| Pan-cancer analysis clarified the similarities of different types of cancers,found out the same characteristics of tumors,and helped us apply an effective method for tumor to other tumors with the same characteristics.The incidence of osteosarcoma is low,but it is mortal.There is no data related to osteosarcoma in the The Cancer Genome Atlas(TCGA)database.Pan-cancer analysis found out that KIF4 A and STIL were overexpressed in various types of tumors.Therefore,this study aimed to explore Clinical significance and biological function of KIF4 A and STIL in osteosarcoma,provide new biological targets for the diagnosis and treatment of osteosarcoma.Objective: To explore the clinical significance and biological mechanism of KIF4 A and STIL in osteosarcoma.Methods:(1)Download all available samples by TCGAbiolink and explore the interaction between KIF4 A and STIL based on physical interaction,co-expression,co-localization,pathways,shared protein domains and predicted interactions by Gene MANIA;Cluster Profiler package was used to analyze central genes Gene Ontology biological pathway enrichment.(2)51 paired specimens of osteosarcoma tissues and normal tissues were collected,clinicopathologic parameters were collected from patients.The expression of KIF4 A and STIL were tested by Immunohistochemistry(IHC),the expression of KIF4 A and STIL in osteosarcoma tissues and the clinicopathologic parameters such as gender,age,tumor location,tumor diameter,degree of differentiation,and 5-year survival rate of patients were analyzed.(3)real-time quantitative PCR(q RT-PCR)and Western blot(WB)methods were used to explore the differences in the expression of KIF4 A and STIL in U2 OS,MG63,HOS osteosarcoma cells and h FOB1.19 cell.Two osteosarcoma cells U2 OS,MG63 were collected,and si RNA and overexpression plasmid were transfected into MG63 and U20 S cells to detect their viability,proliferation,migration,invasion,cycle,apoptosis by CCK-8 assay,colony formation,wound healing assay,transwell assays,and flow cytometry.WB was initiated to explore it’s signaling pathway in osteosarcoma.Subsequently,the biological behavior of KIF4 A was explored in vivo experiments.Results:(1)Cell-Cycle-Related genes were overexpressed in various types of tumors,A total of 28 DEGs were enriched.(2)Overexpression of KIF4 A and STIL can be treated as diagnostic and prognostic indicators for many tumors.(3)KIF4A was co-expressed and interacted with STIL in tumors,the overexpression of KIF4 A in poor prognostic tumor types were exactly the same as that of STIL,the m RNA expression of KIF4 A and STIL were highly correlated in all 12 tumors.(4)Expression of KIF4 A and STIL in osteosarcoma tissues were significantly higher than those in normal tissues(P<0.05).(5)There was no correlation between the expression level of KIF4 A with the sex and age with osteosarcoma,but the diameter(P<0.05)and differentiation(P<0.05),and Enneking stage(P<0.05)of osteosarcoma.There was no significant correlation between the expression level of STIL with sex,age,location,and diameter,but the degree of differentiation(P<0.05),Enneking stage(P<0.05)of osteosarcoma.(6)Kaplan-Meier survival analysis showed that the five-year survival rate of osteosarcoma patients with low expression of KIF4 A and STIL were significantly higher than that of patients with high expression(P < 0.05).(7)Cox multivariate regression analysis showed that KIF4 A and STIL were independent risk factors for lower five-year survival rate in osteosarcoma patients.(8)The results of q RT-PCR and WB showed that the relative m RNA exprssion of KIF4 A and relative protein expression in U2 OS,HOS,and MG63 cells were significantly higher than that of h FOB1.19 cell(P<0.05),and U2 OS and MG63 cells were higher than those in HOS cell(P<0.05).The results of q RT-PCR and WB showed that the efficiency of downregulation of KIF4 A and upregulation of KIF4 A in U2 OS and MG63 were satisfactory(P<0.05).(9)CCK-8 assay,colony formation,wound healing assay,transwell assays results showed that the viability,proliferation,migration,and invasion capacity of MG63 and U2 OS were significantly reduced in si-KIF4 A group(P<0.05).The viability,proliferation,migration,and invasion capacity of MG63 and U2 OS were significantly enhanced in oe-KIF4 A group(P<0.05).(10)The results of flow cytometry showed that cells blocked at G0/G1 phase were significantly increased when silenced KIF4A(P<0.05).Cells blocked at G0/G1 phase were significantly decreased when overexpressed KIF4A(P<0.05).(11)Downregulation of KIF4 A induced osteosarcoma cell apoptosis,and upregulation of KIF4 A inhibited osteosarcoma cell apoptosis(P<0.05).WB results showed that KIF4 A activated with Wnt/β-catenin signaling pathway through PRC1.(12)Downregulation of KIF4 A leaded to reduced tumor size,weight,and growth curve in vivo.IHC results confirmed that downregulation of KIF4 A inhibited proliferation capacity of tumor.IHC and WB results showed that KIF4 A activated with Wnt/β-catenin signaling pathway.Conclusions:(1)Cell cycle-related genes,KIF4 A and STIL,were overexpressed and co-expressed in many types of tumors.KIF4 A and STIL overexpression could be treated as diagnostic and prognostic indicators for many tumors.(2)The expression of KIF4 A and STIL in osteosarcoma tissues were higher than those of normal tissues.The expression levels of KIF4 A and STIL were significantly correlated the severity of the disease in osteosarcoma patients.KIF4 A and STIL were independent risk factors for five-year survival rate in osteosarcoma patients.(3)The expression of KIF4 A in osteosarcoma cells was significantly higher than that of normal cells.KIF4 A promoted the viability,proliferation and invasion of osteosarcoma cells;affected the cell cycle and inhibited the apoptosis of osteosarcoma cells.(4)KIF4A activated the Wnt/β-catenin pathway through PRC1.(5)KIF4A promoted the growth of osteosarcoma in nude mice. |
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