The Mechanism Of RNA-binding Protein PTBP1 Promotes The Malignant Biological Behavior Of Osteosarcoma

INTRODUCTIONOsteosarcoma(OS)is the leading primary malignant tumour of bone of mesenchymal origin,characterised by the formation of bone or bone-like tissue with high malignancy,early metastasis and rapid progression.It is most common in adolescents and elderly people aged 60-70 years,and is most likely to occur near the epiphysis of the long bones of the limbs.The current treatment model of definitive diagnosis by puncture biopsy,neoadjuvant chemotherapy,followed by surgery and intensive chemotherapy based on tumour pathology has become the standard of care for osteosarcoma,resulting in an overall5-year survival rate of 68% for new osteosarcoma patients,but the prognosis for patients with metastatic or recurrent osteosarcoma remains poor.Currently the etiology of osteosarcoma is unclear,and existing studies have identified multiple mutations in the tumour,represented by TP53,as well as a significant increase in the incidence of osteosarcoma in patients with related genetic disorders,revealing the complexity of the genetic aspects of osteosarcoma development.Due to the complex and widespread heterogeneity and highly malignant biological behaviour exhibited by osteosarcoma patients,the identification of new therapeutic targets remains a challenge and an urgent need to form the basis for new therapeutic approaches.Polypyrimidine Tract-Binding Protein 1(PTBP1)is a widely expressed RNA-binding protein(RBP)involved in RNA maturation,translocation,localization and translation,and its broadest physiological role is as a splicing factor involved in the regulation of alternative splicing(AS)of pre-m RNA.Dysregulated expression of PTBP1 is also closely associated with the development of many tumours.In addition,PTBP1 can influence cell cycle,differentiation,metabolism,motility and immunity through the regulation of AS events.It can enhance the Warburg effect through AS regulation in a variety of tumours,thereby promoting tumour cell proliferation and enhancing tumour migration and invasion in glioma,hepatocellular carcinoma and breast cancer.The AS events induced by PTBP1 in tumours are complex and diverse,and previous studies have not been able to fully characterise all AS events in a particular tumour.Our analysis of clinical tissue samples,cell lines and public databases revealed that PTBP1 expression is elevated in osteosarcoma and significantly correlates with poor patient prognosis,which may be a new target for osteosarcoma treatment.Considering the high expression of PTBP1 in osteosarcoma and its broad role in a variety of tumours,its important role as a proto-oncogene in the malignant biological behaviour of osteosarcoma warrants further in-depth investigation.OBJECTIVE1.To clarify the expression of PTBP1 in osteosarcoma and its correlation with patient prognosis.2.To clarify the effect of PTBP1 on the malignant biological behaviour of osteosarcoma.3.To explore the possible mechanisms by which PTBP1 promotes the malignant biological behaviour of osteosarcoma.4.To explore the mechanism by which PTBP1 plays a pro-cancer role in osteosarcoma by regulating variable splicing.5.To explore the possible mechanisms by which PTBP1 affects the malignant biological behaviour of tumours at the pan-cancer level and its potential as a prognostic marker and new therapeutic target.MATERIALS and METHODS1.Tumour samples from osteosarcoma patients operated at the Department of Orthopaedic Oncology,Second Affiliated Hospital of Naval Medical University were used to detect the expression level of PTBP1 in osteosarcoma by immunohistochemistry(IHC)and Western Blot(WB)experiments.The expression levels of PTBP1 in various human osteosarcoma cell lines(MG63,Sao S2,U2 OS,HOS)and human mesenchymal stem cell lines(h BMSC)were measured using WB.2.To investigate the correlation between PTBP1 expression levels and clinical prognosis of osteosarcoma patients through the OS dataset in the TARGET database.The collected human osteosarcoma dataset(GSE152048)from the GEO public database and self-assessed human cancellous bone single cell transcriptome data were used to further reveal the differences in PTBP1 expression between tumour and normal tissue and specific cell types within the tumour at the single cell level and to preliminarily explore possible mechanisms.3.PTBP1 knockdown and overexpression cell lines were constructed by CRISPR/Cas9 and overexpression plasmids,and the efficiency of PTBP1 knockdown and overexpression was verified by q RT-PCR(Quantitative Real Time Polymerase Chain Reaction)and WB experiments.The effect of PTBP1 expression level on the malignant biological behavior of osteosarcoma cells was clarified by CCK-8 cell proliferation,Transwell cell migration and invasion experiments.4.Multi-omics data including expression,copy number variation,and tumour mutational load in tumour and normal tissues were collected from multiple databases.Focusing on sarcoma(SARC),we explored the common effects of PTBP1 on the malignant biological behaviour of multiple tumours in terms of PTBP1 expression at the pan-cancer level and its impact on tumour prognosis,self-mutation frequency,its correlation with tumour immune infiltration,genetic instability and drug sensitivity,in order to further explore its mechanism of action in osteosarcoma.5.We analyzed gene expression differences by triple full-length transcriptome sequencing and combined with Gene Regulatory Network(GRN)analysis to find key genes in the mechanism of PTBP1 affecting tumor function,identified AS events significantly regulated by PTBP1 and verified the differences in target gene expression levels before and after AS by q RT-PCR.The potential signalling pathways regulated by PTBP1 in osteosarcoma were analysed in conjunction with the TARGET-OS dataset.6.To explore the possible direct binding of PTBP1 protein to target genes in osteosarcoma by RNA Binding Protein Immunoprecipitation Assay(RIP)and the main functions regulated by these genes,and to explore its new binding sites in osteosarcoma by Motif analysis.RESULTS1.PTBP1 expression is elevated in osteosarcoma and is significantly associated with adverse outcome for patients.At the single-cell level,it is widely expressed in tumours,particularly in stem-associated cells with high proliferative capacity,and is associated with cell cycle regulation and maintenance of cell proliferation activity.2.Regulation of PTBP1 expression can significantly affect the proliferation,migration and invasive ability of osteosarcoma.Overexpression of PTBP1 significantly enhances the malignant biological behaviour of osteosarcoma,while knockdown of PTBP1 results in the opposite.3.PTBP1 mutations are most commonly found in sarcomas and are significantly overexpressed in a variety of tumours,including sarcomas,and are associated with poor patient prognosis,cell cycle,spliceosomes,TGFB and WNT pathway regulation.Its expression at the pan-cancer level is strongly associated with immunosuppression,reduction of tumour stromal components,tumour mutations,genetic instability and partial drug sensitivity.4.Key genes such as EGR1,TNF,TP73 and DDIT3 play a key role in the promotion of malignant biological behaviour in osteosarcoma by PTBP1.PTBP1 promotes malignant biological behaviour in osteosarcoma through activation of cell cycle,variable splicing,metabolism and inhibition of tumour immunity,NF-KB,JAK-STAT and other related pathways.5.PTBP1 regulates AS events in osteosarcoma mainly by promoting exon skipping(ES)and identified 574 genes that may bind directly to this protein,of which 13 genes underwent significant AS events and 8 genes underwent alterations at the m RNA level.The target genes are mainly involved in regulating cell differentiation,proliferation,migration,cell cycle and signalling pathways such as HIF-1.The newly identified motifs provide new perspectives to further explore their cancer-promoting mechanisms in osteosarcoma.CONCLUSION1.PTBP1 is highly expressed in osteosarcoma and is associated with adverse outcome and maintenance of tumour cell proliferative activity.Its high expression is significantly associated with malignant biological behavior of tumors,and reducing PTBP1 expression levels significantly inhibits tumor proliferation,migration,and invasive ability.2.In osteosarcoma,PTBP1 contributes to the malignant biology of osteosarcoma through activation of cell cycle,variable splicing,metabolism and inhibition of tumour immunity,NF-KB,JAK-STAT and other related pathways.3.PTBP1 regulates AS events in osteosarcoma mainly by promoting ES,and regulates AS events of multiple genes and differential expression of transcripts through direct gene binding by its protein.The newly discovered motif provides insight into the pro-cancer mechanism of PTBP1 and is expected to be a new target for precise regulation of pro-cancer splicing events.4.PTBP1 has prognostic value at the pan-cancer level,regulates cell cycle,TGFB,WNT and other related pathways,and is associated with inhibition of tumor immunity,tumor mutation,genomic instability and some drug sensitivity.5.In the context of immuno-oncology,PTBP1 plays a critical pathogenic role in osteosarcoma and a variety of tumours,and is expected to guide the prognosis of osteosarcoma patients and become a new therapeutic target.