| Osteosarcoma(OS),a cancer which is derived from early lineage bone-forming mesenchymal cells,predominantly occurs in young adults and children.Although the incidence of osteosarcoma is low,the degree of malignancy is high,and OS patients with distant metastases have a high mortality rate,no effective treatment is currently available.In order to improve the prognosis of OS patients and the quality of life,it is necessary to explore the molecular mechanism of OS.Eukaryotic translation initiation factors(e IFs)participate in regulate tumorigenesis,invasion and metastasis of cancer through different pathways,but the mechanism in osteosarcoma is still unclear.The site of protein biosynthesis is in the ribosome,which is the organizer that recognizes m RNA and ribosomal RNA.Ribosomal protein(RP)is the main component of the ribosomal core of the organism,and plays an important role in the process of protein biosynthesis.Ribosomal protein L34(RPL34)belongs to the ribosomal protein L34 E family member.Studies have found that RPL34 is related to the pathogenesis of OS and the prognosis of patients,and bioinformatics analysis revealed that the upstream transcription factor c-Myc regulates downstream RPL34 gene expression,and RPL34 then regulates downstream e IF3 gene expression.Therefore,we speculate that RPL34 might regulate the proliferation of human osteosarcoma through c-MYC/RPL34/e IF3 Signaling Axis,but this hypothesis has not been experimentally verified.Objective: To study the potential mechanism of RPL34 regulating the proliferation of OS through the c-MYC/RPL34/e IF3 signal axis,this will help to study the pathogenesis of OS.Methods: The overexpression model of c-Myc was constructed.The m RNA and protein expression levels of c-Myc and RPL34 were detected by q PCR and western blot.Chromatin immunoprecipitation was used to detect whether the upstream transcription factor c-Myc was binding to the downstream gene RPL34,and the binding site of c-Myc to RPL34 was detected by double-luciferase report.Real-time quantitative PCR was used to detect the expression levels of RPL34 and downstream genes e IF3 A,e IF3 G,e IF3 F,UBA52,UBC and FAU in SAOS-2 and h FOB1.19 cells respectively.RPL34 lentivirus vector was constructed and transfected into Saos-2 cells.Western blot and real-time quantitative PCR were used to detect the knockdown rate of RPL34 and the expression of downstream related genes.Results:1.Regulation mechanism of upstream transcription factor c-Myc and downstream target gene RPL341.1 The expression level of c-Myc in Sao-2 and U2 OS cells transfected with p CDNA3.1(+)-c-Myc was significantly increased.1.2 c-Myc transcription factor can up-regulate the expression of RPL34 gene.1.3 In the detection of c-Myc chip product,the enrichment degree of RPL34 is similar to that of LEF1.2.Study on the interaction mechanism between RPL34 and downstream target gene e IF32.1 Compared with human osteoblasts h FOB 1.19 in the control group,RPL34,UBA52 and FAU in Saos-2 cells were significantly up-regulated,e IF3 A and e IF3 G were significantly up-regulated,UBC was significantly up-regulated,and e IF3 F was significantly down-regulated.2.2 The expression of RPL34 m RNA was significantly down-regulated in the sh-RPL34 transfected cells compared to the cells transfected with NC-sh RNA.2.3 Compared with NC-sh RNA,lentivirus RPL34-sh RNA interference vector can significantly knock down the expression of RPL34 gene in Saos-2cells,the expression levels of e IF3 A and e IF3 F genes related to RPL34 are significantly reduced,and the expression levels of FAU gene are significantly increased.Conclusion:1.The transcription factor c-Myc can regulate the promoter activity of gene RPL34.2.c-Myc combines with RPL34 promoter sequence to regulate its expression.3.RPL34 may regulate the proliferation of OS via c-MYC/RPL34/e IF3 signaling axis. |
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