miR-634调控趋化因子受体-4蛋白对鼻咽癌细胞生物活性的作用机制

Mechanism of miR-634 regulating CXCR4 protein on biological activity of nasopharyngeal carcinoma cells

目的 探究miR-634调控趋化因子受体-4[chemokine(C-X-C motif)receptor 4,CXCR4]蛋白对鼻咽癌细胞生物活性的作用机制。方法 过表达miR-634转染至CNE1细胞,RT-PCR检测miR-634和CXCR4 mRNA的表达;荧光素酶报告基因检测miR-634和CXCR4的靶向关系;同时转染miR-634和CXCR4至鼻咽癌细胞,蛋白质印记检测CXCR4蛋白的表达;CCK8检测细胞增殖;Transwell检测细胞侵袭;流式细胞仪检测细胞凋亡。结果 与NP69细胞相比,CNE1细胞中miR-634 mRNA(0.48±0.05)表达显著降低,CXCR4 mRNA(0.96±0.09)表达显著升高(P<0.05);空白组与miR-NC组CNE1细胞中miR-634和CXCR4 mRNA表达无明显的差异(P>0.05);和空白组、miR-NC组相比,miR-634组CNE1细胞中miR-634 mRNA(1.72±0.12)的表达显著升高,CXCR4 mRNA(0.51±0.07)表达得到显著抑制(P<0.05)。荧光素酶报告实验结果显示,过表达miR-634显著降低CXCR4野生型质粒荧光素酶的活性(P<0.05);和空白组相比,miR-634组CXCR4(0.37±0.04)蛋白显著降低(P<0.05),CXCR4-1组CXCR4(1.26±0.11)蛋白明显升高(P<0.05),和CXCR4-1组相比,CXCR4-2组中CXCR4蛋白表达得到显著抑制(P<0.05),CXCR4-2组中CXCR4蛋白水平高于miR-634组(P<0.05)。和空白组相比,miR-634组细胞的增殖速率及侵袭数目显著降低,而CXCR4-1组细胞的增殖速率显著升高(P<0.05);和CXCR4-1组相比,CXCR4-2组细胞的增殖速率及侵袭数目(46.21±5.83)得到明显抑制(P<0.05);和空白组相比,miR-634组细胞凋亡率(17.68±3.21)显著增加,而CXCR4-1组细胞凋亡率明显降低(P<0.05);和CXCR4-1组细胞凋亡率相比较,CXCR4-2组细胞的凋亡率明显升高(P<0.05)。miR-634组和顺铂组细胞增殖、侵袭、凋亡能力均没有显著差异(P>0.05)。结论 miR-634抑制鼻咽癌CNE1细胞的增殖和侵袭,促进鼻咽癌细胞的凋亡,其作用机制与调控CXCR4蛋白有关。

OBJECTIVE To explore the mechanism of miR-634 regulating chemokine receptor-4[chemokine(C-X-C motif) receptor 4,CXCR4]protein on the biological activity of nasopharyngeal carcinoma cells.METHODSOverexpression of miR-634 was transfected into CNE1 cells,RT-PCR was used to detect the mRNA expression of miR-634 and CXCR4.Luciferase reporter gene was used to detect the targeting relationship between miR-634 and CXCR4.At the same time,miR-634 and CXCR4 were transfected into nasopharyngeal carcinoma cells,the expression of CXCR4 protein was detected by Western-blot,cell proliferation was detected by CCK8,cell invasion ability was detected by Transwell and apoptosis was detected by flow cytometry.RESULTS Compared with NP69 cells,the expression of miR-634 mRNA(0.48±0.05) in CNE1 cells was significantly reduced,and the expression of CXCR4 mRNA(0.96±0.09) was significantly increased(P<0.05).miR-634 and CXCR4 mRNA expression in CNE1 cells in the blank group and miR-NC group were not significantly different(P>0.05).Compared with the blank group and miR-NC group,the expression of miR-634 mRNA(1.72±0.12) in CNE1 cells in the miR-634 group was significantly increased,and CXCR4 mRNA(0.51±0.07) was significantly inhibited(P<0.05).The results of the luciferase report experiment showed that overexpression of miR-634 significantly reduced the activity of CXCR4 wild-type plasmid luciferase(P<0.05).Compared with the blank group,the CXCR4(0.37±0.04) protein in the miR-634 group was significantly reduced(P<0.05),and the CXCR4(1.26±0.11) protein in the CXCR4-1 group was significantly increased(P<0.05).Compared with the CXCR4-1group,the expression of CXCR4 protein in the CXCR4-2groups was significantly inhibited(P<0.05),and the level of CXCR4 protein in the CXCR4-2 group was higher than that in the miR-634 group(P<0.05).Compared with the blank group,the proliferation rate of the miR-634 group was significantly lower,and the number of invasions was significantly reduced,while the proliferation rate of cells in the CXCR4-1 group was significantly increased(P<0.05).Compared with the CXCR4-1 group,the proliferation rate and the number of invasions(46.21±5.83) of the cells in the CXCR4-2 group were significantly decreased.Compared with the blank group,the apoptotic rate of cells in the miR-634group(17.68±3.21) was significantly increased,while the apoptotic rate of cells in the CXCR4-1 group was significantly reduced(P<0.05).Compared with the apoptosis rate of CXCR4-1 group,the apoptosis rate of CXCR4-2 group was significantly increased(P<0.05).There was no significant difference in cell proliferation,invasion and apoptosis in miR-634 group and cisplatin group(P>0.05).CONCLUSIONmiR-634 inhibits the proliferation and invasion of nasopharyngeal carcinoma CNE1 cells and promotes the apoptosis of nasopharyngeal carcinoma cells.Its mechanism my be related to the regulation of CXCR4 protein.