miR-498靶向磷酸烯醇丙酮酸羧激酶1对口腔鳞状细胞癌细胞生长的影响

Effect of miR-498 targeting PCK1 on growth of oral squamous cell carcinoma cells

目的探讨miR⁃498靶向磷酸烯醇丙酮酸羧激酶1(PCK1)对口腔鳞状细胞癌(OSCC)细胞生长的影响。方法qRT⁃PCR检测150例OSCC患者肿瘤组织及OSCC细胞系HSC⁃3、SCC⁃15、SCC⁃9中miR⁃498表达;将miR⁃498 mimics和anti⁃miR⁃498分别转染于SCC⁃15细胞,qRT⁃PCR检测细胞中miR⁃498水平,western blot检测细胞中PCK1蛋白表达,CCK⁃8检测细胞增殖,流式细胞术检测细胞凋亡,Transwell实验检测细胞迁移与侵袭;双荧光素酶报告基因检测miR⁃498与PCK1的靶向关系;共转染miR⁃498和PCK1进一步验证miR⁃498和PCK1对SCC⁃15细胞增殖、凋亡、迁移与侵袭的影响。结果与人正常口腔角质细胞HOK(1.03±0.14)比较,人OSCC细胞系HSC⁃3(2.05±0.21)、SCC⁃15(2.75±0.31)、SCC⁃9(2.31±0.28)中miR⁃498的表达水平显著升高(F=53.639,P<0.001),且SCC⁃15细胞中miR⁃498的表达量最高,因此,选择SCC⁃15细胞进行后续研究。与对照组和miR⁃NC组比较,miR⁃498 mimics组SCC⁃15细胞迁移与侵袭数目显著升高(P<0.05),细胞凋亡率显著降低(P<0.05);与对照组和anti⁃miR⁃NC组比较,anti⁃miR⁃498组SCC⁃15细胞迁移与侵袭数目显著降低,细胞凋亡率显著升高(P<0.05)。miR⁃498 mimics组(0.23±0.02)SCC⁃15细胞中PCK1蛋白表达水平显著低于对照组(0.51±0.08)和miR⁃NC组(0.49±0.06,P<0.05);anti⁃miR⁃498组(1.03±0.05)SCC⁃15细胞中PCK1蛋白表达水平显著高于对照组(0.51±0.08)和anti⁃miR⁃NC组(0.48±0.07,P<0.05)。miR⁃498靶向负调控PCK1表达,上调PCK1表达可逆转过表达miR⁃498对SCC⁃15细胞增殖、凋亡、迁移和侵袭的影响。结论miR⁃498通过靶向抑制PCK1表达促进SCC⁃15细胞增殖、迁移、侵袭,抑制细胞凋亡,可能为OSCC的临床诊治提供新的分子靶标。

Objective To investigate the effect of miR⁃498 targeting phosphoenolpyruvate carboxykinase 1(PCK1)on growth of oral squamous cell carcinoma(OSCC)cells.Methods QRT⁃PCR was used to detect expression of miR⁃498 in tumor tissues from 150 OSCC patients and different OSCC cell lines HSC⁃3,SCC⁃15 and SCC⁃9.LipofectamineTM2000 transfection kit was used to transfect miR⁃498 mimics and anti⁃miR⁃498 into SCC⁃15 cells respectively.qRT⁃PCR was used to detect expression of miR⁃498 in the cells,so were Western blot to detect expression of PCK1 protein in the cells,CCK⁃8 to detect cell proliferation,flow cytometry to detect cell apoptosis,Transwell migration assay to detect cell migration and invasion,and dual⁃luciferase reporter gene assay to detect the targeting relationship between miR⁃498 and PCK1.Co⁃transfection of miR⁃498 and PCK1 was used to further verify the effects of miR⁃498 and PCK1 on proliferation,apoptosis,migration and invasion of SCC⁃15 cells.Results Compared with human normal oral keratinocyte HOK(1.03±0.14),the expression level of miR⁃498 in human OSCC cell lines HSC⁃3(2.05±0.21),SCC⁃15(2.75±0.31),and SCC⁃9(2.31±0.28)was significantly higher(F=53.639,P<0.001),and expression of miR⁃498 was the highest in SCC⁃15 cells.SCC⁃15 cells were then selected for the subsequent research.Compared with the control group and miR⁃NC group,the number of migration and invasion of SCC⁃15 cells in miR⁃498 mimics group increased significantly(P<0.05),and the apopto⁃sis rate decreased significantly(P<0.05);compared with the control group and anti⁃miR⁃NC group,the number of migration and invasion of SCC⁃15 cells in anti⁃miR⁃498 group decreased significantly(P<0.05),and the apoptosis rate increased significantly(P<0.05).The expression level of PCK1 protein in SCC⁃15 cells in the miR⁃498 mimics group(0.23±0.02)was significantly lower than that the control group(0.51±0.08)and miR⁃NC group(0.49±0.06,P<0.05);The expression level of PCK1 protein in SCC⁃15 cells in the anti⁃miR⁃498 group(1.03±0.05)was significantly higher than that in the control group(0.51±0.08)and anti⁃miR⁃NC group(0.48±0.07,P<0.05).miR⁃498 negatively regulated PCK1 expression.Up⁃regulation of PCK1 expression reversed the effects of overex⁃pression of miR⁃498 on proliferation,apoptosis,migration and invasion of SCC⁃15 cells.Conclusions MiR⁃498 can accelerate the proliferation,migration and invasion of SCC⁃15 cells and inhibit apoptosis of SCC⁃15 by inhibit⁃ing the expression of PCK1,probably providing new molecular targets for the clinical diagnosis and treatment of OSCC.