MiR-34a Inhibits The Apoptosis Of MDSCs By Regulating The Expression Of N-myc

MicroRNAs (miRNAs) are endogenous ～22nt non-coding RNAs which have a regulatory function in eukaryotes. Recent studies have shown that miRNAs are involved in the regulation of many biological processes, including cell development, ontogenesis, organogenesis, hematopoiesis, cell proliferation and apoptosis, fat metabolism, bacteria and virus infection and body defense, development of inflammation and so on. Much effort was investigated in exploring the function of microRNAs in immune system. Previous studies have shown that microRNAs played an important role in the differentiation and development of T cells, B cells, DC, NK cell, and thus participated in the body’s innate and acquired immune responses. Rafael Casellas’s lab has reported that miR-34a exhibited different expression in various types of immune cells. This implies that miR-34a might play a vital role in the differentiation and development of lymphocytes. To explore the function of miR-34a in immune system, we first established miR-34a chimeric mice and transgenic mice models and investigated the role of miR-34a in lymphocytes development using these two mice models. First, we constructed miR-34a over-expressed plasmid pMDH1-PGK-GFP-miR-34a and the expression of miR-34a was confirmed by qPCR and FACS analysis. Then, we injected retrovirus infected bone marrow hematopoietic stem cell into irradiated mice to establish the miR-34a chimeric mice. The successful establishment of miR-34a chimeric mice was verified by FACS analysis and qPCR. On the other hand, we linearized the plasmid of pMDH1-PGK-GFP-miR-34a by digestion enzyme Hind III and injected this linearized plasmid into embryonic stem cell to establish miR-34a transgenic mice. FACS analysis showed that the percentage of B cells was significantly reduced both in BM and in spleen, in miR-34a overexpressed chimeric mice and transgenic mice. This was consistent with Baltimore’s study, which further confirmed the successful establishment of mice model. Besides B cells reduction, we also found that MDSCs (myeloid-derived suppressor cells) was increased no matter in chimeric or transgenic mice while miR-34a was overexpressed. The absolute number of MDSCs was elevated in the transgenic mice model. But there was no significant difference in BrdU positive cells proportion in BM and spleen by BrdU staining between WT and miR-34a TG mice, which suggested that miR-34a did not affect the self-renewal or proliferation of MDSCs. Analysis of early progenitor cells revealed that there was no prominent difference in the percentage of MDPs and GMPs, between miR-34a TG mice and WT mice. However, Annexin V staining experiment suggested that the apoptosis of MDSCs from transgenic mice decreased significantly, compared to that in WT mice, suggesting that the increasing of percentage and absolute number of miR-34a MDSCs might be due to the apoptosis inhibition. Subsequently, to further investigate the mechanism of miR-34a in apoptosis inhibition of MDSCs, we used the miRWalk software to predict the probable target gene of miR-34a and combined with luciferase assay, RT-PCR and Western-blot to further consider the miR-34a target genes in MDSCs. All together suggest that miR-34a might regulate the apoptosis of MDSCs by down-regulating the expression of N-myc et al. genes.In order to further study whether miR-34a affect the biological function of MDSCs, we first purified MDSCs by FACS sorting, then evaluated the cytokine expression change of miR-34a overexpressed MDSCs by real time PCR. The results showed that miR-34a overexpressed MDSCs from spleen exhibited higher amounts of TNF, iNOS and IL-17, accompanied with decreased expression of Arg and TGF-p. However, it showed an increased expression of TNF, iNOS, IL-6, IL-10 and IL-12 in BM MDSCs from miR-34a TG mice compared to that in WT mice. To verify whether this cytokine expression profile change will have an influence on the function of MDSCs in tumor growth, we established LLC mice model in WT and miR-34a TG mice respectively and found no difference in tumor growth in these two groups. Considering that miR-34a was also overexpressed in other cells and to exclude interference of other cells, we purified MDSCs from miR-34a TG mice and injected into mice through the ophthalmic vein before tumor injection. Comparing to WT MDSCs infusion group, miR-34a MDSCs did not significantly alter the tumor growth in vivo. These results indicated that although miR-34a changed the cytokine expression profile of MDSCs, it had no influence on the tumor growth.Taken together, we have successfully established the miR-34a over-expressed chimeric mice and transgenic mice models. Our study revealed that miR-34a inhibited MDSCs apoptosis by regulating the expression of N-myc. Despite the altered cytokine expression profile after miR-34a overexpression, miR-34a had no effect on the function of MDSCs in tumor growth. On the one hand, this series of studies revealed the effect of miR-34a in the differentiation and apoptosis of MDSCs. On the other hand, it also provided a new basis for microRNAs in lymphocyte biological process.