敲低DUSP2对结直肠癌细胞SW480的影响及机制研究

Effect of Knocking Down DUSP2 Expression on Colorectal Cancer Cells Line SW480 and Its Mechanism

目的探讨双特异性磷酸酶2(DUSP2)对结直肠癌(CRC)细胞SW480的影响及其可能的机制。方法利用癌症基因组图谱(TCGA)数据库分析DUSP2在CRC患者中的表达情况对预后的影响。通过对CRC细胞SW480转染慢病毒并用嘌呤霉素筛选构建出DUSP2稳定敲低的细胞株为实验组,以空载慢病毒转染并筛选后的SW480细胞为对照组。利用实时定量聚合酶链式反应(qRT-PCR)技术及蛋白质印迹法检测DUSP2在mRNA水平及蛋白水平的表达。采用细胞划痕试验及细胞迁移试验评估SW480细胞迁移能力。采用CCK-8细胞增殖试验检测DUSP2敲低表达对SW480细胞增殖能力的影响。通过蛋白质印迹法检测丝裂原活化蛋白激酶(MAPK)通路中ERK、p-ERK、p38、p-p38蛋白表达水平及多聚二磷酸腺苷-核糖聚合酶-1(PARP1)蛋白表达水平。结果DUSP2基因表达水平与CRC患者的生存率无关(P>0.05)。蛋白质印迹法及qRT-PCR检测结果均显示DUSP2稳定敲低的SW480细胞(实验组)中DUSP2表达较对照组下调(P<0.05)。DUSP2稳定敲低的SW480细胞株构建成功。与对照组比较,实验组细胞划痕区域闭合速度较快,迁移能力较强,增长速度较快(P<0.05)。实验组细胞中p-ERK、p-p38及PARP1蛋白表达较对照组上调(P<0.05)。结论敲低DUSP2的表达可显著增强SW480细胞的增殖及迁移并抑制其凋亡,这与DUSP2可促进ERK、p38的磷酸化有关。

Objective To investigate the effect of dual specificity phosphatase 2(DUSP2)on the cell SW480 of colorectal cancer(CRC)and its possible mechanism.Methods The cancer genome atlas(TCGA)database was used to analyze the effect of DUSP2 expression in CRC patients on prognosis.By transfecting CRC cells SW480 with lentivirus and screening with puromycin,a cell line with stable knockdown of DUSP2 was constructed as the experimental group.The SW480 cells transfected with empty lentivirus were screened as control group.The quantitative real-time polymerase chain reaction(qRT-PCR)and western blot were used to detect the expression of DUSP2 at mRNA level and protein level.The effect of SW480 cell migration was assessed by wound healing assay and transwell assay.The effect of DUSP2 downregulation on the proliferation ability of SW480 cells was evaluated by CCK-8 cell proliferation assay.The protein expressions of ERK,p-ERK,p38,p-p38 and poly adenosine diphosphate-ribose polymerase-1(PARP1)were tested by the western blot analysis.Results There was no correlation between DUSP2 gene expression level and survival rate of CRC patients(P>0.05).The results of western blot and qRT-PCR showed that the expression of DUSP2 in SW480 cells with stable knock-down of DUSP2(experimental group)was lower than that in control group(P<0.05).The cell line SW480 with stable knock-down of DUSP2 was successfully constructed.Compared with their control group,the experimental group had faster closure of wound area,stronger migration ability and increased faster(P<0.05).The expressions of p-ERK,p-p38 and PARP1 protein in experimental group were up-regulated compared with those in control group(P<0.05).Conclusion Knocking down the expression of DUSP2 can significantly enhance the proliferation and migration of SW480 cells and inhibit their apoptosis.This is related to the fact that DUSP2 can promote the phosphorylation of ERK and p38.