| BackgroundThe analysis of cancer situation in China in 2020 shows that colon cancer still ranks in the top of malignant tumors in China,seriously endangering the physical and mental health of citizens.It is of great scientific significance and clinical research value to study the occurrence,development and metastasis of colon cancer and clarify the molecular regulation mechanism of colon cancer metastasis.CK1(casein kinase)is a conserved silk/threonine protein kinase that participates in the regulation of various physiological and pathological signals.However,whether CK1 kinase is involved in the regulation of colon cancer metastasis has not been reported.MTSS1(tumor metastasis suppressor)plays an important role in colon cancer metastasis,but whether MTSS1 protein is targeted by other proteins or kinases in the cell and its mechanism of action have not been reported.ObjectiveTo elucidate the role of CK1ε in colon cancer metastasis,and to verify the possible mechanism that CK1ε regulates the migration and invasion of colon cancer cells by phosphorylation of MTSS1 protein,thus providing a direction for finding new targets for clinical control of colon cancer metastasis.Methods1.Culture normal colonic epithelial cells(NCM460)and colorectal cancer cell lines(HCT-8,RKO,HCT-116,Lo Vo,HT29,DLD1,CACO2,SW480)in vitro,and detect the expression of CK1δ and CK1ε protein in the cells by Western blotting.2.Construct shRNA CK1ε colon cancer cell lines(RKO,HCT-116,LOVO).3.Scratch test to detect the effect of inhibiting CK1ε on the migration ability of colon cancer cells.4.Transwell experiment detects the effect of scratch inhibiting CK1ε on the invasion ability of colon cancer cells.5.Scratch test to detect the effect of CK1 kinase inhibitor on the migration ability of colon cancer cells.6.Western blotting detects the expression of metastatic related proteins(E-cadherin,Twist,Snail,MTSS1).7.Western blotting was used to detect the expression of MTSS1 in normal colonic epithelial cells(NCM460)and colorectal cancer cell lines cultured in vitro.8.Scratch test to detect the effect of MTSS1 siRNA on the decline of migration caused by CK1 inhibitor.9.qPCR detection inhibits the expression of CK1ε on MTSS1 mRNA.10.Phosphorylation test detects CK1ε phosphorylation modified MTSS1 protein.11.Establish an animal orthotopic model of colon cancer to observe the effect of inhibiting CK1ε on liver metastasis of colon cancer.Results1.The expression level of CK1δ in normal colonic epithelial cells and colon cancer cell lines has no significant difference,while CK1ε has a significantly high expression in colon cancer cells.2.Successfully constructed a stable and low-expressing CK1ε colon cancer cell line(RKO,HCT-116,LOVO).The difference was statistically significant compared with the control group,P <0.05.3.shRNA CK1ε can significantly reduce the invasion ability of colon cancer cells.The difference was statistically significant compared with the control group,P <0.05.4.shRNA CK1ε can significantly reduce the migration ability of colon cancer cells.The difference was statistically significant compared with the control group,P <0.05.5.Different concentrations of CK1 inhibitors(IC261,D4476)inhibited the migration ability of colon cancer cells(HCT-116,RKO,LOVO).The difference was statistically significant compared with the control group,P <0.05.6.Inhibition of CK1ε,the expression of metastasis-related proteins E-cadherin,Twist,and Snail did not change much,while the expression of protein MTSS1 increased.The difference was statistically significant compared with the control group,P <0.05.7.The expression of MTSS1 in normal colonic epithelial cells is higher,while the expression of MTSS1 in colon cancer cell lines is significantly decreased.8.MTSS1 siRNA can attenuate the migration inhibitory effect of colon cancer cell HCT-116 caused by CK1 inhibitor IC261.After statistical analysis,the difference results were statistically significant,P <0.05.9.Inhibition of CK1ε has no significant effect on the mRNA of MTSS1.10.Transfection of CK1ε can reduce the expression of MTSS1 in HCT116 cells,and increase the expression of MTSS1 after administration of dephosphorylase,suggesting that CK1ε may cause a decrease in protein levels through phosphorylation of MTSS1.Statistical analysis shows that the difference was statistically significant compared with the control group,P <0.05.11.In the animal orthotopic model of colon cancer,compared with all 3 animals in the control group with liver metastases,all 3 animals in the CK1ε inhibited group had liver metastases.ConclusionInhibition of CK1ε can significantly reduce the invasion and migration ability of colon cancer cells.The mechanism may be that CK1ε targets the expression of MTSS1 protein through phosphorylation modification to inhibit the metastasis ability of colon cancer. |
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