SPDL1 Affects The Invasion Ability Of Colorectal Cancer And Regulates EMT Of Colorectal Cancer Through EGFR/ERK Signaling Pathway

Objectives Colorectal cancer has become a global public health problem due to its high morbidity and mortality.It has become an urgent clinical problem to explore the mechanism of invasion and metastasis of colorectal cancer and to find effective targets for diagnosis and treatment.Studies have shown that SPDL1 is closely related to the occurrence and development of tumors,and may become a new target for tumor diagnosis and treatment.However,the role and molecular mechanism of SPDL1 in the invasion and metastasis of colorectal cancer are not clear.Therefore,this study intends to verify the effect and mechanism of SPDL1 on the invasion and metastasis of colorectal cancer by mediating EGFR/ERK signaling pathway and regulating EMT through bioinformatics,clinicopathological prognosis analysis,phenotype and mechanism analysis in vitro.Methods 1.Bioinformatics analysis: Search for the difference of SPDL1 expression in normal tissues,cell lines and colorectal cancer in GTEx,CCLE,TCGA,uniport,HPA databases.The biological function of SPDL1 was analyzed by RNA-seq,sc RNA-seq,subcellular localization,clinicopathological and prognostic analysis.Complete GO/KEGG enrichment analysis.2.Clinicopathological and prognostic analysis: The clinical data of 129 cases of colorectal cancer and 20 cases of normal colorectal tissues confirmed by operation and pathology were collected.The expression of SPDL1 in tissues was detected by immunohistochemistry,and the relationship between its expression and clinical prognosis was analyzed.3.Phenotypic analysis: A colorectal cancer cell line with low expression of SPDL1 was established.Cell cycle,cell proliferation,invasion and migration were detected by flow cytometry,CCK8 and transwell chamber.4.Explore the mechanism and verification: After interfering with SPDL1 expression,transcriptome sequencing was performed and verified by GEPIA2 database.U0126,an inhibitor of MAPK pathway,was used to treat the transfected cells.The proliferation,invasion and metastasis of cells were detected,and the expression of MAPK signal pathway and EMT-related proteins were detected by Western blot.Result 1.Bioinformatics analysis: The screened SPDL1 was differentially expressed in normal tissues and cells.sc RNA-seq analysis showed that the expression of SPDL1 in normal colon tissue was mainly concentrated in distal colon glandular epithelial cells and undifferentiated cells.The subcellular localization of SPDL1 is mainly in the cytoplasm.The expression of SPDL1 was low in colorectal cancer tissues and cells line,and the low expression of SPDL1 was associated with poor prognosis.GO/KEGG enrichment analysis showed that SPDL1 was involved in cell mitosis and affected cell cycle.2.Clinicopathological and prognostic analysis: SPDL1 is low expressed in colorectal cancer.The expression of SPDL1 was significantly correlated with the degree of differentiation,TNM stage and lymph node metastasis.There was no significant correlation with age,sex,tumor size,depth of invasion and distant metastasis.Prognostic analysis showed that the shorter the survival time of low expression of SPDL1.3.Phenotypic analysis: Flow cytometry showed that the S phase was inhibited after the low expression of SPDL1,and the proportion of cells in G2/M phase increased.The ability of proliferation,invasion and migration of HCT116 cells was enhanced after interfering with the expression of SPDL1.4.Explore the mechanism and verification: RNA-seq showed that the low expression of SPDL1 was mainly concentrated in MAPK signal pathway and cell cycle.GEPIA2 database verification showed that SPDL1 was related to MAPK signal pathway protein.Western blot results showed that after interfering with the expression of SPDL1,the expression of EGFR,p-ERK,Snail and Vimentin increased,while the expression of E-cadherin decreased,and this effect could be inhibited by U0126.The enhancement of HCT116 proliferation,invasion and migration caused by low expression of SPDL1 can be inhibited by U0126.Conclusion 1.The low expression of SPDL1 in colorectal cancer is related to the degree of differentiation,lymph node metastasis and poor prognosis of colorectal cancer.2.The low expression of SPDL1 may promote the invasion and migration of CRC,which may be related to the activation of EMT by EGFR/ERK signal pathway.