| BackgroundOsteosarcoma(OS)is the most common primary malignant bone tumor in children and young adults,with an incidence of about 3 in one million all over the world,accounting for 15% of all bone tumors.It is the second leading cause of cancer-related death among children and adolescents.Osteosarcoma is highly malignant,clinically characterized by local invasion and early distant hematogenous metastasis with a poor prognosis.Currently,high-dose methotrexate,cisplatin and doxorubicin(MAP),as the first-line chemotherapy regimens for osteosarcoma,have greatly improved the prognosis of patients on the basis of traditional surgery.However,chemotherapy resistance is common in osteosarcoma due to heterogeneity,which makes the therapeutic effect and prognosis of OS enter the bottleneck in recent decades.Elucidating the mechanism and overcoming the resistance mechanism of chemoresistance to different antitumor drugs is an urgent problem to be solved.Protein tyrosine phosphatase receptor type N(PTPRN),also known as islet antigen 2,encodes a protein belonging to a member of the protein tyrosine phosphatase(PTP)family.In the past,PTPRN has been linked to diseases including insulinoma and type 1 diabetes.With the deepening understanding of the whole human genome,the role of the gene in various tumors has gradually emerged.It has been reported that PTPRN is closely related to proliferation,invasion,metastasis and drug resistance of multiple tumors,including lung cancer and glioma,suggesting that PTPRN also plays a potential role in the biological behavior of tumors.However,PTPRN related research is still in its infancy in osteosarcoma,at the same time,the effect of PTPRN on methotrexate drug sensitivity is not clear,which is worth exploring and discovering.Objective1.To forward screen the important genes affecting the sensitivity of OS to methotrexate at the level of the whole human genome by CRISPR-Cas9.2.To determine the expression level of PTPRN in OS tissues and cells.3.To explore the effects of PTPRN on OS biological behaviors such as cell proliferation,migration,apoptosis and drug sensitivity.4.To explore the molecular mechanism of PTPRN affecting sensitivity of OS to methotrexate.Research methods1.Through CRISPR-Cas9 genome-wide library screening and second-generation sequencing analysis,the genes with large expression differences between the experimental group and the control group were obtained and sequenced according to the degree of differences.After comprehensive analysis,specific molecules(PTPRN)were selected for subsequent experiments.The expression level of PTPRN in OS cell lines relative to other tumor cells lines were analyzed through CCLE database.The relationship between the expression level of PTPRN and the survival and prognosis of patients in pan-carcinoma were analyzed through GEPIA database.RT-PCR and Western Blot were used to detect the expression of PTPRN in tumor tissues and corresponding paratumoral tissues of 5 clinical OS samples in order to analyze the differential expression of PTPRN between OS tissues and paratumoral tissues.MTX-resistant OS cell lines were constructed,and the expression of PTPRN in OS parent cell lines and drug-resistant cell lines was detected by Western Blot,and the differential expression of PTPRN between OS parent cells and drug-resistant cell lines was analyzed.Western Blot was used to detect the change of PTPRN expression in OS after a short time of MTX stimulation.2.PTPRN over-expressed and interfered OS cell lines were constructed,and the efficiency of over-expression and interference were detected by Western Blot.In vitro,CCK-8 and clonal formation assay were performed to detect the proliferation ability of cells.Cell migration ability was detected by wound healing assay and transwell assay.ATP assay was used to detect the sensitivity of cells to methotrexate.The apoptosis of cells was detected by flow cytometry.In vivo,the effect of PTPRN in OS cells on methotrexate sensitivity was further verified by cell line-derived xenograft model in nude mice.3.The differential expression of genes and metabolites in OS parent cell lines and drug-resistant cell lines was determined by transcriptomic and metabolomic high-throughput sequencing,and the differential genes and metabolites were analyzed jointly to enrich the KEGG signaling pathway.Based on the joint analysis results of transcriptomics and metabolomics.Western Blot was used to verify the expression and phosphorylation level of PI3 K,AKT,m TOR and other important molecules in the PI3K/AKT/m TOR signaling pathway and the expression of glycolysis-related proteins(PKM2,LDHA,HK2,GLUTS)as well as metabolites such as lactic acid and pyruvate during PTPRN intervention in order to clarify the main molecular mechanism of PTPRN affecting OS sensitivity to methotrexate.Then we explore the regulatory relationship between PI3K/AKT/m TOR signaling pathway and glycolytic metabolic pathway and the interaction way between PTPRN and PI3K/AKT/m TOR.Cisplatin was used to detect the drug sensitivity of OS cell lines with PTPRN overexpression and interference,so as to clarify the relationship between PTPRN-mediated OS cell resistance and multidrug resistance.Research results1.The result of CRISPR-Cas9 genome-wide screening and second-generation sequencing analysis showed that a total of 211 genes had significant differences in the negative screening between the experimental group and the control group,among which PTPRN ranked the top according to the comprehensive influence degree.Therefore,it may be an important gene affecting MTX drug sensitivity in OS cell line,which is worthy of further study.The data analysis results of CCLE and GEPIA databases show that among all tumor cell lines,PTPRN is relatively highly expressed in OS cell lines,and the expression of PTPRN in pan-carcinoma is closely related to the poor prognosis of patients.The results of RT-PCR and Western Blot showed that the expression of PTPRN in tumor tissues was higher than that in OS adjacent normal tissues.The OS resistant cell lines were successfully constructed and the RF were 6.19 and 4.04,respectively.The results of Western Blot showed that the expression of PTPRN in methotrexate resistant OS cell lines was higher than that in parent OS cell lines.The expression of PTPRN in OS cell line increased after short-term stimulation with different concentrations of MTX.2.In vitro cell experiments confirmed that up-regulated PTPRN expression could increase the resistance of OS to MTX and promote the migration of OS cells.On the contrary,interference of PTPRN can reduce the resistance of OS to MTX and inhibit the migration of OS cells and increase the pro-apoptotic effect of MTX on OS cells.The change of PTPRN had no significant effect on the proliferation of OS.Vivo experiments showed that under the condition of MTX treatment,the tumor growth rate and tumor volume of nude mice implanted with sh PTPRN OS cells lines were significantly inhibited compared with ordinary OS cells lines.3.Transcriptomic sequencing results showed that 939 genes were up-regulated and655 genes were down-regulated in MTX-resistant Saos2 cell lines compared with parent cell lines.In MTX-resistant HOS cell lines,2367 genes were up-regulated and2542 genes were down-regulated.Metabolomics sequencing results showed that compared with parental cell lines,the expression of 219 metabolites was up-regulated and 203 metabolites was down-regulated in MTX-resistant Saos2 cell lines,and in MTX-resistant HOS cell lines,874 metabolites were up-regulated and931 were down-regulated.According to differential genes and metabolites obtained by sequencing,the KEGG signaling pathway was enriched by multi-omics analysis.The related genes and metabolites involved in the PI3K/AKT/m TOR signaling pathway and glycolysis were significantly different.The results of Western Blot showed that after the over-expression of PTPRN in OS cells,the expressions of PI3 K,p-AKT and p-m TOR increased,and the PI3K/AKT/m TOR signaling pathway was activated.However,after knockdown of PTPRN in OS cells,the expressions of PI3 K,p-AKT and p-m TOR decreased and the PI3K/AKT/m TOR signaling pathway is inhibited.After over-expression of PTPRN in OS cells,lactic acid and pyruvate production and glycolysis related proteins PKM2,LDHA,HK2,and GLUT1 increased to varying degrees.On the contrary,after knockdown of the expression of PTPRN in OS cells,lactic acid and pyruvate production and glycolysis related proteins PKM2,LDHA,HK2 and GLUT1 also decreased to varying degrees.After the add of AKT inhibitor MK-2206 or m TOR inhibitor rapamycin,the increase of glycolysis metabolites content and glycolysis related proteins caused by PTPRN over-expression were reversed,and drug resistance of OS cells was decreased.The result of gene correlation analysis in database showed that there was a positive correlation between SNAP25 and PTPRN in bone tissue.The results of Western Blot showed that the expression of SNAP25 increased after PTPRN was over-expressed in OS cells and vice versa.Over-expression of PTPRN could enhance the resistance of OS cells to DDP.While knockdown of PTPRN can reduce the resistance of OS cells to DDP.ConclusionPTPRN plays an important role in the regulation of the sensitivity of OS cells to MTX.Relatively high expression is showed in OS tissues and cell lines,and the expression was more obvious in MTX-resistant cell lines.Over-expression of PTPRN can increase the resistance of OS cells to MTX and promote the migration ability of cells.While knockdown of PTPRN can weaken the resistance of OS cells to MTX and inhibit the migration ability of cells,and increase the apoptosis induced by MTX.In terms of mechanism,PTPRN promotes glycolytic metabolism by activating PI3K/AKT/m TOR signaling pathway,eventually increasing the resistance of OS cells to MTX.At the same time.The influence of PTPRN on drug sensitivity of OS is related to multi-drug resistance of tumor to a certain extent. |
