK-8法检测鼻咽癌细胞活性;划痕实验、Transwell细胞体外迁移侵袭实验观察Nef对鼻咽癌CNE-1、5-8F细胞侵袭、迁移能力的影响;Western blot法检测上皮-间质转化(epithelial to mesenchymal transition, EMT)相关蛋白及其转录因子表达水平;miRNA基因芯片检测Nef处理过的5-8F细胞与对照组miRNA差异基因表达谱,对差异表达基因进行生物信息学分析及筛选,同时研究其表达与鼻咽癌侵袭转移的相关性。实验结果显示:30μmol·L^-1 Nef对鼻咽癌CNE-1、5-8F细胞活性无明显影响, Nef能显著抑制鼻咽癌细胞的侵袭与转移,Western blot结果显示Nef使鼻咽癌CNE-1、5-8F细胞中神经型钙黏蛋白(N-cadherin)、波形蛋白(vimentin)表达下调,上皮型钙黏蛋白(E-cadherin)表达上调, Twist、Snail、Slug转录因子无显著表达差异;miRNA基因芯片结果显示Nef作用后的5-8F鼻咽癌细胞与对照组相比共10个miRNA有2倍以上表达差异,其中hsa-let-7c-5p与hsa-miR-423-5p表达差异最显著,分别下调至30.6%、28.6%。生物信息分析显示hsa-let-7c-5p与hsa-miR-423-5p有共同的下游靶基因:小细胞膜蛋白3 (small integral membrane protein 3, SMIM3)、神经生长因子(nerve growth factor, NGF)。鼻咽癌5-8F细胞转染hsa-let-7c-5p mimic与hsa-miR-423-5p mimic后增强其侵袭与转移能力, SMIM3、NGF表达下调。以上研究结果表明, Nef可能通过抑制hsa-let-7c-5p与hsa-miR-423-5p的表达,作用于下游靶基因SMIM3、NGF,抑制EMT相关蛋白的表达,从而抑制鼻咽癌细胞的侵袭转移。研究结果为Nef抑制肿瘤侵袭转移提供实验依据。

This study was designed to investigate the inhibitory effect and mechanism of neferine(Nef) on invasion and metastasis of nasopharyngeal carcinoma cells(NPC). The viability of CNE-1 and 5-8 F cells was detected by CCK-8 assay after treatment with different concentrations of Nef. The effects of Nef on cell migration and invasion were detected by the scratch test and Transwell assay. Western blot analysis was used to detect the effects of Nef on levels of epithelial-mesenchymal transition(EMT)-associated proteins and transcription factors.The differentially expressed gene profiles between control group and Nef group were analyzed by microRNA microarray, combined with bioinformation analysis. It was observed that 30 μmol·L^-1 Nef had no significant effect on the viability of CNE-1 and 5-8 F cells. Western blot assay showed that the expression level of neurotroponin cadherin(N-cadherin) and vimentin decreased after treatment with Nef, while the expression of epithelial cadherin(E-cadherin) increased. The expression of transcription factors including Twist, Snail, and Slug exhibited no significant difference. Results of the microRNA microarray suggest that 10 microRNAs showed significant differences when compared with the control group. Bioinformatics analysis showed that hsa-let-7 c-5 p and hsamicroRNA-423-5 p targeted the same downstream genes: small integral membrane protein 3(SMIM3) and nerve growth factor(NGF). Overexpression of hsa-let-7 c-5 p and hsa-miR-423-5 p promoted the invasion and migration ability of 5-8 F cells and decreased the expression of SMIM3 and NGF. The results from this study suggest that Nef may inhibit the invasion and metastasis of NPC cells by inhibiting the expression of hsa-let-7 c-5 p and hsamiR-423-5 p followed by the upregulation of SMIM3 and NGF;thus, regulating the expression of EMT-associated proteins. Our data have provided experimental evidence for the inhibition of tumor invasion and metastasis by Nef.