The Role And Mechanism Of Cripto-1 In Invasion Of Colorectal Cancer

Objective: To observe the expression characteristics of Cripto-1 in colorectal cancer,and to investigate the effects of targeted silencing Cripto-1 and 5Fu on the proliferation and migration of human colorectal cancer cells,and to explore the role and mechanism of Cripto-1 in colorectal cancer invasion.Diagnostic and molecular targeted therapies provide research directions.Methods:(1)Immunohistochemistry was used to detect the expression of Cripto-1 and EMT-related factors(E-cadherin,Snail)in colorectal cancer tissues and adjacent normal tissues in colorectal cancer tissue microarrays.(2)By in vitro culture of colorectal cancer Lovo cells,the synthetic Cripto-1 si RNA was transfected into cells by Lipofectamine TM2000.Western blot and real-time fluorescent quantitative PCR were used to screen the best interference effects from protein and m RNA levels.Cripto-1 si RNA.(3)Targeting silencing Cripto-1 gene expression and the effect of5 Fu on proliferation and invasion of human colorectal cancer cells,human colorectal cancer cell Lovo cells were divided into 4 groups: Lovo cells(normal control group),Lovo cells + si RNA Cripto-1(Interference group),Lovo cells + 5-Fu 10 mg/L(drug group),Lovo cells + si RNACripto-1 + 5-Fu(10 mg/L)(interference + drug group).CCK-8 was used to detect the effect of Cripto-1 si RNA on the proliferation of colorectal cancer cells.Transwell was used to detect the invasion ability of colorectal cancer cells.Western blot was used to detect the expression of Cripto-1,E-cadherin and Snail in colorectal cancer cells.Results:(1)The results of immunohistochemistry showed that Cripto-1 was expressed in colorectal cancer tissues and adjacent tissues,but it was highly expressed in cancer tissues.Overexpression of Cripto-1 protein was significantly associated with clinical stage and lymph node metastasis of colorectal cancer(P<0.05),and was not significantly correlated with age,gender and distant metastasis(P>0.05).The expression of E-cadherin in colorectal cancer tissues was lower than that in adjacent tissues(P<0.05),and the expression of Snail was higher than that in adjacent tissues(P<0.05).The expression of Cripto-1 and E-in colorectal cancer tissues The expression of cadherin was negatively correlated(r=-0.524,P<0.05),and positivelycorrelated with the expression of Snail protein(r=0.710,P<0.05).(2)(2)Three si RNA interference sequences(Cripto-1 si RNA-187,368,494)were detected by RT-PCR.Three si RNA sequences were effective for Cripto-1 gene silencing,and Cripto-1 si RNA-494 had the most significant silencing effect.The results of Western blot were consistent with those of RT-PCR.(3)By targeting silencing Cripto-1expression and 5-Fu in human colorectal cancer Lovo cells,the results of CCK-8detection of cell proliferation showed that the proliferation rates were(96.96±2.69)%,(79.42±2.46)%,respectively.(74.39±4.79)%,(62.12±2.80)%,compared with the normal control group,the proliferation rate of the other three groups decreased(P<0.05),and the cell proliferation rate of the combined group was higher than that of the Cripto-1 si RNA group,There was no significant difference in the 5-Fu group(P<0.05).There was no significant difference between the Cripto-1 si RNA group and the 5-Fu group(P>0.05).The results of Transwell cell migration assay showed that the number of transmembrane cells in the normal control group,Cripto-1 si RNA group,5-Fu group and combination group were(185.80±4.76),(143.60±3.36),(128.60±4.51),respectively.111.60±4.28),compared with the normal control group,the number of transmembrane cells decreased in the other three groups(P<0.05).The number of transmembrane cells in the combined group was lower than that in the Cripto-1 si RNA group and 5-Fu group(all P<0.05).The number of transmembrane cells in the 5-Fu group was lower than that in the Cripto-1Si RNA group(P<0.05),and the difference was statistically significant.The expression of Cripto-1,E-cadherin and Snail protein in each group was detected by Western blot: Cripto-1 si RNA group,5-Fu group,combined group Cripto-1 and Snail protein expression were lower than normal control group,E-cadherin protein expression The expression of Cripto-1 and Snail was lower than that of Cripto-1 si RNA group and 5-Fu group,and the expression of E-cadherin protein was higher than that of Cripto-1 si RNA group and 5-Fu.The group(P<0.05);there was no significant difference in the expression of Cripto-1 si RNA group and 5-Fu group(P>0.05).Conclusion:Cripto-1 is highly expressed in human colorectal cancer;targeting silencing of Cripto-1 gene significantly inhibits the proliferation and migration of human colorectal cancer cell line Lovo in vitro;Cripto-1 may be involved incolorectal cancer invasion.