| Objective:Hypoxia-inducible factor-1(HIF-1ɑ)is a key molecule of solid tumor adaptation to hypoxia microenvironment,which is closely related to tumor invasion and metastasis,metabolic reprogramming,chemoradiotherapy tolerance,and immunosuppression.The immunosuppressive state of tumor patients also hinders the efficacy of radiotherapy.Therefore,We explored the screening of candidate mir-212-3p that can bind HIF-1ɑ,immunosuppressive molecules PD-L1 and TIM-3through bioinformatics,and verified the binding mir-212-3p to the target gene sites through experiments.Furthermore,this study systematically investigated whether the effects of mir-212-3p on HIF-1ɑ,PD-L1 and tim-3 of melanoma cells B16-F10 can change the radiotherapy sensitivity of tumor cells,induce immunogenic death of tumor cells,and enhance the anti-tumor immune response of dendritic cell-mediated specific cytotoxic T cells by regulating the expression of mir-212-3p.Methods:1.miRNAs with predicted binding targets of HIF-1ɑ,PD-L1 and TIM-3 were screened by mir Walk,Targetscan and other miRNA databases on bioinformatics websites,and the targeting relationship was verified by Dual-luciferase reporter gene assay;2.QPCR was used to detect the expression of mir-212-3p in mouse melanoma cells B16-F10 and mouse epidermal melanocytes,and the expression changes of mir-212-3p,HIF-1ɑ,PD-L1 and TIM-3 before and after radiotherapy;3.Lipofectamine2000 was used to transfect mouse mir-212-3p mimic into B16-F10 cells.The expressions of mir-212-3p,HIF-1ɑ,PD-L1 and TIM-3 were detected by qPCR and Western blot.CCk-8,Transwell,Western blot and cell scratches were used to observe the changes of biological behaviors such as proliferation,invasion,migration and apoptosis of B16-F10 cells after mir-212-3p elevation.4.Immunofluorescence assay was performed to observe the DNA damage of B16-F10 cells after transfection with mir-212-3p mimic for 48h.After 5GY radiotherapy,Confocal microscopy was used to detect DNA damage.5.The effects of mir-212-3p overexpression combined with tumor cell antigen treated with radiotherapy on DC biology and immunological behavior were investigated.Experimental groups:blank Control group(Control group),mir-212-3p mimic transfection group(transfection group),radiotherapy group,mir-212-3p mimic transfection combined with radiotherapy group(combined group).B16-F10 cells were transfected with NC mimic and mir-212-3p mimic or treated with 5GY electron ray radiotherapy,respectively,and freeze-thaw antigens were extracted from each group of tumor cells repeatedly.On the sixth day of culture,the bone marrow derived mouse DCs were stimulated by freeze-thaw antigen of tumor cells of different treatments.On the seventh day,the cells were collected and the mature phenotypes(MHCⅡ,CD40,CD86,CD80)and the expression of immunosuppressive molecular ligand PD-L1 on the surface of the DCs were detected by flow cytometry.The expression of CDC-stimulated T cell proliferation,CD4~+T cells,CD8~+T cells,Treg(CD4~+CD25~+Fox P3~+)and T cell depletion(BTLA,PD-1 and TIM-3 in CD4~+T cells and CD8~+T cells)were detected by heterogenous mixed lymphocytic reaction6.Animal experiment:Melanoma B16-F10 cells were subcutaneously inoculated to establish a tumor-bearing mouse model,which was randomly divided into four groups.On the fifth day after tumor implantation,they received intratumoral injection of mir-212-3p Angomir and 5GY radiotherapy alone,as well as the combination of radiotherapy and intratumoral injection of mir-212-3p Angomir.Tumor growth was recorded.7.On day 21 after tumor inoculation,the mice were sacrificed and the tumor tissues were stripped and weighed.Single cell suspension was prepared from spleen tissue,and the maturation of spleen DC,activation and depletion of T cells were detected by flow cytometry;8.The expressions of mir-212-3p,HIF-1ɑ,PD-L1 and TIM-3 in mouse tumor and spleen were detected by qPCR.Results:1.Bioinformatics results showed that the target miRNA was mir-212-3p.mir-212-3p was predicted to have two hexabase binding sites with HIF-1ɑ,one 6-base binding site with PD-L1,and one 7-base binding site and two hexabase binding sites with TIM-3.This prediction was verified by Dual-luciferase reporter gene assay.The expression of HIF-1ɑ,PD-L1 and TIM-3 is down-regulated by mir-212-3p in mice.2.QPCR results showed that the expression level of mir-212-3p in mouse melanoma cells was significantly lower than that in mouse epidermal melanoma cells(p<0.05),and the expression level of mir-212-3p in B16-F10 cells was significantly increased after radiotherapy,while the mRNA expression levels of HIF-1ɑ,PD-L1and TIM-3 were decreased correspondingly(p<0.05).3.The results of qPCR and Western Blot showed that mir-212-3p significantly increased in B16-F10 cells transfected with mir-212-3p mimic by Lipofectamine2000(p<0.05),while the mRNA and protein expression levels of HIF-1ɑ,PD-L1 and TIM-3 in transfected cells were decreased(p<0.05);CCk-8 results showed that the proliferation of cells transfected with mir-212-3p mimic was weaker than that of the control group(p<0.05);The results of Transwell chamber assay showed that the invasion and migration of transfected cells were significantly reduced compared with the control group(p<0.01);Flow cytometry results showed that Annexin V~+/PI~+apoptosis was significantly higher in the transfected group than in the normal group(p<0.01).4.Confocal fluorescence microscopy showed that the degree of DNA damage in cells transfected with mir-212-3p mimic after radiotherapy was significantly higher than that in radiotherapy alone or mir-212-3p alone(p<0.01).5.Flow cytometry results showed that the expression levels of mature phenotype and functional indexes CD11c,MHCⅡ,CD40,CD80 and CD86 were higher in the group of radiotherapy combined with mir-212-3p compared with the group of radiotherapy alone or mir-212-3p alone,while the expression level of immunonegative regulatory molecule PD-L1 was significantly decreased.6.Flow cytometry results showed that mixed culture of DC and T cells stimulated by freeze-thaw antigen after radiotherapy combined with mir-212-3p increased the expression of CD8~+IFN-γ~+T cells and CD8~+Granzyme B~+T cells,while the depletion markers of T cells(BTLA,PD-1 and TIM-3 in CD4~+T cells and CD8~+T cells)decreased,while the expression of Treg cells was relatively reduced(p<0.05).7.Animal experiments:The tumor growth rate and size of B16-F10tumor-bearing mice treated with radiotherapy combined with mir-212-3p agonist were significantly reduced compared with the control group(p<0.05).8.Flow cytometry results showed that the expression of Flow cytometry results showed that the expression of CD80,CD86,MHCⅡand CD40 on the surface of DCS with positive CD11C expression in spleen of mice in the combination of radiotherapy and mir-212-3p increase group was significantly different from that in the blank group.At the same time,combined therapy can down-regulate the expression of PD-1,TIM-3 and BTLA in spleen T cells of tumor-bearing mice,and increase the expression of CD8~+IFN-γ~+T and CD8~+Granzyme B~+T(p<0.05),CD40,CD80 and CD86 on the surface of DCS with positive CD11C expression in spleen of mice in the combination of radiotherapy and mir-212-3p increase group was significantly different from that in the blank group.At the same time,combined therapy can down-regulate the expression of PD-1,TIM-3 and BTLA in spleen T cells of tumor-bearing mice,and increase the expression of CD4~+IFN-γ~+T and CD4~+Granzyme B~+T(p<0.05).9.The expression of mir-212-3p in spleen and tumor of mice was increased by qPCR,while the expression of HIF-1ɑ,PD-L1 and TIM-3 mRNA was slightly decreased compared with the control groupConclusions:1.mir-212-3p affects b16-F10 black by regulating the expressions of HIF-1ɑ,PD-L1,and TIM3Proliferation,invasion,migration and apoptosis of melanoma cells;2.mir-212-3p can significantly improve the radiotherapy sensitivity of mouse melanoma B16-F10 and promote cell apoptosis.DC uptake of freeze-thaw antigen of tumor cells with high mir-212-3p expression after radiotherapy can improve the mature phenotype,reduce the expression of PD-L1,stimulate the proliferation and activation of T cells,and improve the depletion state of T cells,suggesting that mir-212-3p can promote the anti-tumor immune response of T cells. |
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