Effects Of Lentivirus-mediated CHPF Silencing Through CCL2 On Proliferation And Invasion Of Human Glioma U87 Cells

Background and Objective:Chondroitin polymerizing factor（CHPF）is a type II transmembrane protein.Recent studies show that the expressions of CHPF in colorectal cancer,laryngeal cancer,head and neck squamous cell carcinoma are significantly increased,and CHPF may be closely related to the occurrence and development of tumors.In the present study,we demonstrated that CHPF is highly expressed in human glioma tissues and four glioma cell lines.Consequently,we constructed a lentivirus vector mediating RNAi targeting of CHPF（LV-CHPF-shRNA）.We employed the LV-CHPF-shRNA to figure out whether CHPF has effects on the progression of human glioma in vitro,As well as clarifying the possible mechanism of CHPF’s regulation of monocyte chemoattractant protein 1（MCP-1,also known as CCL2）in glioma.Methods:1.The glioma U87 cells were cultured in DMEM（Dulbecco’s modified Eagle’s medium）at 37°C with 5%CO₂ containing 10%FBS（fetal bovine serum）.A lentiviral vector expressing CHPF shRNA was constructed and transfeeted into the U87 cells.U87 cells were placed under an inverted fluorescence microscope and transfection efficiency was observed.2.The experiments were divided into three groups:blank group,non-sense sequence control（NC-shRNA）group and CHPF-shRNA group.3.The expression of CHPF at mRNA and protein levels was detected by real-time PCR and Western blotting after infected with CHPF-shRNA,respectively.4.After infected with CHPF-shRNA,the cell proliferation of glioma U87 cells was measured by CCK8 assay.The changes in cell cycle and apoptosis of glioma U87cells were accessed by flow cytometry.5.After infected with CHPF-shRNA,cell invasion was evaluated by Transwell assay.6.The expression levels o CCL2 in glioma cell line U87 after transfected with CHPF shRNA was measured.7.The changes of CCL2 and CHPF expression levels in U87 cells after transfection with CCL2 siRNA was detect by real-time PCR.Results:1.Recombinant lentivims was successfully transfected into U87 cells.More than80%cells showed positive green fluorescence in shRNA-CHPF group,indicating the successful lentivirus infection.The expression of CHPF in U87 cells after transfection with the CHPF-shRNA was significantly down-regulated（P<0.05）.2.The results of the present study showed that CHPF knockdown significantly inhibited growth of the U87 cells.CHPF regulates U87 cells growth and blocks cell cycle progression in the G0/G1 phase.The apoptosis percentage of U87 cells in shRNA-CHPF group was significantly higher.The results indicated that CHPF silencing inhibited the proliferation of U87 cells by inducing G0/G1 phase cell cycle arrest and apoptosis.3.The abilities of invasion of U87 cells after transfection with CHPF-shRNA were inhibited（P<0.05）.4.The expression of CCL2 in U87 cells was significantly down-regulated,and with significant difference as compared with the cells transfected with NC-shRNA cells（P<0.05）.5.The relative expression levels of CCL2 in CCL2 siRNA group wassignificantly difference with those in negative control group,but there was no change of expression of CHPF in U87 cells after CCL2 express inhibition（P>0.05）.Conclusion:A high level of expression of CHPF was found in human glioma tissues.Downregulation of CHPF expression by CHPF-shRNA lentiviral vector inhibited glioma U87 cells proliferation and induced cell apoptosis through CCL2.It implicated that CHPF can be a novel target in clinical treatment for gliomas.