The Mechanism Of HUC-MSCs Supernatants Against Glioma Via The IL-6/JAK/STAT3 Signaling Pathway

Objective: Gliomas originate from a wide range of glial cells in the brain and are the most common primary thought intracranial tumours.Glioblastoma(GBM)is one of the most devastating and aggressive WHO grade 4 gliomas.In recent years,significant progress has been achieved in the clinical care of GBM in terms of surgery,radiation,and chemotherapy.Mesenchymal stem cells(MSCs)have shown their superiority as a new therapeutic strategy for tumours.Studies have shown that MSCs are rich in chemokines that interact with various cytokines secreted by GBM and migrate to the tumour site.It also has immunomodulatory capabilities,inhibiting tumour growth by modulating immune cells without causing harm to healthy tissues.However,the use of human umbilical cord mesenchymal stell cells(HUC-MSCs)for the treatment of GBM is rarely investigated.Therefore,we investigated the efficacy and feasibility of the tumour-suppressive effects of supernatant secreted by HUC-MSCs on four GBM cell lines,RG-2,U251,U87-MG and LN-428.Further,we investigated the molecular mechanism by which HUC-MSCs supernatant targets the IL-6/JAK2/STAT3 signaling pathway to mediate apoptosis and autophagy in GBM cells.Methods: Umbilical cord tissues from full-term healthy fetuses delivered by caesarean section were isolated and cultured using the tissue block isolation method to obtain HUC-MSCs and ensure their purity.The supernatant of 3rd-8th generation HUC-MSCs were collected and prepared into a lyophilised powder using the lyophilisation technique.HUC-MSCs supernatants were collected using two serum-free methods,which were set to five medium-high and low different concentrations to treat U251,U87-MG,LN-428 and RG-2 cells respectively.By CCK-8 assay,the tumor suppression feasibility of HUC-MSCs supernatant acting on GBM cells was determined.The CCK-8 assay combined with HE morphological staining determined that 9 mg/m L HUC-MSCs supernatant was the effective concentration for subsequent experimental studies..We examined the cell cycle changes of RG-2,U251,U87-MG and LN-428 cells after treatment with HUC-MSCs supernatants using flow cytometry and Western Blot assays.Flow cytometry,TUNEL,Western Blot combined with Immunocytochemistry(ICC)assays were used to detect apoptotic changes in RG-2,U251,U87-MG and LN-428 cells after 48 h treatment of GBM cells with HUC-MSCs supernatants.Transwell assay detects the ability of HUC-MSCs to migrate towards the four GBM cells.Tranwell assay and Western Blot combined with ICC assay were performed to detect the effects of migration and invasion of RG-2,U251,U87-MG and LN-428 cells after treatment with HUC-MSCs supernatants.The IL-6/JAK/STAT3 signaling pathway and its downstream associated proteins(Survivin,VEGFA,MCL-1,Bcl-2,and c-Myc)expression alterations were identified by Western Blot paired with ICC assay after GBM cells were treated with HUC-MSCs supernatant for 48 h.Western Blot combined with Immunofluorescent Labeling(IF)assay was performed to detect the changes of autophagy-related protein expression in GBM cells treated with HUC-MSCs supernatant for 48 h.The effect of STAT3 inhibitor C188-9 on the proliferation of four GBM cells and the effect of IL-6/JAK2/STAT3 signaling pathway-related proteins was examined by CCK-8 assay,Transwell migration assay combined with Western Blot assay,to elucidate the inhibitory effect of HUC-MSCs supernatant on GBM cells and the related mechanism.Results: The proliferation capacity of RG-2,U251,U87-MG and LN-428 cells was inhibited in vitro after treatment with HUC-MSCs supernatant.RG-2,U251 and U87-MG cell cycles showed a significant S phase block,while LN-428 cells were blocked in the G0/G1 phase.The migratory and invasive capacities of all four GBM cells were inhibited to varying degrees,and the rates of apoptosis and autophagy were increased.The results also showed that the changes in apoptosis and autophagy in GBM cells treated with HUC-MSCs supernatants were mediated by the IL-6/JAK2/STAT3 signalling pathway.The results also showed that the changes in apoptosis and autophagy in GBM cells treated with HUC-MSCs supernatants were mediated by the IL-6/JAK/STAT3 signalling pathway.In addition,overexpression of the STAT3 protein inhibitor of activated STAT3(PIAS3)inhibited STAT3 activity.In four GBM cell lines,STAT3 and its downstream protein expression was further inhibited by the use of STAT3 inhibitor C188-9.However,MCL-1 protein expression was not inhibited by C188-9 and was even promoted,which may be related to the targeting of the structural domain of STAT3 by C188-9 in GBM cells.Disruption of the p Y peptide binding site in the SH2 region of STAT3 in GBM cells did not affect MCL-1 expression.Conclusions: HUC-MSCs supernatant has an anti-GBM effect.After treatment with HUC-MSCs supernatants,the proliferation,migration and invasion of GBM cells were significantly inhibited and their apoptosis was promoted.Negative regulation of the IL-6/JAK2/STAT3 signaling pathway enhances tumor cell apoptosis and autophagy,thereby improving the therapeutic effect on GBM.