Knockdown Of GPR56 Inhibits The Proliferation?Invasion And Migration In Human Colorectal Cancer Cells

[Background]Colorectal cancer(CRC)ranked the fifth in the incidence and fifth in mortality rate of all kinds of malignant tumors in our country.As the most common malignant tumor of gastrointestinal tract,the threat of which to the health of human beings is increasing year by year.However,due to the limitation of current examination methods,the majority of patients diagnosed with colorectal cancer have been in the advanced stage,which directly affects the patient's survival prognosis.Therefore,the search for new early diagnostic markers and therapeutic targets in colorectal cancer for the improvement of prognosis of patients is with great clinical significance.[Objective]The purpose of this study was to investigate the expression of GPR56 in colorectal cancer and its effect on the proliferation,invasion and migration of colorectal cancer cell lines.In addition,the possible related mechanisms were investigated and analyzed.[Methods]1.The expression of GPR56 in CRC tissues and corresponding normal tissues were tested by quantitative real-time PCR(qRT-PCR).The relationship betweem GPR56 expression level and the clinicopathological features of patients with CRC was analysed by Chi-square test.The mRNA and protein level of GPR56 in CRC cell lines were tested by qRT-PCR and Western blot.2.The influence of GPR56 on tumor cell proliferation were investigated via a Cell Counting Kit-8;apotosis rate and cell cycle distribution via flow cytometry.Western blot and qRT-PCRwas used to detect the expression of apoptosis-related proteins and cell cycle-related proteins after the GPR56 expression was inhibited.3.Scratch experiments and Transwell experiments were used to observe the effect of down-regulation of GPR56 on the invasion and migration of colorectal cancer cells.4.Western blot and qRT-PCR were performed to detect the expression of Epithelial-mesenchymal Transition(EMT)and PI3K-AKT pathway related protein in GPR56 interference group and control group.After using the PI3K-AKT pathway-specific inhibitor LY294002,Transwell experiment were performed to observe the changes in cell migration among control group ?GPR56 overexpression group ?GPR56 overexpression with LY294002 group and single LY294002 group.5.Western blot and qRT-PCR was performed to detect the expression of EMT related protein among control group ?GPR56 overexpression group and GPR56 overexpression with LY294002 group.[Results]1.The expression of GPR56 was remarkably higher in tumor tissues than adjacent non-tumorous tissues and GPR56 expression was correlated with the following clinicopathological: TNM stage,lymph node metastasis,tumor invasion depth and distant metastasis.Western blot and qRT-PCR showed that GPR56?s mRNA and protein expression levels were markedly higher in all CRC cell lines apart from HT29 when compared with NCM460 cells.2.Knocking down GPR56 in HCT116 and DLD-1 cell lines reduce the proliferation;decrease S phase cell counts and increase the apoptosis of colorectal cancer cells.The expression of Bax protein was increased and the expression of Bcl-2 was decreased in GPR56-disturbed cell lines and the expression of CylinD?CyclinE and Cmyc in GPR56-disturbed group were both decreased when compared to the control group.3.Knockdown of GPR56 absolutely inhibited the migration and invasion effect in both HCT116 and DLD-1 cells4.GPR56 knockdown increased E-cadherin and decreased N-cadherin and Vimentin;decreased phosphorylated PI3 K and AKT levels.Overexpression of GPR56 significantly increased cell migration in a transwell assay;however,after addition of PI3 K / AKT-specific inhibitor LY294002,the cell migration capacity decreased.5.GPR56 overexpression upregulated Vimentin and N-cadherin expression and downregulated E-cadherin expression.Conversely,when cells overexpressing GPR56 were treated with LY294002,the typical change in EMT was reversed.[Conclusion]GPR56 expression was markedly up-regulated in CRC tissues and cell lines compared to corresponding normal controls.Depletion of GPR56 dramatically suppressed cell proliferation,migration,and invasion.GPR56 overexpression promoted CRC cell metastasis by expediting epithelial-mesenchymal transition through activating PI3K/AKT signaling.GPR56 plays an important role in CRC progression and may represent a new therapeutic target to reduce CRC metastasis.