Exosome-mediated Transfer Of MIF Confers Temozolomide Resistance By Regulating TIMP3/PI3K/AKT Axis In Gliomas

Background Glioma(Gliobla)is the primary malignant tumor with the highest incidence in the brain,account for more than 50%of primary intracranial tumors,of which,and among them,more than 50%of intracranial gliomas are WHO grade IV Glioblastoma Multiforme(GBM).At present,the standard clinical treatment of glioma is surgical resection,followed by concurrent radiotherapy and chemotherapy.Although there have been significant advances in the research of surgical treatment,radiotherapy,chemotherapy,gene therapy,immunotherapy and other treatments for glioma in recent years,clinically it still shows that patients with glioma have a poor prognosis,high recurrence rate and high mortality rate.Temozolomide(TMZ)is the first-line chemotherapy drug for glioma,but TMZ resistance in gliomas has become an important cause of treatment failure.Therefore,further research on the molecular mechanism of glioma drug resistance and finding possible specific treatment methods need to be solved urgently.In 2013,3 reseachers from the United States and Germany were awarded the 2013 Nobel Prize in Physiology or Physiology for their "discovery of the main transport system in cells-the regulatory mechanism of vesicle transport".Medical awards.The exosomes are vesicles with a diameter of 30-150 nm,,which can be secreted by various types of cells.Exosomes contain protein,RNA,DNA and other substances,which can participate in the exchange of materials and information transmission between cells.As an important type of transport vesicles in the human body,the role of exosomes is receiving more and more research and attention.Scientists have found that exosomes can participate in important biological processes,such as tumor cell growth and proliferation,angiogenesis and chemotherapy resistance.Tumor cells can secrete exosomes and transfer the signal molecules contained in them to distant tissues.And in the cell,and then play a regulatory role.Macrophage migration inhibitory factor(MIF),a pro-inflammatory multifunctional cytokine,was first discovered in 1966.Previous studies have shown that MIF is overexpressed in many tumors.Recent studies have shown that MIF is related to tumor resistance to chemotherapy.As a secreted cytokine,MIF is found to be highly expressed in plasma exosomes of tumor patients and can promote the malignant progression of tumors.However,whether exosomal MIF contributes to TMZ resistance in gliomas remains to be elucidated.Therefore,the purpose of this study is to explore whether MIF is involved in the formation of TMZ resistance in gliomas,and whether it is through the exosomal pathway to target and regulate TIMP3/PI3K/AKT pathways to mediate TMZ resistance in gliomas.By studying these issues,we try to provide new drug targets and treatment strategies for the treatment of glioblastoma.Objective To clarify the role of MIF in exosomes in the development of TMZ resistance in gliomas and its clinical significance,and to explore the possible mechanism and potential clinical application of MIF-mediated TIMP3 regulation of PI3K/AKT pathway in TMZ resistance in brain gliomas.Methods In the early stage,the drug-resistant cell line TR(Temozolomide Resistant)cells were constructed by gradually increasing the concentration of TMZ in the culture medium,and the MIF content in the supernatant of TS(Temozolomide Sensitive)and TR cells and the expression of MIF in the cells were detected by ELISA and WB,respectively Circumstances,and changes in the expression of MIF under different concentrations of TMZ treatment.CCK8 method,EdU staining and flow cytometry were used to clarify the effects of MIF on the proliferation,apoptosis and TMZ sensitivity of glioma cells;extract exosomes and detect the expression of MIF;add TR exosomes to PKH26 after labelling The TS cells were co-cultured in the supernatant,and confocal was used to photograph their engraftment,and to verify the changes in MIF content,proliferation,apoptosis,and chemotherapy sensitivity of TS cells.Stable knockdown of MIF in U87 was performed by RNA sequencing,and the differentially highly expressed gene TIMP3 was screened out,and the regulatory relationship between MIF and TIMP3 was verified by RT-Qpcr and WB.Further cell function analysis to determine whether MIF participates in TMZ resistance by regulating TIMP3 and analyze the specific molecular mechanism.The cell function recovery certificate verifies that MIF regulates the expression of TIMP3/PI3K/AKT to promote proliferation,inhibit apoptosis and mediate the process of TMZ resistance.Animal experiments verified that exosomes mediate MIF to regulate the TMZ drug resistance process,and in vivo experiments verified the chemotherapeutic sensitivity of the MIF small molecule inhibitor ISO-1 to TR cells.Results 1.MIF is highly expressed in patients with high-grade glioma and recurrent glioma,and its high expression is negatively correlated with clinical prognosis.2.MIF can promote drug resistance in gliomas.Stable knockdown of MIF can significantly reduce the proliferation of TR cells,promote TR cell apoptosis,and restore the chemotherapy sensitivity of TR cells;and overexpression of MIF in TS cells can promote TS cell proliferation and reduce TS cell apoptosis,leading to TS cell chemotherapy Resistance.3.MIF can enter TS cells from TR cells through exosomes.The exosomes extracted from TR cells were stained with PKH26 and added to the U87-GFP and U251-GFP cell supernatants for co-cultivation.Confocal fluorescence microscopy analysis revealed that the exosomes of U87TR and U251TR entered U87 and U251 through endocytosis.And up-regulate its MIF gene and protein levels.4.Exosomal MIF can cause TMZ drug resistance in sensitive cells by promoting proliferation and inhibiting apoptosis.Different types of exosomes were added to the TS cell supernatant and co-cultured for 2 days.It was found that TR EXO can promote TS cell proliferation and inhibit TS cell apoptosis,leading to chemotherapy resistance of TS cells.TR-MIF-sh EXO can proliferate TS cells.The effects of,apoptosis and chemotherapy sensitivity were significantly weakened.In addition,TR EXO and TR-NC EXO significantly reduced the expression levels of apoptosisrelated proteins(caspase-3 and caspase-9)in receptor TS cells.5.TIMP3 may be the downstream target of MIF.After stable knockdown of MIF in U87,RNA-Seq sequencing analysis revealed that Tissue inhibitors of metalloproteinases-3(TIMP3)may be a very likely downstream target of MIF in glioma cells.TCGA and CGGA cohort analysis showed that MIF and TIMP3 were significantly negatively correlated.Further experiments confirmed that after stable knockdown of MIF in TR cells,TIMP3 was up-regulated at both Mrna and protein levels.6.MIF mediates TMZ resistance in gliomas by regulating the TIMP3/PI3K/AKT signaling pathway.Stable knockdown of TIMP3 can significantly inhibit cell apoptosis in TR-MIF-sh cells.EdU test showed that after knocking out TIMP3 in TRMIF-sh cells,the percentage of proliferating cells increased.The recovery experiment showed that stable knockdown of TIMP3 can reverse the decrease of IC50 value caused by stable knockdown of MIF.WB analysis showed that stable knockdown of MIF expression can inhibit the activation of PI3K/Akt signaling pathway,and knockdown of TIMP3 in TR-MIF-sh cells can reverse the changes of these proteins.7.MIF in exosomes promotes TMZ resistance in gliomas in vivo.The results show that MIF in exosomes can promote TMZ resistance in gliomas in vivo,and the MIF small molecule inhibitor ISO-1 can reverse this TMZ resistance.Conclusion 1.Up-regulation of MIF expression can promote TMZ resistance in gliomas and lead to poor prognosis of patients;2.MIF can promote cell proliferation and inhibit cell apoptosis to promote TMZ resistance in gliomas;3.MIF transfers TMZ drug resistance from TR cells into TS cells through exosomes;4.Exosomal pathway mediated MIF to inhibit TIMP3 expression and activate PI3K/AKT pathway to promote TMZ resistance in gliomas.