| Background:Colorectal cancer is one of the most common malignancies worldwide with a high mortality rate.At present,the molecular basis of colorectal tumors is still not fully understood.In recent years,with the development of modern molecular biology,the exploration of the molecular mechanism of colorectal cancer and targeted gene therapy from the gene level has become a research hotspot.With the development of tumor molecular biology,more and more attention has been paid to the mechanism of cell pathways in colorectal carcinogenesis,such as Hippo/YAP1 and Wnt/β-catenin signaling pathways.Hippo pathway is a kinase cascade reaction pathway composed of many molecules,which regulates cell proliferation and apoptosis,and then regulates tissue growth and organ size.YAP1 is an important factor in Hippo pathway kinase cascade,and its main role is to promote cell proliferation and inhibit apoptosis.Wnt signaling pathway is also involved in the regulation of cell proliferation,β-catenin is the key element in the process of Wnt pathways pathologic activation.After activation of the Wnt/β-catenin signaling pathway,the undegraded β-catenin enters the nucleus and binds to target genes,leading to excessive cell proliferation and eventual formation of tumors,which contribute to the metastasis of the primary cancer focus and the maintenance of invasive tumor stem cell characteristics.SFRPs(Secreted Frizzle related proteins)are antagonists that bind directly to Wnt signaling,are expressed at significantly reduced levels in a variety of cancers,are associated with disease progression and poor prognosis,and regulate cancer stem cell survival and cell proliferation.At present,the mechanisms and interactions of Hippo and Wnt pathways in colorectal tumors are still unclear and need to be further studied.Object:To explore the molecular mechanism and interaction of Hippo/YAP1 pathway and Wnt/β-catenin pathway in the occurrence of colorectal cancer and the effects of YAP1 and SFRP2 on the biological behavior of colorectal cancer cells.Methods:Colon cancer cell line SW480 was used as promoter screening to construct YAP 1 gene and SFRP2 gene overexpression vectors,which were packaged into lentivirus to infect SW480 cells.The overexpression efficiency was verified by qPCR and Western blot to obtain SW480 cell line with stable overexpression of YAP1 and SFRP2.The experimental groups were as follows:1)SW480 cell group(Control);2)SW480 cells+no-load lentivirus group(NC);3)SW480 cells+YAP1 overexpression lentivirus group;4)SW480 cells+SFRP2 overexpression lentivirus group,Western blot was used to detect the expression of YAP 1,SFRP2 and β-catenin in SW480 cells infected with YAP1 and SFRP2 overexpressed lentivirus,respectively.The expression and localization of YAP1 and SFRP2 in cells of group 1)to group 4 were detected by immunofluorescence.The interaction between YAP1,SFRP2 and β-catenin was detected by immunoprecipitation assay.The proliferation of SW480 cells in groups 1)to 4)was detected by CCK8 assay.Flow cytometry was used to detect the apoptosis of SW480 cells in the 1)-4)group.Results:1.The results of promoter screening showed that the fluorescence signal of SW480 cells+PLVX-EFla-IRES-EGFP-PGK-PURO plasmid group was the strongest,and this vector could be selected for the construction of subsequent experiments.2.YAP1,SFRP2 overexpression of SW480 cell line authentication:qPCR and Western blot test results showed that expression of slow virus has been infected with the YAP1 relative expression of YAP 1 in cells of the group is significantly higher than blank group and slow virus no-load,infected with the virus that had SFRP2 express slow cell group relative expression of SFRP2 no-load group is significantly higher than blank group and slow virus,the differences were statistically significant(P<0.05).3.Western blot results showed that the relative expression level of SFRP2 protein in YAP1-overexpressed SW480 cells was higher than that in blank group and no-load group,and the difference was not statistically significant(P>0.05).4.Western blot results showed that the relative expression level of YAP1 protein in SFRP2 overexpressed SW480 cells was significantly higher than that in blank group and no-load group,with statistical significance(P<0.05).5.Western blot analysis showed that the relative expression levels of β-catenin protein in YAP1 overexpression group and SFRP2 overexpression group were significantly higher than those in blank group and no-load group,with statistical significance(P<0.05).6.Immunofluorescence showed that YAP1 was expressed in cytoplasm and nucleus of cells in each group,and the YAP1 overexpression group had the highest relative expression,while SFRP2 was not expressed in cells in all groups.7.The results of immunoprecipitation showed that the protein complexes of SW480 cells were immunoprecipitated with YAP 1 antibody and SFRP2 antibody respectively,and β-catenin bands were detected by Western blot analysis withβ-catenin antibody.8.CCK8 results showed that YAP1 overexpression promoted the proliferation of SW480 cells,while SFRP2 overexpression inhibited the proliferation of SW480 cells.9.Flow cytometry results showed that YAP1 overexpression inhibited the apoptosis of SW480 cells,but the difference was not statistically significant(P>0.05),while SFRP2 overexpression promoted the apoptosis of SW480 cells.Conclusion:1.In colorectal cancer SW480 cell line,overexpression of YAP 1 promotes the expression of β-catenin protein,and overexpression of SFRP2 promotes the expression of YAP1 and β-catenin protein,and both YAP1 and SFRP2 interact with β-catenin in SW480 cell line.The Hippo/YAP1 pathway and Wnt/β-catenin pathway play an important role in the occurrence of colorectal cancer,and they interact with each other in the pathogenesis of colorectal cancer.2.Overexpression of YAP1 promotes cell proliferation,while overexpression of SFRP2 has the opposite effect.YAP1 and SFRP2 are involved in the occurrence and development of colorectal cancer. |
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