A Preliminary Research On The Role Of PNO1 In Osteosarcoma

Background and objectiveOsteosarcoma is the most common primary malignant bone tumor,which occurs in the metaphysis of long bone in adolescents.Before 1970s,the treatment of osteosarcoma is mainly surgery,while the survival rate of patients who accepted surgery were less than 20%.With the development of surgery and chemotherapy,about 70%osteosarcoma patients could gain a long term survival.However,such a rate haven’t changed in the past 40 years,as for those who suffered recurrence and metastasis,their 5-year survival rate is still at about 20%.The revolution of genomics,molecular biology and bioinformatics reveals numbers of genes that play an important role in the tumorigenesis and progression in malignant tumors.The research aiming to target therapy has developed several new drugs which showed an inspiring effect in lung cancer and breast cancer.While the current targeted agents haven’t obtained a significantly effect in osteosarcoma,which asking for a further research to seek potential targets in osteosarcoma therapy.PNO1(partner of NOB 1 homolog,PNO1)is one of RNA-binding proteins(RBPs),which has been proved involved in process of pre-18S rRNA and ribosome biogenesis.Current studies show that RBPs can regulate the progression of malignant tumors,and previous research has reported that the expression of PNO1 was upregulated in colorectal cancer,lung adenocarcinoma and bladder cancer and played a key role in their tumorigenesis and progression.Besides,PNO1 has not been reported in osteosarcoma,exploring the role of PNO1 in osteosarcoma may provide new potential target for osteosarcoma treatment.Our research is focus on bioinformatics,to investigate the expression of PNO1 and its possible biological function in osteosarcoma.Moreover,we will also study the effect of PNO1 on proliferation,apoptosis,invasion and migration in osteosarcoma via transfecting shPNO1 lentivirus into MG-63 osteosarcoma cell line.Methods1.GEPIA,CCLE and GEO database were employed to analyze the mRNA expression of PNO1 in osteosarcoma;2.We downloaded the transcriptome sequencing data from TARGET database,and used GSEA software to obtain the PNO1-related pathways in osteosarcoma;3.We used CIBERSOFT to calculate the proportion of immunocytes in TARGET dataset.Furthermore,we obtained the correlation between the expression of PNO1 and immunocytes by Spearman analysis;4.We collected osteosarcoma tissues and adjacent tissues,then used qRT-PCR to examine the mRNA expression level of PNO1 in osteosarcoma;5.Lentivirus shPNO1 was used to knockdown the expression of PNO1 in MG-63 cell line,the efficiency was validated by qRT-PCR and Western blot;6.MTT assay was used to investigate the effect of PNO1 on the proliferation in osteosarcoma;7.We performed flow cytometry cell apoptosis assay and cell cycle to detect the influence of PNO1 in apoptosis and cell cycle;8.We used Transwell invasion and migration assays to verify the invasive and migratory ability of shPNO1 cells;9.qRT-PCR was employed to detect the mRNA expression level of EMT biomarkers.Results1.The expression of PNO1 was upregulated in osteosarcoma;2.PNO1 might play its role in osteosarcoma via energy metabolism pathways;3.PNO1 was involved in immune infiltration of osteosarcoma,and might have correlation with neutrophils and eosinophils;4.Lentivirus could downregulate the expression of PNO1 effectively in MG-63 cell line;5.MTT assay showed that downregulation of PNO1 inhibited the proliferation of MG-63 cell line;6.Flow cytometry assays suggested downregulation of PNO1 induced the apoptosis and cell cycle arrest of MG-63 cell line;7.Transwell assays showed that downregulation of PNO1 inhibited the invasion and migration of MG-63 cell line.qRT-PCR result indicated that PNO1 was involve in the progression of EMT in osteosarcoma.ConclusionThe results demonstrated that PNO1 was upregulated in osteosarcoma and involved in energy metabolism and immune infiltration in osteosarcoma.Downregulated expression of PNO1 could inhibit the proliferation and induce the apoptosis,cell cycle in osteosarcoma cell line.In addition,downregulation of PNO1 also decreased the ability of invasion and migration in osteosarcoma cell line,moreover,inhibited the progression of EMT.