Spondin-2基因沉默对人结直肠癌SW480细胞的侵袭及上皮-间充质转化能力的影响

Effect of spondin-2 gene silencing on invasion and epithelial-mesenchymal transition ability of human colorectal cancer SW480 cells

目的探讨Spondin-2(SPON2)基因沉默对人结直肠癌SW480细胞侵袭及上皮-间充质转化(EMT)能力的影响。方法体外培养人结直肠癌SW480细胞, 对细胞进行慢病毒转染, 并将细胞分为对照组、阴性转染组和shSPON2转染组。采用实时荧光定量聚合酶链反应(PCR)法检测各组细胞中Spondin-2 mRNA的表达;细胞计数试剂盒8(CCK-8)法检测培养24、48、72 h后各组细胞增殖情况, Transwell法检测培养48 h后各组细胞侵袭情况;蛋白免疫印迹法检测培养48 h后各组细胞中蛋白细胞外信号调节激酶1/2(ERK1/2)、pERK1/2、含ETS域蛋白1(ELK-1)、pELK-1及EMT相关蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)的表达。研究数据采用单因素方差分析进行比较。结果 shSPON2转染组SPON2 mRNA表达水平(0.43±0.04)低于对照组(1.00±0.05, t=21.805, P<0.05), 低于阴性转染组(0.97±0.03, t=26.454, P<0.05)。培养24、48、72 h后, 3组间同一时间吸光度(A)值比较, 差异有统计学意义(F=27.982、31.052、28.484, P<0.05)。同一时间下, 与对照组比较, shSPON2转染组细胞A值降低, 且低于阴性转染组(P<0.05)。shSPON2转染组SW480细胞侵袭数目[(55.93±11.24)个]低于对照组[(118.75±19.61)个, t=6.808, P<0.05], 低于阴性转染组[(105.87±15.36)个, t=6.427, P<0.05]。3组间SW480细胞蛋白pERK1/2、pELK-1表达水平比较, 差异有统计学意义(F=357.899、339.313, P<0.05)。与对照组比较, shSPON2转染组细胞蛋白pERK1/2、pELK-1表达降低, 且低于阴性转染组(P<0.05)。3组间E-cadherin、N-cadherin、Vimentin蛋白表达水平比较, 差异有统计学意义(F=316.867、533.522、416.446, P<0.05)。与对照组比较, shSPON2转染组细胞蛋白E-cadherin表达升高, N-cadherin、Vimentin蛋白表达降低(P<0.05)。结论 SPON2基因沉默能够抑制SW480细胞增殖、侵袭, 抑制EMT过程, 可能与ERK/ELK-1信号通路的抑制有关。

Objective To investigate the effect of Spondin-2(SPON2)gene silencing on invasion and epithelial-mesenchymal transition(EMT)ability of human colorectal cancer SW480 cells.Methods SW480 cells were cultured in vitro,transfected with lentivirus and divided into control group,negative transfection group and shSPON2 transfection group.The expression of SPON2 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction(PCR).The cell proliferation after culture for 24,48 and 72 h was detected by cell counting kit-8(CCK-8)method.The cell invasion after culture for 48 h was detected by Transwell method.Western blotting was used to detect the expression of extracellular regu-lated protein kinases 1/2(ERK1/2),pERK1/2,ETS-domain containing protein-1(ELK-1),pELK-1 and EMT related proteins E-cadherin,N-cadherin and vimentin of cells in each group after culture for 48 h.The data were compared by one-way ANOVA.Results The SPON2 mRNA expression level in the shSPON2 transfection group(0.43±0.04)was lower than that in the control group(1.00±0.05,t=21.805,P<0.05),and in the negative transfection group(0.97±0.03,t=26.454,P<0.05).After 24,48 and 72 h of incubation,there was statistically significant difference in the absorbance(A)values at the same time among the three groups(F=27.982,31.052,28.484,P<0.05).At the same time,the A value was decreased in the shSPON2 transfection group as compared with that in the control group,and lower than that in the negative transfection group(P<0.05).The number of invasive cells in the shSPON2 transfection group(55.93±11.24)was less than that in the control group(118.75±19.61,t=6.808,P<0.05),and the negative transfection group(105.87±15.36,t=6.427,P<0.05).Moreover,the expression of pERK1/2 and pELK-1 showed significant difference among the three groups(F=357.899,339.313,P<0.05).The expression of pERK1/2 and pELK-1 in shSPON2 transfection group decreased as compared with that in the negative group(P<0.05).This study also demonstrated that SPON2 regulated colorectal cancer invasion and metastasis by modulating EMT by up-regulating E-cadherin,N-cadherin,and Vimentin(F=316.867,533.522,416.446,P<0.05).In detail,the shSPON2 group showed increased expression of E-cadherin,while decreased expression of N-cadherin and Vimentin as compared with the control group(P<0.05).Conclusion Silencing SPON2 can inhibit the proliferation and invasion ability of SW480 cells and the EMT process,which may be related to the inhibition of ERK/ELK-1 signaling pathway.