To Explore The Mechanism Of The GRM4 Gene In Osteosarcoma Cell Line By RNAi And RNA-seq Technology

BACKGROUND: Osteosarcoma is the most common primary malignant tumor of mesenchymal origin in children and adolescents.Because of its low incidence,and the exact pathogenesis of OS still less understanding.Moreover,the mortality was high in OS patients with metastatic to the lung.Hence,it is necessary to explore the molecular mechanisms of OS in order to improve therapeutic treatments.Glutamate metabotropic receptor 4(GRM4)has been correlated with the pathogenesis of OS.But its role in OS cell and the underlying molecular mechanisms of GRM4 remain largely unknown.OBJECTIVE: The objective of this study is to investigate the expression level of the GRM4 m RNA in different human OS cell lines and human osteoblast h FOB1.19 cell lines.RNA sequencing technique was used to obtain the differentially expressed genes after GRM4 silencing in U2 OS cells.It was also committed to explore the underlying molecular mechanism of GRM4 in osteosarcoma.METHODS: 1?The expression level of GRM4 m RNA in four human osteosarcoma cell lines(HOS,MG-63,U2 OS and Saos-2)and human osteoblasts h FOB1.19 cell lines were detected by real-time quantitative PCR(RT-q PCR).2?The sequences of three si RNAs and one negative control si RNA were synthesized according to the sequence information of GRM4 m RNA,and the best silencing effect sequence of si RNAs were screened out to construct the lentiviral vector for GRM4.3?The U2 OS cells of GRM4 highest expression were transfected with lentivirus-mediated small interfering RNA(si RNA).Stable transformants were obtained by puromycin selection.4 ? The knockdown efficacy of GRM4 was confirmed by real-time quantitative PCR and western blot.5?The differentially expressed genes(DEGs)after GRM4 silencing were screened through RNA sequencing,and analysed by bioinformatics.6?Additionally,the transcription factors(TFs)targeting to GRM4 were selected and the downstream protein-protein interaction(PPI)network was constructed by the bioinformatics technology.RESULTS: 1?The expression level of GRM4 m RNA in human OS cells(MG-63,U2 OS,HOS and Saos-2)was higher than that of human osteoblasts h FOB1.19,among which U2 OS cell lines had the highest expression level.2?The silencing effect of si RNA-1 was the best among the three sequences,which was used for later experiments.3?The expression of GRM4 m RNA and protein level in U2 OS cells were significantly inhibited by lentivirus mediated GRM4-si RNA.4?A total of 51 significant DEGs were obtained,including 14 up-regulated and 37 down-regulated DEGs.The Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of the DEGs indicated that four significant enrichment pathways were obtained.5?A total of six TFs that could be involved in the transcriptional regulation of GRM4 were detected.The 182 genes in PPI network were significantly enriched in 14 pathways.The chemokines and chemokine receptors were found to be significantly enriched in three pathways.Moreover,in the GO category of MF,the chemokines and chemokine receptors were mainly enriched in the chemokine receptor binding,chemokine activity,chemokine receptor activity,chemokine binding,C-C chemokine receptor activity,C-C chemokine binding and cytokine activity.CONCLUSION: 1?Compared to human osteoblasts h FOB1.19 cell lines,GRM4 is highly expressed in human OS cell lines.2?The DEGs in the four significant enrichment pathways might participate in the development and progression of OS through GRM4.3?EGR1 and CTCF are probably involved in the transcriptional regulation of GRM4,which participates in the progress of osteosarcoma by interacting with chemokines and their receptors.