MiR-21 Regulates Immunosuppression Mediated By MDSCS By Affecting RUNX1-YAP Interaction In Lung Cancer

Background:Lung cancer is one of the widely-diagnosed cancers around the worl d,and remains one of the leading causes of cancer-associated mortality i n both men and women.Moreover,less than 7%of patients survive 10years after diagnosis acrossall stages of lung cancer.Metastasis to other or gans,especially the brain,is considered to be responsible for the high m ortality rate associated with lung cancer.Nowadays,the implementation of immunotherapy has become more and more encouraging and promising in the treatment and management of lung cancer.Meanwhile,myeloid-der ived suppressor cells(MDSCs),a heterogeneous group of immune cells f rom the myeloid lineage,which serve as suppressors of antitumor immun ity,have been found to contribute to the immunosuppressive microenviro nment during tumor development.Therapeutic strategies targeting MDSCs in combination with primary mammary tumor resection has also been re ported to reduce and retard metastatic growth in the lungs in mice beari ng orthotopic murine mammary tumors.Additionally,studies have shown that targeting MDSCs recruitment and enhancing antitumor immunity can augment the therapeutic efficacy of ablative hypofractionated radiation th erapy in subcutaneous tumors using syngeneic lung cancer.Considering t he emerging role of MDSCs in lung cancer,improved therapeutic regime ns targeting MDSCs could prove useful in controlling the development a nd progression of lung cancer.Increasing evidence has further shown that micro RNAs(miRNAs),such as miR-494 and miR-155,influence tumor-ex panded MDSCs accumulation,and also function in tumor microenvironme nt and favor solid tumor growth.MiRNAs are a group of endogenous RNAs of about 21 nucleotides in length and play numerous regulatory roles in animals and plants.The novel ability of miRNAs to regulate the expression of oncogenic pathway s and their vital roles in lung cancer progression indicate that miRNAs c ould potentially serve as prognostic biomarkers or targets for treatment of cancers.Moreover,studies have demonstrated that over-expression of miR-21-5p by mesenchymal stem cell-secreted extracellular vesicles(MSC-E Vs)promotes the development of lung cancer.In addition,suppressing m i R-21 inhibited cell migration and invasion in non-small cell lung carcino ma(NSCLC)cells.Bioinformatics analysis revealed the RUNX1 transcrip tion factor as one of the downstream targets of miR-21.The runt-related transcription factor(RUNX)family(RUNX1,RUNX2,and RUNX3),w hich is involved in many cell lineages,has been widely-associated with t he development of human cancers,such as acute myeloid leukemia,colo n carcinoma,and hepatocellular carcinoma.Even more so,RUNX1,a cor e-binding factor in transcription families,is one of the most commonly mutated genes found in various hematological malignancies.A prior study also revealed shown that RUNX1 can inhibit Yes-associated protein(YA P)to accelerate the occurrence of tumor.Therapeutic activation of YAP,a Hippo pathway effector,is known to bring about severe side effects in human cancer development.Besides,miR-129 has also been found to di rectly-suppress the expression of RUNX1 and mediate the transcriptional modulation by RUNX1.In this regard,we hypothesized that a regulatory network of the miR-21/RUNX1/YAP axis may be implicated in the progression of lung cancer.Therefore,the current study was conducted with the aim to verify the expected involvement of the miR-21/RUNX1/YAP axis in lung cancer,and to elucidate the underlying molecular mechanisms.Methods:(1)Bioinformatics methods were used to predict the expression of miR-21in lung cancer and its related regulatory pathways.(2)The immunosuppressive effect and mechanism of miR-21/RUNX1/YAP axis on mouse lung cancer cells.The proportions of MDSCs,T helper cells(Th),and cytotoxic T lymphocytes(CTL)were evaluated by flow cytometric analyses.T cell proliferation assay was performed in CD4~+or CD8~+T cells cocultured with MDSCs.MDSCs apoptosis was examined by flow cytometric analysis.The level of miR-21 was determined by RT-qPCR.The levels of IL-10,TGF-?,and GM-CSF were determined by ELISA.miR-21 targeting RUNX1and RUNX1 interaction with YAP were evaluated by RIP,dual-luciferase reporter gene,and Ch IP assays.(3)The immunosuppressive effect and mechanism of miR-21/RUNX1/YAP axis on the growth of nude mice tumor transplantation.Animal models were established and experimental groups were carried out.The experiment was divided into eight groups:miR-21antagomir negative control(NC)?miR-21antagomir?miR-21 antagomir NC+sh-NC?miR-21 antagomir+sh-NC?miR-21antagomir+sh-RUNX1?miR-21 antagomir NC+oe-NC?miR-21antagomir+oe-NC?miR-21 antagomir+oe-YAP.Cell proliferation and MDSCs sorting of peripheral blood was detected by flow cytometry.The proportion of MDSCs?Th and CTL in peripheral blood and tumor tissues of mice were detected by flow cytometry.The inhibitory effects of MDSCs on TH and CTL were detected by T cell proliferation analysis.The expression of RUNX1 and YAP in tumor tissues were measured by immunohistochemistry.The expression of IL-10,TGF-?and GM-CSF were detected by ELISA.The expression of ARG-1 and i NOS were analyzed by RT-qPCR.The expression of RUNX1,YAP,ARG-1 and i NOS were measured by Western Blot analysis.(4)Statistical analyses:Statistical analyses were conducted using the SPSS21.0 statistical software(IBM,Armonk,NY,USA).Measurement data were summarized by mean±standard deviation.When conforming to normal distribution and homogeneity,data between two unpaired groups were compared by unpaired t-test.Measurement data among multiple groups were compared by one-way analysis of variance(ANOVA)with Tukey's post hoc test.Data comparison among multiple groups at different time points were conducted using repeated measurement ANOVA with Bonferroni's post hoc test.Pearson's correlation analysis was used to analyze the relationship between indicators.Measurement data were represented by examples,and verified using the Chi square test.A value of P<0.05 indicated statistical significance.Results:(1)Bioinformatics methods were used to predict the expression of miR-21in lung cancer and its related regulatory pathways.(1)The R language was used for differential analysis of GEO database chip GSE63805,and 19 miRNAs with significant differences were obtained,among which the expression difference of miR-21 was the top(P<0.05).(2)In order to determine the expression patterns of miR-21 in lung cancer,a box line map was plotted by extracting the expression data of miR-21 of dataset GSE63805,which revealed that miR-21 was over-expressed in the lung cancer samples(P<0.05).(3)The downstream target gene RUNX1 of miR-21 was screened.(4)The downstream target gene YAP1(YAP)of RUNX1 was screened.(5)Through further GEPIA analysis of lung adenocarcinoma and lung squamous cell carcinoma data in the TCGA database,RUNX1 and YAP1 were found to be negatively-correlated.(2)The immunosuppressive effect and mechanism of miR-21/RUNX1/YAP axis in mouse lung cancer cells.(1)The expression of miR-21 was increased in mouse lung cancer cells,miR-21 modulated the immunosuppression of MDSCs.(2)MiR-21 was negatively-correlated with RUNX1.(3)RUNX1 was negatively-correlated with YAP.(4)MiR-21 regulated YAP by regulating the expression of RUNX1,mediated the immunosuppressive ability of MDSCs in lung cancer.(5)The miR-21/RUNX1/YAP axis promoted the cycle and apoptosis of MDSCs,inhibited the key immunosuppressive molecules.(3)The effect and mechanism of miR-21/RUNX1/YAP axis on the immunosuppressive ability of lung cancer xenografted tumor in vivo.(1)Low expression of miR-21 inhibited the immunosuppressive abilit y of MDSCs in lung cancer.(2)MiR?21 promoted the expression of YAP by inhibiting RUNX1.(3)MiR-21 regulated the immunosuppressive ability of MDSCs against lung cancer by promoting the expression of RUNX1-mediated YAP.(4)The effect of miR-21/RUNX1/YAP axis on the immunosuppressive ability of lung cancer xenografted tumor.MiR-21 promoted tumor develo pment by elevating the expression of RUNX1-mediated YAP.In summary,miR-21 inhibition by its antagomir reduced the proportion of MDSCs,increased the proportion of Th and CTL in peripheral blood and tumor tissues of Lewis lung-cancer-bearing mice,protected Th and CTL from the suppression of MDSCs,increased apoptosis of MDSCs,but reduced IL-10,TGF-?and GM-CSF levels in mouse serum.RUNX1 could transcriptionally inhibit the YAP expression,whereas miR-21 targeting RUNX1 led to elevated YAP expression levels.Mechanistic investigation showed that miR-21 maintained MDSCs accumulation in tumor microenvironment and promoted immunosuppressive ability of MDSCs in Lewis lung-cancer-bearing mice by down-regulating RUNX1 and up-regulating YAP.Conclusions:Collectively,our findings demonstrated that up-regulation of miR-21inhibits the expression of the downstream target RUNX1 in lung cancer.RUNX1 subsequently binds to the promoter region of the YAP gene to down regulate its expression.We conclude that miR-21 up-regulated the YAP expression by inhibiting the transcription factor RUNX1 to regulate the immunosuppressive ability of MDSCs against lung cancer.Our findings not only improve the understanding of how miR-21 modulates the immunosuppressive ability of MDSCs in lung cancer,but also offer a potential prognostic marker and a therapeutic target in the form of miR-21 silencing for lung cancer.Taken together,the study provides evidence that targeting miR-21in MDSCs may be developed as an immunotherapeutic approach to combat lung cancer development.