The Role Of GOLM1 On Proliferation,Invasion And Metastasis In Colorectal Cancer And Its Underlying Mechanisms

PART Ⅰ The expression of GOLM1 in colorectal cancer and its relationship with clinicopathological featuresObjective:To detect the expression of GOLM1 in Colorectal cancer(CRC)and analyze its relationship with CRC clinicopathological features.To explore whether GOLM1 acts as a biomarker in CRC diagnosis and a potential target for CRC therapy.Methods:The expression of GOLM1 mRNA in CRC and normal tissue were analyzed by GEPIA.The expression of GOLM1 protein in 11 CRC and matched adjacent normal tissues as well as 14 CRC primary tissues(7 samples with lymph node metastasis and 7 samples without lymph node metastasis)were examined by Western blot.The difference of serum GOLM1 between 21 healthy controls and 25 CRC patients were analyzed by ELISA.The expression of GOLM1 protein in 125 CRC tissues and 43 adjacent normal tissues were examined by Immunohistochemistry(IHC),and the relationships between GOLM1 and CRC clinicopathological features were further evaluated.Results:GEPIA analysis showed that the expression of GOLM1 mRNA in CRC was higher than in normal tissues.The expression of GOLM1 protein was higher in 11 CRC samples than in matched adjacent normal tissues.There were no statistical differences of serum GOLM1 levels between 21 healthy controls and 25 CRC patients.The expression of GOLM1 protein was higher in 7 CRC primary samples with lymph node metastasis than 7 CRC primary samples without lymph node metastasis.IHC analysis further demonstrated that the expression of GOLM1 protein was higher in 125 CRC tissues than in 43 adjacent normal tissues.The expression of GOLM1 was positively associated with lymph node metastasis,vascular invasion and advanced TNM stage.Conclusions:GOLM1 is highly expressed in colorectal cancer and closely correlated with CRC clinicopathological features.GOLM1 can be a useful biomarker in CRC diagnosis and a potential target for CRC therapy.Part Ⅱ To investigate the effect of GOLM1 on proliferation,invasion and metastasis of colorectal cancer cellObjective:To investigate the effect of GOLM1 on proliferation,invasion and metastasis of CRC cell in vitro and in vivo.Methods:The expression of GOLM1 protein in CRC cell lines were detected by Western blot and the GOLM1 stably knockdown and overexpression cell lines were established and verified by real-time PCR and Western blot.The effect of GOLM1 on CRC cell proliferation was evaluated by CCK-8 assay,colony formation assay and subcutaneously implanted tumor model in nude mice.The effect of GOLM1 on CRC cell migration,invasion,metastasis and EMT were evaluated by Transwell assay,lung metastasis model in nude mice and Western blot respectively.Results:GOLM1 was highly expressed in HCT8、HCT116、RKO、LOVO and CACO2 cell lines as well as lowly expressed in SW480 and normal intestinal epithelial HIEC cell line.GOLM1 Stable knockdown cell lines(HCT116 and CACO2)as well as stable overexpression cell line(SW480)were successfully established.The identification results showed that GOLM1 was significantly downregulated/upregulated in above cell lines respectively.Knockdown of GOLM1 inhibited the proliferation of HCT116 and CACO2 cells,overexpression of GOLM1 promoted the proliferation of SW480 cells.Knockdown of GOLM1 inhibited the growth of subcutaneously implanted HCT116 cell-derived tumors in nude mice.Knockdown of GOLM1 inhibited the migration and invasion of HCT116 and CACO2 cells,overexpression of GOLM1 promoted the migration and invasion of SW480 cells.Compared to control groups,knockdown of GOLM1 inhibited migration and invasion of HCT116 and CACO2 cells after TGF-β1 induced.Knockdown of GOLM1 inhibited the formation of lung metastasis nodules in nude mice.Compared to control groups,Knockdown of GOLM1 upregulated E-cadherin as well as downregulated N-cadherin and Snail1 in HCT116 cells,overexpression of GOLM1 downregulated E-cadherin as well as upregulated N-cadherin and Snail1 in SW480 cells.Compared to control groups,Knockdown of GOLM1 upregulated E-cadherin as well as downregulated N-cadherin and Snail1 in HCT116 and CACO2 cells after TGF-β1 induced.Conclusions:GOLM1 facilitates CRC cell proliferation,invasion,metastasis and EMT.Part Ⅲ The study of GOLMl regulates colorectal cancer cell proliferation,invasion and metastasis via AKT/GSK3β signaling pathwayObjective:To study the potential mechanisms of GOLM1-mediated CRC cell proliferation,invasion and metastasis.Methods:The signaling pathway involved in GOLM1-mediated CRC proliferation,invasion and metastasis were analyzed by GSEA analysis.The change of AKT signaling pathway and key downstream effectors induced by GOLM1 knockdown and overexpression in CRC cells were evaluated by Western blot.The change of AKT/GSK3β pathway,EMT proliferation,migration and invasion in GOLM1-overexpression SW480 cells with MK2206 inhibition were evaluated by Western blot,CCK-8 and Transwell assay respectively.Results:GSEA analysis showed that PI3K/AKT signaling pathway played an important role in GOLM1-mediated CRC progression.Knockdown of GOLM1 inhibited phosphorylation of AKT and its key effector GSK3β in HCT116 cells,overexpression of GOLM1 enhanced phosphorylation of AKT and its key effectors GSK3β in SW480 cells.Knockdown of GOLM1 inhibited AKT/GSK3β signaling pathway in TGFβ1-induced HCT1 16 and CACO2 cells.MK2206 significantly inhibited AKT/GSK3(3 signaling pathway,EMT,proliferation,migration and invasion in GOLM1 overexpression SW480 CRC cells.Conclusions:GOLM1 regulated CRC cell EMT,proliferation,invasion and metastasis via AKT/GSK3β signaling pathway.