| Objective:To investigate the mRNA and protein expression levels of BCAP31(B-cell receptor-associated protein 31)in colorectal cancer and adjacent tissues and its correlation with clinicopathological features,and to analyze the relationship between BCAP31 expression and prognosis.To detect the effects of BCAP31 on the proliferation,migration and autophagy of colorectal cancer cells.Methods:(1)TCGA database were searched the mRNA expression of BCAP31 and its correlation with clinicopathological features of colorectal cancer patients;HPA database was used to explore the protein expression level of BCAP31 in colorectal cancer and adjacent protein.Prognoscan database was used to analyze the relationship between BCAP31 expression and prognosis.(2)Real-time fluorescence quantitativepolymerase chain reaction RT-qPCR),Immunohistochemistry(IHC)and Western blot(WB)verify the mRNA and protein expression of BCAP31 in colorectal cancer and adjacent tissues,and the correlation between BCAP31 mRNA expression and clinicopathological features was further analyzed.(3)Colorectal cancer cell line SW620 with transient transfection of BCAP31 small interfering RNA were constructed,and their silencing efficiency was detected by Western blot.The effects of silencing BCAP31 on the proliferation and migration of colorectal cancer cells were detected by CCK8 assay and Scratch assay.The expression changes of autophagy related proteins LC3 and P62 in SW620 cell after BCAP31 knockdown were detected by Western blot.Transfection of adenovirus(mCherry-GFP-LC3)with both green and red fluorescent proteins,and transfection of small interfering RNA of BCAP31 on this basis.The effect of si-BCAP31 on autophagic flow formation(mCherry-GFP-LC3 spot number)in colorectal cancer cells was investigated by immunofluorescence assay.Results:(1)TCGA database analysis showed that BCAP31mRNA was highly expressed in head and neck squamous cell carcinoma(HNSC),esophageal carcinoma(ESCA),hepatocellular adenocarcinoma(LIHC)and other cancers,and the expression of BCAP31mRNA was relatively high in colorectal cancer tissues,which was closely related to lymph node metastasis(p<0.05)and TNM stage(p<0.05).Prognoscan database analysis showed that overall survival(OS)of colorectal cancer patients with high BCAP31 expression was lower than that of patients with low BCAP31 expression.(2)The high expression of BCAP31mRNA in colorectal cancer was verified by RT-qPCR assay of tissue clinical samples,and the expression level was correlated with lymph node metastasis(p<0.05)and TNM stage(p<0.05).Immunohistochemical experiments and Western blot assay demonstrated that BCAP31 protein levels were highly expressed in colorectal cancer tissues compared with adjacent tissues.(3)CCK8 assay showed that silencing BCAP31 inhibited the proliferation of colorectal cancer cells.Scratch assay showed that silencing BCAP31 inhibited the migration of colorectal cancer cells.After silencing BCAP31,the expression levels of autophagy related proteins LC3Ⅱ and P62 decreased.Immunofluorescence analysis showed that compared with the control group,the yellow fluorescence numbers was decreased in the si-BCAP31 group,indicating that BCAP31 inhibited the generation of autophagosomes in the early stage of autophagy,and the red fluorescence numbers was also decreased,and the autophagy flow was blocked.Autophagy level of colorectal cancer cells was inhibited after BCAP31 was silenced.Conclusion:Compared with paracancer tissues,the expression levels of BCAP31 mRNA and protein in colorectal cancer tissues were significantly increased,which was related to lymph node metastasis and TNM stage.Patients with high BCAP31 expression had lower overall survival rate than those with low BCAP31 expression.BCAP31 promotes the proliferation,migration and autophagy of colorectal cancer cells,which may further affect the occurrence and development of colorectal cancer.BCAP31 may be used as a biomarker for staging treatment and diagnosis of colorectal cancer. |
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