Effects Of RNA Interfering CPNE1 Gene Expression On Biological Behavior Of Human Osteosarcoma Saos-2 Cells

Objective: To investigate the impacts of lentivirus-mediated inhibition of CPNE1 on human osteosarcoma Saos-2 cells and explore the relevant molecular mechanism so as to provide a experimental basis for its research in the gene therapy of human osteosarcoma.Methods: CPNE1 expression in human osteosarcoma specimens was was detected using immunohistochemical analysis.The authors constructed lentiviral vectors to deliver small hairpin RNA(sh RNA) targeting CPNE1 into Saos-2 cells. The Saos-2 cells transfected with negative vector was the control group. Under the fluorescent microscopy,the transfection efficiency was determined by detecting the GFP expression. The expression level of CPNE1 was examined by RT-PCR and real-time quantitative polymerase chain reaction(RT-q PCR) Western blotting. The protein expression level of CPNE1 was finally examined by Western blotting for identifing the inhibitory efficiency of RNAi. After RNAi targeting CPNE1,cell counting Cellomics were performed to examine proliferation and flow cytometry were performed to examine cell cycle distribution and apoptosis.The alteration of gene expression profiles after CPNE1 gene silencing was investigated through gene expression array, and bioinformatics was used to analysize data.In order to analyze the impacts of silencing CPNE1 gene on the expression of IRAK2、BIRC3 and TNFSF10, the methods mentioned above was applied to detect the changes in expression of these cytokines respectively.Results: The expression level of CPNE1 is significantly higher in gliomas compared tononcancerous brain tissue. RNAi lentivirus expression vectors targeting to CPNE1 gene were produced,the sequence and correct site of ds oligos inserted were confirmed by sequencing and PCR assay. The lentivirus were packaged with high titer in 293 T cells, small hairpin RNA(sh RNA) targeting CPNE1 was transfected into human osteosarcoma Saos-2 cells by recombinants of lentiviral vectors and the transfection efficiency could reach more than90%.The expression of CPNE1 gene in Saos-2 cells was inhibited by lentiviral recombinants of LV-CPNE1. The inhibitory efficiency of LV-CPNE1 could reach over 80%. Inhibition of CPNE1 expression in Saos-2 cells by RNAi significantly impaired cell proliferation, increased apoptosis and arrested cells in G2/M phase.There were 400 differentially expressed genes in the samples,among which 133 were up-regulation genes and 267 were down-regulation genes. Knockdown of CPNE1 gene by RNAi could down-regulate the expression of BIRC3、IRAK2 and TNFSF10.Conclusions: Lentiviral vectors was successfully constructed. When the CPNE1 gene interfered by the lentivirus vector,the cell growth of Saos-2 was inhibited and apoptosis level of cells increased in vitro. Many genes had a significantly differential expression after the silence of CPNE1 gene. These genes are involved in many pathways. However, the present study still can not completely reveal the CPNE1 signal pathways and molecular mechanism.CPNE1 might be a potential adjuvant gene therapeutic target for human osteosarcoma.