| BackgroundNeural cadherin(N-cadherin)is a member of cadherin family,which was first discovered in the neural tube of chick embryo.It mainly mediats cell adhesion in the nervous system,and affects the sorting,migration and location of neurons,and the growth path selection of axons.Inside the cell,β-catenin is encoded by CTNNB1.β-catenin is not only interacts with cadherin effect cell-cell adhesion,but also a key regulatory factors in canonical Wnt signaling pathway and plays an important role in cell proliferation and adhesion.At present,many scholars have focused on the functional study of cadherin andβ-catenin,but the relationship betweenβ-catenin and cadherin is rarely reported.Therefore,this study by overexpression of N-cadherin explores the effect on cell adhesion and the member of catenin family.Combined with CRISPR-Cas9 technology,we studied the effect of CTNNB1 gene knockout on cell adhesion and canonical Wnt signaling pathway,and then analyzed the relationship between N-cadherin andβ-catenin.Objective1.To explore the effect of N-cadherin overexpression on cell adhesion and expression of catenin family members.2.To research the effect of the interaction of N-cadherin andβ-catenin on cell adhesion.3.To study the effect of CTNNB1 gene knockout on cell proliferation and canonical Wnt signaling pathwayMethods1.N-cadherin overexpression vector(pCAG-N-cad-EGFP)was constructed.Cell adhesion assay was used to detect the effect of N-cadherin overexpression on cell adhesion.Western blotting technology was used to detect the effect of N-cadherin overexpression on the expression ofβ-catenin and other catenin family members.2.CTNNB1-sgRNA vector(pGL3-U6-sgRNA-PGK-puromycin)was constructed,and cotransfected in HEL 293T cells with pST1374-NLS-flag-linker-Cas9 vector by Lipofectamine 3000 liposome kit.The HEK 293T cell lines ofβ-catenin gene knockout were screened,and HEK 293T cells of CTNNB1~KOO and CTNNB1~WTT would be used for subsequent experiments.3.MTT test,cell adhesion assay and cell flow pattern were used to detect the proliferation activity,cell adhesion,cell cycle and apoptosis of CTNNB1~KOO HEK 293T and CTNNB1~WTT HEK 293T,respectively.Then the effect ofβ-catenin knockout on cell behavior was analyze.4.qPCR and western blotting technology were used to detect the expression of related cadherin and Wnt signal pathway from transcriptional level and protein expression level,respectively.The effects ofβ-catenin gene knockout on N-cadherin and Wnt signaling pathways were investigated.Results1.Compared with the control group,N-cadherin overexpression enhances cell adhesion and inhibits migration.N-cadherin overexpression resulted in the expression level ofα-catenin,β-catenin and p120-catenin protein significantly increased(P<0.05),whileγ-catenin expression was decreased without significant difference(P>0.05).2.CTNNB1-sgRNA vector was successfully transfected into HEK 293T cells,CTNNB1 gene knockout HEK 293T cells were screened.DNA test resuls showed 7-nucleotide deletion in the third exon of CTNNB1 gene,which leaded to frameshift mutation,and maked the code terminate in advance.3.The MTT assay indicated that the adhesion ability of CTNNB1~KOO HEK 293T cells was significantly weakened compared with CTNNB1~WTT HEK 293T cells.The MTT assay also indicated that the proliferation of CTNNB1~KOO HEK 293T was significantly lower than CTNNB1~WTT HEK 293T cells as a time-dependent manner.However,the flow cytometry with the Annexin V-FITC/PI dual labeling indicated that there was no significant difference between CTNNB1~KOO and CTNNB1~WTT HEK 293T cells in apoptosis rates.4.qPCR results showed that the expression of GSK-3β,c-Myc,CCND1(Encoding cell cycle protein D1,cyclin E1),CCNE1(Encoding cell cycle protein E1,cyclin E1),CDK4 and CDK6 were all lower than that of the CTNNB1~WTT HEK 293T cells.Among them,GSK-3β,CCND1 and CCNE1 were statistically significant(P<0.001).There was no significant difference in the expression level of CDK2 compared with the control group(P>0.05).Western blotting results showed that the protein expression level of N-cadherin and cyclin D1 was significantly decreased(P<0.01),and the expression level ofγ-catenin was significantly increased(P<0.001).But E-cadherin,α-catenin,GSK-3β,c-Myc,cyclin E1,CDK2,CDK4,and CDK6 protein expression level did not change significantly(P>0.05).These results suggest thatβ-catenin gene knockout inhibits proliferation through canonical Wnt signaling pathway.Conclusion1.N-cadherin overexpression promoted cell protrusions formation and cell adhesion,and affected the expression of the members of the catenin family,in which the change ofβ-catenin was the most obvious.2.The absence ofβ-catenin prevented the formation of cadherin·β-catenin·α-catenin complexes,altered cell adhesion,and affected the canonical Wnt signaling pathway and inhibited cell proliferation. |
