miR-4295在鼻咽癌细胞增殖中的作用

The role of miR-4295 in the proliferation of human nasopharyngeal carcinoma

目的探讨miR-4295在鼻咽癌细胞发生发展及增殖过程中的作用及相关的分子机制。方法通过脂质体转染miR-4295 mimic,inhibitor及其NC到鼻咽癌细胞株6-10B,CNE2。分别应用MTT实验、软琼脂集落形成实验检测miR-4295对鼻咽癌细胞增殖能力的影响;WB和QPCR方法检测miR-4295、细胞周期素D1(CyclinD1)、p21cip1、Rb、pRb在细胞中蛋白及mRNA的表达水平。通过Targetscan分析预测miR-4295与SOX9的3’UTR的结合情况,采用双荧光素酶报告实验检测miR-4295与SOX9的结合情况,同时采用WB检测细胞株中SOX9的表达。结果MTT、软琼脂集落形成实验均显示过表达miR-4295后鼻咽癌细胞增殖能力明显增强,细胞生长速率高于NC组,细胞集落形成个数多于NC组,差异均有统计学意义(P<0.05);而miR-4295沉默后鼻咽癌细胞增殖能力明显降低,与NC-in组相比,差异有统计学意义(P<0.05);WB及QPCR结果显示:过表达miR-4295后,细胞周期蛋白CyclinD1表达升高而细胞周期抑制因子p21cip1下调;通过Targetscan预测发现miR-4295靶向结合SOX9的3’UTR,荧光素酶活性检测结果发现miR-4295过表达后荧光素酶活性明显低于NC组及突变组,差异有统计学意义(P<0.05)。结论上调miR-4295可通过抑制SOX9蛋白翻译从而促进鼻咽癌细胞增殖能力,并调节细胞周期相关蛋白,在鼻咽癌发生发展中扮演重要的角色。

Objective To investigate the possible functions and mechanism of miR-4295 in proliferation of human nasopharyngeal carcinoma(NPC).Methods MiR-4295 mimic,inhibitor and its negative control were delivered by lipofectamine 2000 at 6-10 B and CNE2 cell line.Cell proliferation was measured by MTT and soft agar assay.MiR-4295,CyclinD1,p21 cip1,Rb,pRb expression were detected by Western blot(WB)and reverse transcriptase polymerase chain reaction(QPCR),respectively.The binding site between miR-4295 and SOX93’UTR was predicted by Targetscan analysis.The relationship between miR-4295 and SOX9 in the transfected cell lines was analyzed by the luciferase report assay and WB test,respectively.Results MTT and soft agar assay found that the proliferation ability was strongly increased by over expression of miR-4295,the cell growth rate was higher than that of NC control group,and the number of cell colony formation was higher than that of NC control group,the difference was statistically significant(P<0.05).The proliferation ability was obviously decrease while miR-4295 was inhibited,compared with NC-in control group,the difference was statistically significant(P<0.05).Western blot and QPCR analysis showed that the expression level of CyclinD1 was up regulated,while the expression of p21 cip1 was down regulated in the over expression of miR-4295 group,meanwhile pRb had up regulated obviously.Moreover,Targetscan result predicted that miR-4295 would bind to SOX93’UTR.The results of luciferase activity test showed that luciferase activity after overexpression of miR-4295 was significantly lower than that of normal and mutant groups,and the difference was statistically significant(P<0.05).Conclusion Up regulation of miR-4295 might promote the proliferation of nasopharyngeal carcinoma cells by inhibiting SOX9 expression,and regulating the cell cycle related proteins,which might play an important role in the development of nasopharyngeal carcinoma.