SNTB1 Regulates Proliferation And Metastasis Functions Through WNT/β-catenin/MYC Signal Pathway In Colorectal Cancer

Objective: the bioinformatics was performed to filter the critical gene during colorectal cancer(CRC)development.This study aims to invetstigate the roles of SNTB1 in colorectal cancer.Methods: Four sets of colorectal cancer(CRC)microarray datasets(GSE39582,GSE8671,GSE44076,GSE9348)were selected from the GEO database to analyze differentially expressed genes(DEGs),the DEGs were subjected to KEGG pathway enrichment analysis and PPI interaction network analysis;Cox univariate regression analysis was performed on the correlation between the above DEGs and clinical prognosis of patients based on the TCGA database;the expression levels of the top five survival risk factors in colorectal cancer and adjacent tissues were detected by qRT-PCR,and the gene SNTB1 with the largest up-regulated differential fold was selected for follow-up research;HE staining and immunohistochemistry were used to detect the expression of SNTB1 in CRC tumor tissue and adjacent tissue.The sh-SNTB1#1 and sh-SNTB1#2 were constructed to knock down SNTB1 expression and transfected into HCT116 and SW480 cells,qRT-PCR detection interference efficiency;The effect of SNTB1 silencing on the proliferation of CRC cells was detected by CCK-8 assay and clone formation assay;Cell scratch assay and Transwell assay were used to detect the effect of SNTB1 silencing on the invasion and migration of CRC cells;GSEA network analysis was performed on SNTB1co-expressed genes to predict the mechanism of SNTB1 involved in the occurrence and development of colorectal cancer,Western blot was used to detect the related mechanisms of WNT/β-catenin/MYC signaling pathway and key proteins in EMT process.Nude mouse subcutaneous tumorigenic model to observe the effect of SNTB1 on the tumorigenic ability of CRC;The effect of SNTB1 knockdown on tumor lung metastasis was observed in a nude mouse model of CRC lung metastasis.Results: 293 up-regulated and 369 down-regulated genes were identified by GEO datasets;The top five risk factors for patient survival are FKBP10,SFTA2,TIMP1,STC1 and SNTB1,of which SNTB1 has the largest up-regulated fold;SNTB1 is highly expressed in colorectal cancer as evidenced by the bioinformatics and CRC tissue samples,and was significantly correlated with clinical TNM classification,pathological cycle grade(H),and shorter survival of patients with colorectal cancer;HE staining and immunohistochemical detection results confirmed that SNTB1 protein was moderately-strongly positive in CRC tissue.The results of qPCR and Western blot showed that compared with NCM460 cells,the mRNA and protein levels of SNTB1 in CRC cell lines such as HCT116,HCT8,LoVo,SW480 and DLD-1 were significantly up-regulated,and the expression changes of HCT116 and SW480 were the most significant;the results of CCK-8 assay and clone formation assay showed that in HCT116 and SW480 cells,the proliferation activity and clone formation ability of tumor cells were significantly inhibited after knockdown of SNTB1 expression;The results of scratch experiments showed that after knockdown of SNTB1 expression,the migration ability of HCT116 and SW480 was significantly inhibited within 48 hours;The results of Transwell experiments showed that the migration and invasion abilities of tumor cells were significantly inhibited after knocking down the expression of SNTB1;GSEA analysis showed that SNTB1 co-expressed genes were positively enriched in Wnt/β-catenin/MYC pathway and Hedgehog pathway related to proliferation and metastasis;The expression levels of Wnt1,C-Jun,C-Myc,TCF7 and cyclin D1 were significantly decreased,as well as EMT-related proteins E-Cadherin was incrased while N-Cadherin,vimentin,Snail and Slug were decreased after knockdown of SNTB1.Nude mice with subcutaneous tumor and lung metastases confirmed that knockdown of SNTB1 inhibited the growth and lung metastasis of colorectal cancer cells by downregulating the Wnt/β-catenin signaling pathway.Conclusion: SNTB1 is highly expressed in colorectal cancer and is closely related to poor clinical prognosis.Knockdown of SNTB1 inhibits tumor cell growth and metastasis by down-regulating Wnt/β-catenin/MYC pathway and EMT process.