| Objective:G-quadruplexes are specific secondary structures formed by guanine-rich single-stranded nucleotides.In the presence of metal ions,guanine-rich mononucleotides form G-quadruplexes with G-quartet plan as the basic composition.Several articles have reported that G-quadruplexes are associated with immune cells,immune response,or immune escape.In addition,the G-quadruplex AS1411 is well known as a ligand for nucleolin(NCL),which has been reported to be involved in various antiviral processes.In order to further understand the relationship between G-quadruplex AS411 and immune cell regulation,this study will verify whether AS1411 forms a G-quadruplex and the effects on the immune characteristics of T cells.And the effect of T cell antitumor effect,and its potential mechanism.Method:1.Circular dichroism(CD)and thermal difference spectroscopy(TDS)were used to analyze 19 G-quadruplex structures with NMR structures determined,and a new method was established to analyze G-quadruplex structures.2.In vitro,we induce proliferation of T cells,and use MTT to detect the effect of AS1411 on the proliferation of CD8+T cells:select the optimal dose and time,complete the subsequent experiments.And we use flow cytometry(FCS)to detect the surface antigen expression of T cells and flow sorting of CD8+T cells.In vitro,Western Blot was used to detect protein expression levels in CD8+T cells,and ELISA was used to detect the levels of cytokines secreted by CD8+T cells.3.The effect of AS1411 and CD8+T on the proliferation of cervical cancer cells was detected by using the optimal concentration and time of CD8+T cells to act on cervical cancer cell line Hela and clone formation assay;TUNEL was used to detect the apoptosis of cervical cancer cells;Cell scratch assay was used to detect the migration of cervical cancer cells.Results:1.We use CD spectroscopy complementary TDS to study the structural characteristics of 19 G-quadruplexes in different ionic environments.The sequences were divided into parallel,antiparallel and hybrid groups by CD spectroscopy.And then,the antiparallel group can be subdivided into chair and basket structures by TDS,which can be compared with the NMR results to form a new way to study the G-quadruplex structures.2.The effect of G-quadruplex AS1411 on T cell proliferation was time-and dose-related;after the effect of AS1411,the proportion of CD8+CD28+T cells increased significantly;the proportion of naive T cells decreased and the proportion of TemRA cells increased;The proportion of Treg cells of CD4+CD25+CD127-was significantly reduced after with AS1411;AS1411 can up-regulate TLR4 of CD8+T cells,while down-regulates FasL;the pro-inflammatory factors TNF-α,IL-1 β and IL-6 was increased,and the expression of antiinflammatory factor IL-10 was decreased.It was speculated by next-generation sequencing analysis that AS1411 may positively regulate CD8+T cells through the NF-kB pathway.We validate AS 1411 regulate CD8+T cells through NF-κB pathway with control sequence,AS 1411,NF-κB inhibitor+AS 1411,and NF-κB activator betulinic acid,after worked AS 1411 and NF-κB activator got the same results.3.In vitro co-culture of CD8+T and tumor cells and CD8+T culture supernatant culture of tumor cells,using MTT method,scratch test,cell cloning test,TUNEL apoptosis detection to verify that AS 1411 can enhance the anti-tumor proliferative effect of cell CD8+T.Conclusion:1.TDS can resolve antiparallel chair structures from all antiparallel G-quadruplexes.This study may provide a simple and feasible method for the detection of G-quadruplexes in the future.2.AS1411 may positively regulate the secretion of TNF-α,IL1-β,IL-6 of CD8+T cells through the NF-κB pathway,and positively regulate the proportion of immune effector cells.3.The co-culture of CD8+T and tumor cells in vitro and the method of culturing tumor cells with CD8+T culture supernatant prove that AS1411+CD8+T has the effect of inhibiting proliferation,migration and invasion and promoting apoptosis in cervical cancer. |
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