| Background:Glioma stem cells(GSC)are closely related to the occurrence,development and recurrence of malignant glioma.In recent years,the use of promoting the activation of the immunogenic function of dendritic cells(DCs)to effectively present tumor antigens to T cells,especially glioma stem cells(GSCs),is considered to be a more effective strategy for glioma immunotherapy.High levels of Signal Transducer and Activator of Transcription(STAT3)usually increase the release of tolerogenic mediators during the development and differentiation of DC,make tumor escape immune detection,reduce the transcription of immune stimulator molecules,inhibit the maturation of DC,and thus reduce the innate and adaptive antitumor immunity,However,the mechanism of regulating DC differentiation in glioma immunotherapy targeting GSC is still unclear.As one of the important signal molecules in the development,differentiation,survival and functional maturation of immune cells such as DC,B/T lymphocytes and macrophages,whether miRNAs also participate in the immune regulation of DC against GSC.Therefore,this study intends to explore the regulatory mechanism between miR-106a/20b and STAT3 signal pathway,aiming to maximize the immune activity of DCs against GSC,so as to more effectively stimulate the immune response of the body to GSC,and to find a new breakthrough in the immunotherapy of glioma.Objective:Our study was to evaluate the effect of the miRNA-106a/20b cluster on the efficacy of DCs loaded with GSCs as an antigen source in activating GSCspecific T-cell responses in vitro and in vivo.It aims to study a new therapeutic strategy for glioma.Methods:First,GSC was induced by GL261 andimmature DC and T cells was obtained from C57BL/6 mice.We compared the effects of three common GSC antigen extraction methods(freeze-thaw lysis,apoptosis and total RNA extraction)on DC maturation by PCR,flow cytometry and ELISA,then choosed the best one.Secondly,We used the most efficient way to obtain GSC antigen and shock the imDC to detect the expression of stat3 in DC.Next,we interfered with STAT3 expressionby siRNA,observed the changes of DC maturation index and cytokine secretion,as well as the differentiation and IFN of homologous CD8+,CD4+and Foxp3+T cells-γEffect of secretion.Finally,We predicted the possible miRNAs clusters targeting STAT3 by bioinformatics analysis,and selected one of them to explore its regulatory relationship with STAT3 by transfected DC,In addition,the specific molecular mechanism of miRNA cluster in regulating DC targeting GSC immune killing was elucidated by detecting its effect of inducing T cells to reduce GSC in vitro and inhibiting the growth of subcutaneous tumors in tumor-bearing mice in vivo.Results:Compared with freeze-thaw group and RNA group,the expression of CD80,CD86,MHC-Ⅱ and the secretion of TNF-α were higher in apoptosis group,but lower IL-10.In addition,the secretion of IL-6 in apoptotic group was also significantly higher than that in the control group and LPS group.Secondly,compared with other groups,the expression of STAT3 increased but the surface molecules CD80,CD86,MHC-II were significantly decreased in siRNA group DC loaded with GSC,while the secretion of IL-12,IL-6 was increased and the secretion of IL-10 was decreased.Correspondingly the abundance of CD8+T cells in siRNA group increased significantly,CD4+T cell abundance of Foxp3+was significantly reduced,and The secretion level of INF-γ was increase.Finally,Eight miRNAs was predicted targeting STAT3,and miR-106a/20b was selected as the research object.PCR showed that the expression of miR-106a/20b in DC loaded with GSC was low,and there was a negative correlation with STAT3.Increasing the level of miR-106a/20b in DC can significantly promote the expression of CD80,CD86,MHC-Ⅱ in DC and increase the inhibition of GSC proliferation by T cells in vitro.Nextly,The tumor-bearing mice form smaller tumors with injection of DC transfected with miR-106a/20b mimic in vivo.At the same time,The cells of Ki-67 staining was significantly reduced and CD8+T cell infiltration was significantly increased in tumor tissue by immunohistochemistry.Conclusions:Obtaining GSC antigen by apoptosis is the best way to sensitize DC.STAT3 is highly expressed in DC loaded with GSC antigen.The knockdown of STAT3 by siRNA can promote the maturation of DC,and induce the increase of CD8+cytotoxic T cells and the decrease of FOXP3+helper CD4+ T cells.It was further found that miR-106a/20b gene cluster negatively regulated STAT3 expression.Up-regulating the level of miR-106a/20b in DC is beneficial to reduce the expression of STAT3,and promote the maturation of DC,enhance the killing effect of induced T cells on GSC in vitro,and inhibit the tumorigenesis rate of GSC in tumor-bearing mice.Therefore,regulating STAT3 signal pathway by miR-106a/20b to enhance the immune activity of DC in anti-tumor targeting GSC may become a new strategy for glioma immunotherapy. |
