Effects Of ACTN4 On The Growth,Invasion And Metastasis Of Osteosarcoma

Background Osteosarcoma(OS)is the most common primary malignant bone tumor.The difficulty in treatment is recurrence and metastasis.Therefore,it is important to clarify the molecular mechanisms of OS development and metastasis and to discover new therapeutic targets to reduce OS recurrence and metastasis,thereby improving patient survival.?-actinin4(ACTN4)is an actin-binding protein belonging to the spectrin superfamily.Our previous study found that the expression of ACTN4 was significantly increased in cases with high osteosarcoma metastasis rate.Therefore,the subject further studies the ACTN4 gene to explore its role and mechanism in osteosarcoma.Nuclear factor-?B(NF-?B)is a protein complex that combines various subunits to form heterodimers with different functions.Among them,P50/65 heterodimer is the most widely distributed.After phosphorylation,P65 binds to P50 to form a heterodimer and is transferred to the nucleus to initiate transcription of downstream genes.Studies have shown that abnormally activated P65 can lead to tumor development,and there is co-immunoprecipitation between ACTN4 and P65.So there is a certain relationship between ACTN4 and P65.This study will further explore the role of NF-?B signaling pathway in the development of ACTN4 affecting OS.Objective To investigate the effects of ACTN4 on the growth,migration,invasion and metastasis of osteosarcoma,and further explore its possible molecular mechanism.It provides a theoretical basis for finding new therapeutic targets and reducing the recurrence and metastasis of osteosarcoma.Methods The m RNA expression differences of ACTN4 in 20 pairs of osteosarcoma and adjacent tissues were analyzed by RT-q PCR.Two osteosarcoma cell lines were selected to up-regulate or down-regulate the expression of ACTN4 in osteosarcoma cells by lentiviral-mediated transfection of osteosarcoma cell lines.The transfected cells were divided into four groups: interference group and control one,overexpression group and control two;the effect of ACTN4 expression on the proliferation of osteosarcoma cells was detected by CCK8 and EDU kits;the effect of ACTN4 expression on the migration ability of osteosarcoma cells was detected by scratch test.The effect of ACTN4 on the invasive ability of osteosarcoma cells was analyzed using Transwell chamber.The transplant tumor model was further prepared by injecting osteosarcoma cells into the legs of nude mice.The expression of ACTN4 in osteosarcoma cells of nude mice was confirmed by immunofluorescence assay.The effect of ACTN4 expression on the growth of osteosarcoma was observed by measuring the size of leg xenografts.After 6 weeks,the lungs of nude mice were removed and HE stained.The effect of ACTN4 on osteosarcoma lung metastasis was determined by observing the size and number of lung metastatic nodules.In addition,this study also used Western Blot and Transwell experiments to analyze the role of NF-?B in the development of ACTN4 in osteosarcoma.Results RT-q PCR results showed that the expression of ACTN4 m RNA in osteosarcoma was significantly higher than that in adjacent tissues.The proliferation experiment showed that the cell proliferation ability of the interference group was lower than that of the control group;the migration experiment showed that the scratch healing rate of the interference group was slower than that of the control group;the number of transmembrane cells in the interference group was significantly less than that of the control group;conversely,raising ACTN4 can give the opposite result.In nude mice,the growth rate and tumor volume of the transplanted tumors in the nude mice were smaller than those in the control group;the number and size of metastatic nodules in the lungs decreased compared with the control group;conversely,up-regulating ACTN4 can give the opposite result.In the mechanism study,overexpression of ACTN4 can enhance the invasive ability of osteosarcoma cells,and interference with the expression of P65 can reverse this promotion.Conclusion In vitro experiments confirmed that ACTN4 can promote the proliferation,migration and invasion of osteosarcoma cells.The transplanted tumor model in nude mice confirmed that ACTN4 can promote the growth and metastasis of osteosarcoma.ACTN4 may enhance the invasion ability of osteosarcoma through the NF-B signaling pathway,thus promoting the development of osteosarcoma.