| Background: Osteosarcoma is the most common malignant bone tumor.It occurs in the fast-growing metaphysis of long bones in adolescents,especially in the lower end of the femur and upper end of the tibia.It is composed of immature bone or osteoid tissue produced by mesenchymal tissue.Typical clinical symptoms and signs are local pain,swelling,and limited joint mobility.According to researchers,in young osteosarcoma patients,the 5-year survival rate without metastasis is 60%,but the 5-year survival rate of patients with metastasis is only 20%.This data has not improved in the past 30 years.At present,the main method of clinical osteosarcoma treatment is surgical resection combined with neoadjuvant chemotherapy and radiotherapy,but the progress of this systemic therapy is also at a stagnant stage.By studying the pathological and molecular mechanisms of osteosarcoma,finding new molecular targets for diagnosis and treatment has more clinical and social value.Pyroptosis is a mechanism of regulated cell death in response to inflammation.Morphologically,pyroptosis is similar to apoptosis,in that nucleus condensation and chromatin DNA fragmentation also occur.Studies have found that pyroptosis plays an important regulatory role in tumors.Pyroptosis is closely related to the occurrence and development of a variety of malignant tumors,and common malignant tumors such as lung cancer,liver cancer,and colorectal cancer are research hotspots.Pyroptosis gene TP63 is a member of the tumor suppressor gene TP53 family.TP63 can regulate the metastasis potential of cancer cells and maintain the pluripotency of stem cells through various mechanisms.It plays an important functional regulatory role in the progression of human tumors.However,the molecular mechanism of TP63 regulating pyroptosis in osteosarcoma is still unclear,and the study of regulating TP63 through ceRNA network has not been reported.Therefore,studying the mechanism of TP63 regulating pyroptosis in osteosarcoma is of great significance for exploring new models of osteosarcoma treatment.Objective: Screening the risk pyroptosis related genes were used to study the molecular regulation mechanism of pyroptosis in osteosarcoma,and the ceRNA regulatory pathway of pyroptosis gene TP63 was studied.To screen the clinical risk factors by analyzing the SEER clinical data of osteosarcoma patients,and use the clinical risk factors to construct a clinical prediction model for osteosarcoma patients,and to intervene early for high-risk patients to improve clinical treatment options for osteosarcoma.Method:Part I: The gene expression matrix data,clinical information and survival followup data of osteosarcoma patients were downloaded from the TARGET database and the GEO database,and the gene expression matrix data of normal musculoskeletal tissues were downloaded from the GTEx database.Based on 52 pyroptosis related genes expression of 141 osteosarcoma and 396 normal specimens,differential expression analysis,consensus clustering,enrichment analysis,tumor microenvironment assessment,pyroptosis risk model construction and ceRNA network construction and other data analysis were performed.To study the mechanism of pyroptosis in regulating the progression of osteosarcoma,we screened the hub pyroptosis related genes,and built a pyroptosis gene ceRNA network to study the molecular regulatory pathways of pyroptosis in osteosarcoma.Part II: After screening the ceRNA network pathway of the lnc RNA-mi RNAm RNA through the Star Base v2.0 database,the transient cell lines of 143 B and U2 OS osteosarcoma cells knocked down by TP63 si RNA and LAMTOR5-AS1 si RNA were constructed.CCK-8 experiments,cell colony experiments,Ed U experiments,migration and invasion experiments were used to explore and evaluate the effects of TP63 and LAMTOR5-AS1 on the proliferation,migration and invasion functions of osteosarcoma.Western blot was used to detect the changes of some pyroptosis-related proteins in cells,including cleaved-caspase-1,cleaved-caspase-3,cleaved-caspase-4,cleaved-caspase-8,GSDME,GSDMD and GSDMD-N.After LAMTOR5-AS1 knockdown,the expression levels of TP63,LAMTOR5-AS1 and hsa-mi R-23a-3p in osteosarcoma cells and normal osteoblasts were detected by PCR experiments,and the ceRNA network was initially verified,and the ceRNA was further verified by luciferase reporter gene experiments role in osteosarcoma.Part III: The study used the SEER database to collect clinical variable data of 1029 patients diagnosed as osteosarcoma at the first disease from 2010 to 2015,including gender,age,race,tumor size,T stage,N stage,M stage,chemotherapy,survival outcome and survival time,etc.Through univariate and multivariate Cox analysis,a nomogram was established to predict the 1-year,3-year and 5-year survival rates of osteosarcoma patients;at the same time,1029 patients were divided into modeling group and verification group to verify the stability of the model,and the accuracy of the model was evaluated by ROC curve,DCA curve and calibration curve.Result:Part I: 141 samples of osteosarcoma patients were obtained,and the expression matrix and copy number variation data of 52 pyroptotic genes were extracted from them.Based on the expression characteristics of 52 pyroptotic genes,osteosarcoma patients were divided into two pyroptotic subtypes by consensus clustering analysis.After differential analysis of the two pyroptotic subtypes,453 differential genes were obtained,and according to the expression characteristics of the differential genes,osteosarcoma patients were further divided into three gene subtypes.The pyroptosis risk scoring model and survival prediction model were constructed according to the differential genes of pyroptosis subtypes.And the regulation of pyroptosis in immune cell infiltration,tumor microenvironment,biological function and pathway regulation,and drug sensitivity was analyzed.In addition,the ceRNA network regulatory pathway was constructed based on7 hub pyroptosis related genes,and the ceRNA regulatory pathway of the pyroptosis related gene TP63 was further screened out by using the Star Base v2.0 online website:LAMTOR5-AS1/hsami R-23a-3p/TP63 regulatory axis.Part II: After the successful construction of TP63 and LAMTOR5-AS1 knockdown osteosarcoma cell line,PCR experiments showed that the expression of TP63 and LAMTOR5-AS1 in the cells was significantly reduced.In vitro experiments,after knockdown or overexpression of TP63 and LAMTOR5-AS1,the proliferation,migration and invasion of osteosarcoma cells were enhanced or decreased.Western blot results showed that after knocking down TP63 and LAMTOR5-AS1,the levels of cleavedcaspase-1,cleaved-caspase-3,cleaved-caspase-4,cleaved-caspase-8,GSDME and GSDMD-N in 143 B and U2 OS cells all decreased,but the level of GSDMD increased.After knocking down LAMTOR5-AS1 in the 143 B cell line,PCR results showed that compared with the un-transfected group and the NC group,the expression levels of LAMTOR5-AS1 and TP63 in the knockdown group were significantly reduced,while hsa-mi R-23a-3p expression levels were significantly increased.Further double-luciferase experiments showed that has-mi R-23a-3p can target TP63 3’UTR to promote TP63 degradation,and LAMTOR5-AS1 competitively binds to hsami R-23a-3p so that TP63 is not degraded by mi RNA and plays a role cancer suppressor effect.Part III: Chi-square test and univariate logistic regression analysis were performed on the risk factors for tumor metastasis.It was found that gender,chemotherapy,tumor size,T stage and N stage were risk factors for tumor metastasis,and further multivariate logistic regression analysis was performed.The results showed that gender,T stage and N stage are correlated with tumor metastasis.Then,chi-square test and univariate Cox regression analysis were performed on the risk factors for patient survival,and it was found that gender,chemotherapy,tumor size,T stage and N stage were risk factors for patient survival.Further multivariate Cox regression analysis found that sex,gender,race,age,tumor size,chemotherapy,T stage,N stage and M stage were all correlated.These risk factors were used to establish a clinical prediction model to predict the survival rate of patients.The AUC values of the modeling group and the verification group were both greater than 0.7.The calibration curve showed that the prediction model had good consistency in predicting the 1-year,3-year and 5-year survival rates.Conclusion:1.Pyroptosis plays an important regulatory role in osteosarcoma,which mainly affects the survival and prognosis of patients by regulating the immune microenvironment and tumor microenvironment of osteosarcoma.2.As the hub pyroptotic gene in osteosarcoma,TP63 can inhibit the proliferation of osteosarcoma cells and promote the pyroptosis process of osteosarcoma cells.3.LAMTOR5-AS1 has the same regulatory effect as TP63 gene,can inhibit the proliferation of osteosarcoma cells,and promote the pyroptosis process of osteosarcoma cells.4.TP63 can be regulated by the LAMTOR5-AS1/hsami R-23a-3p ceRNA network and affect the pyroptosis process of osteosarcoma cells.5.Gender,T stage and N stage are independent risk factors affecting tumor metastasis.In addition,age,gender,race,N stage,M stage and tumor size are independent risk factors affecting patient survival time. |
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