The Role Of MiR-16in The Selective Activation Of M2Macrophages

Objective:Our previous study applied the microRNA microarray analysis to find the differences between microRNAs expressed in HCC. MiR-16was found down-regulated in HCC. It was also found that miR-16was highly expressed in monocytes. But it was down-regulated in M2macrophages induced by IL-4in vitro. Suggesting that miR-16may be related with the differentiation of M2macrophages. To explore the possible regulatory role of miR-16to the differentiation of M2macrophages, the project intends to explore the effects on the phenotype and functional differentiation of macrophages by increasing the expression level of miR-16through in vitro cell culture. This study reveals the key microRNA molecule which regulate the differentiation of macrophages and provides a target to block the activation of macrophages to the M2type. The study also provides new ideas to regulate the anti-tumor immunity.Methods:1. In order to clarify the expression of miR-16in the process of differentiation of M2macrophages, The45ng/ml of IL-4was added into the THP-1cell line. Then alternative activation of macrophages (M2or TAM2) were induced by IL-4. The secretion levels of IL-10were detected by ELISA before and after the induction of macrophage. Real-time PCR was used to detect the expression of miR-16.2. In order to establish the over-expression cell lines we first used a lentiviral vector to increase expression of miR-16. Secondly, we chose the THP-1cell line, which was induced into M2macrophages in vitro, adding an auxiliary reagent which named polybreen and LV-hsa-mir-16-1virus to enhance infection. The cells were observed after48hours in culture condition. Thirdly, they were replaced with fresh medium and added puromycin to enhance the efficiency of infection. After3or4days, the fluorescence was observed of the infected cells.5days after infection, downstream experiments were done using flow cytometry and Real-time PCR to detect the infection efficiency and evaluate the over-expression of miR-16.3. We used the following methods to find the effects on the phenotype and functional differentiation of macrophages by increasing the expression level of miR-16:The cell cycles of M2macrophages after infection were detected with PI staining. The secretion levels of IL-10were detected by ELISA between the over-expression group and the control group in the supernatant of M2macrophages. The intracellular expression of IL-10was detected by flow cytometry simultaneously. The expression levels of arginine protease-1(arginase-1, Argl) between each group were also detected by Western blot.Results:1. The secretion levels of IL-10in the supernatant of THP-1cells were gradually increased after induced by IL-4. The secretion level of IL-10increased to peak after6or7days, suggesting the macrophages activated with IL-4polarized to M2successfully. It was found that the relative expression levels of miR-16declined when the M2macrophages were activated with IL-4.2. The GFP expression rate of M2macrophages detected by flow cytometry increased to97.7%after lentiviral infection and puromycin selection. Statistical results showed that the gene expression levels of miR-16in the over-expression group was7.644±0.110times more than the negative control group (P<0.05).3. After M2macrophages were infected by lentivirus, the proportion of cells in G1/G0phase was54.67%in the negative control group, while it increased to62.25%in the over-expression group. The proportion of cells in S phase in the over-expression group was36.31%which is lower than it in the control group (42.12%). It revealed that the cell cycle was arrested in G1phase after over-expression(P<0.05).4. The ELISA assay indicated that the secretion level of IL-10in the supernatant of M2macrophages was72.15±0.158pg/ml in the over-expression group, which was lower compared with the control group (103.47±0.136pg/ml)(P<0.05). The differences of IL-10staining intracellular of M2macrophages between the over-expression group and the negative control group detected by flow cytometry displayed that the mean fluorescence intensity of IL-10(Gm) was3.47in the over-expression group. It was lower than the control group (Gm=12.40) lower (P<0.05). The expression levels of arginine protease-1(arginase-1, Argl) of M2macrophages were detected by Western blot assay. The relative expression values of Argl are the ratios of gray values of Argl/p-actin. They are respectively as follows,0.4565±3.14%in negative control group and0.1158±2.83%in over-expression group. The differences between the negative control group and over-expression group were statistically significant(P<0.05).Conclusions:The relative expression levels of miR-16declined when the monocytes activated with IL-4were polarized to M2. It made clear that the cell cycle was arrested in G1phase after over-expression with lentiviral infection. And the secretion levels of IL-10in the supernatant and intracellular were both declined. The relative expression values of Argl were also decreased. It demonstrated that the phenotype and function of M2macrophages were affected by miR-16. It may block the differentiation of M2macrophages to a certain extent.