The Role And Mechanism Of Rab31 And GPR116 In The Tumorigenesis And Progression Of Colorectal Cancer

Section I The role and mechanism of Rab31 in the tumorigenesis of colorectal cancerAims:Rab31 belongs to the Ras superfamily of small GTP-binding proteins,which has been found to regulate the vesicle transport from the Golgi apparatus to early and late endosomes.So far,multiple researches have been reported that Rab31 might promote tumor proliferation.However,the role and mechanism of Rab31 in colorectal cancer has not been reported.The aim of our study is to determine the relationship of Rab31,the clinical and pathologic features and prognosis of CRC patients,to clarify the role of Rab31 in the proliferation,apoptosis and cell cycle of CRC and to demonstrate the molecular mechanism of Rab31 in the tumorigenesis of CRC.Methods: 1)We profiled the expression of mRNA and protein level of Rab31 in CRC tissue and adjacent normal tissue using RT-PCR and Western blotting.2)We examined the expression of Rab31 in CRC tissue microarrays using immunohistochemical(IHC)staining to determine the correlation between Rab31,the clinicopathologic features and prognosis of CRC patients.3)To explore the mechanism of Rab31 in the tumorigenesis of CRC,we downregulated the expression of Rab31 in colorectal cancer cell HCT116 and performed RNA-sequence.Then,we analyzed the signal pathway involving Rab31 using Gene score enrichment analysis(GSEA).4)We finally verified the molecular mechanism of Rab31 in CRC using Western blotting.Results:1)Rab31 was overexpressed in CRC tissues compared with their non-cancerous counterparts at both the mRNA and protein levels.2)According to CRC tissue microarrays analysis using IHC,Rab31 expression in CRC specimens was significantly correlated with histological differentiation.The survival time was significantly shorter in patients with higher Rab31 expression than those with lower Rab31 expression group.Rab31 was the independent prognostic indicator for patients with CRC.3)The results of RNA-sequence have shown that Rab31 was closely correlated with mTOR signaling pathway.4)Knockdown of Rab31 reduced the proliferation,arrested the cell cycle progression and increased the apoptosis of HCT116 and LOVO.The results were inverse when we overexpressed the expression of Rab31.5)We verified the expression of downstream proteins of Rab31 using Western blotting and demonstrated that Rab31 promoted colorectal cancer cell proliferation by mTOR-p70S6K-cyclinD1/C-MYC signaling pathway.Conclusions: Rab31 was significantly upregulated in CRC.The expression of Rab31 is correlated with histological differentiation and prognosis of CRC patients.Rab31 may promote CRC proliferation by mTOR-p70S6K-cyclinD1/C-MYC signaling pathway.It may be a reliable prognostic biomarker or a new therapeutic target in CRC.Section II The role and mechanism of GPR116 in the development and progression of colorectal cancerAims:G-protein-coupled receptors(GPCRs)constitute the hugest transmembrane receptors.GPR116 belongs to adhesion G-protein-coupled receptor(a GPCR),which contains adhesion domains in their extracellular region,was potentially involved in cancer metastasis considering that adhesion domains may play vital roles in cell migration and motility.So far,it was reported that GPR116 was associated with breast cancer metastasis.However,the role of GPR116 in colorectal cancer has not been reported.The aim of our study is to determine the correlation of GPR116,the clinicopathologic features and prognosis of CRC patients and to clarify the function of GPR116 in the metastasis of CRC.Methods: 1)We profiled the expression of m RNA and protein level of GPR116 in CRC tissue and adjacent normal tissue using RT-PCR and Western blotting.2)We examined the expression of GPR116 in CRC tissue microarrays using immunohistochemical staining to determine the correlation between GPR116,the clinicopathologic features and prognosis of CRC patients.3)To explore the function of GPR116 in the progression of CRC,we downloaded The Cancer Genome Atlas databases and analyzed the signal pathway involving GPR116 using Gene score enrichment analysis(GSEA).4)Western blotting analysis was performed to validate the expression level of the gene involved in GSEA analysis.Results: 1)GPR116 was overexpessed in CRC tissues compared with their non-cancerous counterparts at both the m RNA and protein levels.2)According to CRC tissue microarrays analysis using IHC,GPR116 expression in CRC specimens was significantly correlated with histological differentiation,distant metastasis and patients prognosis.GPR116 was the independent prognostic indicator for patients with CRC.3)The analysis of GSEA has shown that GPR116 was closely associated with proliferation,metastasis and epithelial to mesenchymal transition(EMT)of CRC.4)Knockdown of GPR116 reduced the proliferation and metastasis of colon cancer cells.5)We verified the expression of downstream proteins of GPR116 using Western blotting and demonstrated that GPR116 promoted EMT of CRC through p-AKT and p-ERK1/2 signaling pathway.Conclusions: GPR116 was significantly correlated with histological differentiation,distant metastasis and prognosis of CRC patients.GPR116 may promote EMT of CRC by p-AKT and p-ERK1/2 signaling pathway.GPR116 may be a new therapeutic target in CRC.