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. 2018 Jan 17;24:16-25.
doi: 10.12659/msmbr.908107.

To Explore the Mechanism of the GRM4 Gene in Osteosarcoma by RNA Sequencing and Bioinformatics Approach

Free PMC article

To Explore the Mechanism of the GRM4 Gene in Osteosarcoma by RNA Sequencing and Bioinformatics Approach

Yunguo Pang et al. Med Sci Monit Basic Res. .
Free PMC article


BACKGROUND Glutamate metabotropic receptor 4 (GRM4) has been correlated with the pathogenesis of osteosarcoma. The objective of this study was to explore the underlying molecular mechanism of GRM4 in osteosarcoma. MATERIAL AND METHODS The expression levels of GRM4 in four human osteosarcoma cell lines and hFOB1.19 cells were examined by real-time quantitative PCR (RT-qPCR). The U2OS cells of the highest GRM4 expression were transfected with lentivirus-mediated small interfering RNA (siRNA). The differentially expressed genes (DEGs) after GRM4 gene silencing were screened through RNA sequencing, and analyzed by bioinformatics. Additionally, the transcription factors (TFs) targeting GRM4 were predicted and the downstream protein-protein interaction (PPI) network was constructed using the bioinformatics approach. RESULTS A total of 51 significant DEGs were obtained, including 14 upregulated and 37 downregulated DEGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs indicated that four significant enrichment pathways were obtained. A total of six TFs that could be involved in the transcriptional regulation of GRM4 were detected. The results showed that 182 genes in the PPI network were significantly enriched in 14 pathways. The chemokines and chemokine receptors were found to be significantly enriched in three pathways. CONCLUSIONS The DEGs in the four significant enrichment pathways might participate in the development and progression of osteosarcoma through GRM4. The results revealed that 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.

Conflict of interest statement

Conflict of interest



Figure 1
Figure 1
Expressions of GRM4 mRNA in OS cell lines and the human osteoblast hFOB1.19 cells and expressions of GRM4 after GRM4 silencing in U2OS cells. (A) GRM4 mRNA in each cell line was detected by RT-qPCR. (B) Expressions of GRM4 mRNA after GRM4 silencing by three siRNAs in U2OS cells. (C) The mRNA level of GRM4 was significantly reduced in GRM4-siRNA treated group compared with NC-siRNA treated group, * p=0.000. (D) Western blot revealing that the protein levels of GRM4 was significantly inhibited by GRM4-siRNA lentivirus transfection.
Figure 2
Figure 2
Heat map and unsupervised hierarchical clustering analysis.
Figure 3
Figure 3
Integrated network of TF-target and PPI. The TFs implicated in the transcriptional regulation of GRM4 and the proteins interacted with GRM4 are presented in green nodes and pink nodes, respectively.
Figure 4
Figure 4
GO analysis of the 182 genes identifed in PPI network. The enriched genes associated with GRM4 were categorized according to the relevant biological functions of molecular function, cellular component and biological process.

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