The Effect Of P38γ On Osteosarcoma Growth And Its Molecular Mechanism

Background and aims:Osteosarcoma(OS)is a malignant tumor originating from mesenchymal cells,which generally occurs in children and young people.Although the current medical methods have significantly improved the 5-years event free survival of patients with osteosarcoma,but the recurrence,metastasis and chemotherapy tolerance of osteosarcoma are still the main reasons that affect the treatment effect.The metastasis of osteosarcoma is the main cause of death in patients.Therefore,finding effective methods to inhibit tumor cell proliferation and migration,and clarifying its underlying molecular mechanism is essential to improve the therapeutic effect and prognosis of osteosarcoma patients.P38γ is one of the four subtypes of p38 MAPK(Mitogen Activated Protein Kinase),which is generally expressed in skeletal muscle tissue.Recent studies have found that p38γ is highly expressed in several malignant tumors,indicating that p38γ plays an important role in the occurrence and development of tumors.The specific mechanism of action is still unclear.This study intends to explore whether p38γ is involved in the occurrence and development of osteosarcoma in human osteosarcoma cells,and to clarify the role of p38γ in the occurrence and development of osteosarcoma by interfering with p38γ expression,to provide a new target for the treatment of osteosarcoma.Methods:The total protein and RNA were extracted from osteosarcoma and its adjacent tissues.The expression of p38γ was detected by Western blot and qRT-PCR.After p38γ silencing(by targeting shRNA)or p38γ knockout(KO)mediated by CRISPR/cas9,the growth,proliferation and migration of stable transfected cells were measured by CCK-8,EdU and Transwell.Apoptosis of stable transfected cells was detected by flow cytometry and Western blot.Secondly,the growth,proliferation and migration of stable transfected cells and the phosphorylated expression of retinoblastoma tumor suppressor proteins(pRb),cyclin E1 and cyclin A were detected by the above methods after overexpression of p38γ in primary OS1 cells.At the same time,after silencing cyclin E1 and cyclin A(by targeting shRNA),the proliferation and migration of stable transfected cells were detected by the above methods.Finally,two kinds of virus stable transfected U2OS cells(shRNA C and p38γ shRNA)were injected subcutaneously into nude mice.Tumor volume and body weight of nude mice were measured every 7 days,and tumor growth rate and body weight were calculated.After 42 days of subcutaneously tumor formation,the expression of p38γ was analyzed by immunohistochemistry.Results:P38γ mRNA and protein levels were detected in OS tissues and surrounding normal tissues,OS cell lines(U2OS),primary human OS cells(OS1,OS2,OS3)and osteoblasts respectively.It was found that p38γ was up-regulated in human OS tissues and cells.In primary human OS cells or OS cell lines,it was found that p38γsilencing or KO inhibited the growth,proliferation and migration of OS cells,and also induced caspase-3 activity and enhanced apoptosis of OS cells.Secondly,we found that in primary human OS cells,the proliferation and migration ability of cells were significantly improved after the p38γ overexpression.At the same time,p38γ affects the expression of cell cycle related proteins,including phosphorylated retinoblastoma tumor suppressor protein(pRb),cyclin E1 and cyclin A.Overexpression of p38γ promotes the expression of pRb,cyclin E1 and cyclin A,and vice versa.At the same time,silencing cyclin E1 and cyclin A inhibited OS cells proliferation and migration.Finally,we found that the knockdown of p38γ significantly inhibited the increase of tumor volume and the proliferation of tumor cells through subcutaneous tumor formation experiment.Conclusion:The results of this study suggest that p38γ plays an important role in the growth,proliferation and migration of OS cells,it is demonstrated that p38γ is involved in mediating the occurrence and development of osteosarcoma through targeted regulation of retinoblastoma tumor suppressor protein and cyclin E1/A,which provides innovative idea for the progression of OS and therapeutic target.