The Expression Of Ribosomal Protein L34 In Osteosarcoma And Its Effects On Proliferation Of Human Osteosarcoma Cell Line

BACKGROUND:Osteosarcoma(OS)is sort of malignant bone tumor,which does great harm to the health of children and adolescents.Since its high tumor heterogeneity and low incidence,Progress in therapy and overall survival of OS has been negligible or non-existent over the past two decades.Ribosomal protein L34(RPL34)has been gradually recognized to possess some extraribosomal functions,apart from its role in ribosome biogenesis.The early studies found that RPL34 was associated with the cell proliferation of prokaryotes,and recent studies have revealed that RPL34 involves in proliferation of some malignant tumors.But its role in human OS cell and the underlying molecular mechanisms of RPL34 regulating malignant cells proliferation remain largely unknown.OBJECTIVE:This project was performed to investigate the expression of RPL34 in human OS tissues and OS cell lines,and its influence on human OS cellproliferation and the prognosis of OS patients.It was also committed to explore the latent mechanisms of RPL34 regulating OS cells proliferation preliminarily.We really expect to provide a new insight into the targeting therapy of OS.METHODS:1、Real time-PCR was conducted to detect the expression of RPL34 m RNA in 11 human OS tissues and corresponding adjacent tissues.2、Immunohistochemistry was used to evaluate the expression of RPL34 protein in 95 OS tissues and 60 normal bone tissues.3、All 95 OS specimens were divided into RPL34 high expression group and RPL34 low expression group according to the average score of immunohistochemical staining.Survival analysis was performed using Kaplan-Meier method and log-rank test.4、Real time-PCR was used to detect the RPL34 m RNA level in human osteoblast cell line h FOB1.19 and human OS cell lines U2 OS,MG-63,HOS and Saos-2.5、We designed and screened for RNAi sequence targeting RPL34 gene,and constructed the lentiviral vector,which was used to transfect Saos-2 cells.The knockdown efficacy of RPL34 was confirmed by Real time-PCR and western blot.6、We used Cellomics high content screening(HCS)、MTT assay、flow cytometry analysis、Annexin V-APC and colony formation assay to detect the impact of RPL34 knockdown on biological behavior of Saos-2.7 、 Transcription factors(TFs)screening and protein-protein interaction(PPI)network construction were performed with UCSC and STRING database separately.Gene Ontology(GO)and KEGG pathway enrichment analysis wereused to determine the TFs regulating RPL34 transcription and downstream proteins interact with RPL34.RESULTS:1、Compared with the adjacent tissues,RPL34 m RNA was shown to be up-regulated in 7/11(63.64%)of the OS tissues and down-regulated in 1/11(9.09%)of the OS tissues,whereas 3/11(27.27%)OS tissues show no significant difference in expression of RPL34.The expression of RPL34 in OS tissues is significant higher than that in adjacent tissues.2 、 The immunohistochemical detection of RPL34 indicate that 75/95(78.95%)OS tissues showed strongly positive staining and the other 20/95(21.05%)OS tissues showed weakly positive staining,whereas all of the normal bone tissues were weakly stained.3、The survival analysis revealed that the overall 3-year survival rate of the high-expression RPL34 patients was 35.42%(17/48),and that of the low-expression RPL34 patients was 61.70%(29/47).OS patients with high level of RPL34 predicts a worse prognosis.4、The m RNA levels of RPL34 in 4 human OS cell lines(U2OS、MG-63、HOS and Saos-2)were higher than that in human osteoblast cell line h FOB1.19.5、Knockdown of RPL34 in Saos-2 cells significantly suppressed cell proliferation and colony formation,and induced cell apoptosis and G2/M phase arrest.6、Bioinformatic analysis showed that MYC、MAX、CEBPB、E2F6、GABP、NFKB、NRSF、SPI1、TAF1、TBP、YY1 possibly involve in the transcriptional control of RPL34,and other 100 downstream proteinsinteracting with RPL34.GO and KEGG pathway enrichment analysis revealed that RPL34 transcription is regulated by proto-oncogene protein MYC,and eukaryotic translation initiation factor 3 subunits A、F or G(EIF3A、EIF3F or EIF3G)mediates the proliferation regulation of human OS cells by RPL34.CONCLUSION:1、RPL34 is highly expressed in OS,and highly expressed RPL34 promote the proliferation of human OS cells.2、OS patients with high level of RPL34 indicates poor prognosis.3、RPL34 participates in the proliferation regulation of human OS cells possibly through MYC/RPL34/EIF3 subunits(EIF3A,EIF3 F or EIF3G)signaling pathway.