RCAD Deficiency Promotes The Resistance Of Osteosarcoma U-2OS Cells To Cisplatin By Regulating Autophagy And Endoplasmic Reticulum Stress

Objective Osteosarcoma is the most common malignant tumor of long bone in children and adolescents,and cisplatin is one of the most commonly used drugs in clinical chemotherapy.However,studies have found that drug resistance of osteosarcoma to cisplatin often occurs and leads to treatment failure.The specific molecular mechanism has not yet been determined.Endoplasmic reticulum stress and autophagy are two biological processes widely existing in cells,which are closely related to the occurrence and development of tumors.RCAD(alias UFL1;KIAA0776;Maxer;NLBP)is the E3 ligase of Ufm1,one ufmylation system,which enhances the stability of DDRGK1 and CDK5RPA3 and plays a role in tumor inhibition by indirectly regulating the NF-k B signaling pathway.The purpose of this study is to investigate the effect of RCAD on osteosarcoma cells and its relationship with autophagy and endoplasmic reticulum stress.Diabetes is the third largest noncommunicable disease after cardiovascular and cerebrovascular diseases and tumors,with a high incidence and growth rate,and osteoporosis is one of its most common complications.At present,there are many and complicated studies on the mechanism of diabetic osteoporosis.In order to verify the universality of the relationship between RCAD and autophagy and endoplasmic reticulum stress,the role and mechanism of RCAD in diabetic osteoporosis were also preliminarily studied.Methods In the study of cisplatin resistance,we used lenti-virus mediated RNAi technology to knock down the RCAD's expression in osteosarcoma cell line U2 OS.After the treatment of cisplatin,we used Western blot,Realtime PCR,flow cytometry and transmission electron microscope and immunofluorescence technology to analyze expression of RCAD,change of autophagy and endoplasmic reticulum stress.In the study of diabetic osteoporosis,we made a BALB/c mouse model of type I diabetes,and confirmed the occurrence of osteoporosis through micro-CT.Then we detected the changes of RCAD,autophagy and endoplasmic reticulum stress by immunohistochemistry,Realtime PCR and Western blot.Results In the study on cisplatin resistance of osteosarcoma,we successfully knocked down the m RNA and protein expressions of RCAD in U-2OS cell line,and found that the biological behavior of tumor cells was improved and the sensitivity to cisplatin was decreased.The mechanism study showed that in the absence of RCAD,GRP78 increased,CHOP decreased,LC3 II increased,p62 decreased,endogenous LC3 expression and the formation of autophagosomes and autophagolysosomes increased.After cisplatin treatment,in the Con-sh RNA group,RCAD decreased,GRP78 increased,CHOP increased,LC3 II increased,p62 decreased,endogenous LC3 expression and autophagosomes and autophagolysosomes formation increased.RCAD was significantly reduced in the RCAD-sh RNA group,and the expression of other indexes was reversed.In the study of diabetic osteoporosis,we detected the expressions of RCAD,LC3,p62,GRP78 and CHOP in the whole bone tissue,and found that the expressions of LC3 II,GRP78 and CHOP in the diabetic osteoporosis group increased,while the expressions of p62 and RCAD decreased.Conclusion Knockdown of RCAD can enhance the resistance of osteosarcoma cells to cisplatin chemotherapy.RCAD deficiency activates endoplasmic reticulum stress and autophagy in osteosarcoma cells.RCAD has a complex effect on autophagy and endoplasmic reticulum stress of osteosarcoma cells in cisplatin chemotherapy.Downregulation of RCAD,activation of ER stress and autophagy,are involved in the pathogenesis of diabetic osteoporosis.