叉头框K1-卷曲螺旋结构域蛋白43轴对大肠癌细胞迁移和上皮-间充质转化的影响

Effects of forkhead box protein K1-coiled coil domain protein 43 axis on migration and epithelial mesenchymal transition of colorectal cancer cells

目的观察叉头框K1(FOXK1)-卷曲螺旋结构域蛋白43(CDC43)轴对大肠癌细胞迁移和上皮-间充质转化(EMT)的影响。方法选取2017年9月到2019年9月南阳医学高等专科学校第一附属医院收治的103例大肠癌和对应的癌旁组织作为研究对象,采用免疫组织化学分析FOXK1蛋白表达水平;采用规律间隔成簇短回文重复序列及其相关核酸酶9系统(CRISPR-Cas9)技术在SW480细胞中建立FOXK1敲除细胞系(FOXK1 KO组)和对照细胞系(对照组),采用细胞计数试剂盒(CCK-8)检测两组细胞增殖;采用Tanswell分析两组细胞迁移;采用蛋白质印迹法(Western blot)检测两组细胞EMT。组间比较采用t检验。结果与癌旁组织FOCK1表达水平(40.32±6.90)比较,大肠癌组织FOXK1蛋白表达水平显著增高(159.21±13.85),差异有统计学意义(t=4.918,P<0.05)。与对照组细胞FOXK1蛋白表达水平(1.09±0.21)比较,FOXK1 KO组细胞FOXK1蛋白表达水平显著下降,差异有统计学意义(t=5.012,P<0.05)。与对照组细胞吸光度值(1.94±0.28)比较,FOXK1 KO组细胞吸光度值显著下调(1.07±0.19),差异有统计学意义(t=3.162,P<0.05)。Transwell结果显示,与对照组细胞迁移数量[(95.28±10.20)个]比较,FOXK1 KO组细胞迁移数量显著下降[(48.39±8.21)个],差异有统计学意义(t=3.091,P<0.05)。与对照组细胞上皮细胞标志物E-钙黏蛋白(E-cadherin)表达水平(0.26±0.08)比较,FOXK1 KO组细胞E-cadherin表达水平显著增高(1.05±0.11),差异有统计学意义(t=3.001,P<0.05)。与对照组细胞间质细胞标志物N-钙黏蛋白(N-cadherin)(0.78±0.13)和波形蛋白(Vimentin)(1.15±0.19)表达水平比较,FOXK1 KO组细胞N-cadherin(0.31±0.11)和Vimentin表达水平显著下调(0.39±0.14),差异有统计学意义(t=2.891、2.318,P<0.05)。与对照组细胞CCDC43表达水平(1.20±0.22)比较,FOXK1 KO组细胞CCDC43蛋白表达水平显著下调(0.57±0.18),差异有统计学意义(t=2.581,P<0.05)。结论FOXK1在大肠癌中高表达,通过调控CCDC43蛋白水平调节大肠癌细胞增殖、迁移和EMT。

Objective To investigate the effects of forkhead box protein K1(FOXK1)-coiled coil domain protein 43(CCDC43)axis on the migration and epithelial mesenchymal transition of colorectal cancer cells.Methods 103 cases of colorectal cancer and adjacent tissues in our hospital from September 2017 to September 2019 were selected as the research objects and the expression level of FOXK1 protein was analyzed by immunohistochemistry.FOXK1 knockout stable cell line and control cell line in SW480 cells were constructed by regularly spaced clustered short palindrome repeats and their related nuclease 9 system(CRISPR-Cas9).Ediated by lentivirus were used;cell proliferation was detected by cell counting kit-8(CCK-8);cell migration was analyzed by Transwell;and Western blotting was used to analyze cell migration.Results Compared with the expression level of FOCK1 in adjacent tissues(40.32±6.90),FOXK1 protein expression in colorectal cancer tissues(159.21±13.85)was significantly increased(t=4.918,P<0.05).Compared with the control group,the expression of FOXK1 protein in FOXK1 KO group was significantly decreased(t=5.012,P<0.05).Compared with the control group(1.94±0.28),the absorbance of FOXK1 KO group was significantly decreased(1.07±0.19),and the difference was statistically significant(t=3.162,P<0.05).Transwell results showed that the number of migration cells in the control group and FOXK1 KO group was and,respectively.Compared with the control group[(95.28±10.20)cells],FOXK1 KO Group[(48.39±8.21)cells]significantly decreased,the difference was statistically significant(t=3.091,P<0.05).The expression level of E-cadherin in FOXK1 KO group was significantly higher than that in control group(0.26±0.08),and that in FOXK1 KO group was(1.05±0.11),the difference was statistically significant(t=3.001,P<0.05).Compared with the control group,the expression levels of N-cadherin(0.78±0.13)and Vimentin(1.15±0.19)were significantly lower in FOXK1 KO group than those in control group(t=2.891,2.318,P<0.05).Compared with the control group(1.20±0.22),the expression level of CCDC43 in FOXK1 KO group was significantly decreased(0.57±0.18),and the difference was statistically significant(t=2.581,P<0.05).Conclusion FOXK1 is highly expressed in colorectal cancer,which regulates the proliferation,migration and epithelial mesenchymal transition of colorectal cancer cells by regulating CCDC43 protein level.