Differentially expressed miRNAs in microglia after SHED-EXs treatment
Array data (Fig. 1A) showed that 27 miRNAs were upregulated and 39 miRNAs were downregulated after SHED-EXs was added to LPS-stimulated BV-2 cells, for which a two-fold change or more was used as the cutoff criterion for consideration of significantly altered miRNAs. We selected the most differentially expressed 10 miRNAs (6 up-regulated and 4 down-regulated miRNAs) and confirmed their expression by Real-time PCR (Fig. 1B, C). We found that changes in miR-330-5p expression were most significantly different. We further quantitated miR-330-5p expression after LPS stimulation of BV-2 cells and found that miR-330-5p expression was significantly downregulated (Fig. 1D). After addition of the EXs inhibitor, GW4869, no significant difference in miR-330-5p expression was shown compared with that of the LPS stimulation group (Fig. 1E). In summary, LPS stimulation significantly downregulated miR-330-5p in microglia. Furthermore, SHED-EXs delivery rescued miR-330-5p expression and may play a role as a key regulator in microglial function.
miR-330-5p inhibits microglial M1 polarization and promotes M2 polarization
In order to elucidate the specific function of miR-330-5p in microglia, miR-330-5p was first upregulated in BV-2 cells. Transfection efficiency was measured 48 h after transfection with miR-330-5p mimics (Fig. 2A). LPS stimulation of BV-2 cells increased the secretions of the pro-inflammatory factors IL-6 and TNF-α while miR-330-5p overexpression significantly reduced the secretions of these two cytokines. In addition, LPS stimulation decreased the secretion of the anti-inflammatory factor IL-10 while miR-330-5p overexpression significantly rescued the secretion (Fig. 2B). Furthermore, Griess assay was used to examine the effects of miR-330-5p overexpression on nitrite concentrations. LPS stimulation significantly increased nitrite concentrations while miR-330-5p overexpression significantly decreased these levels (Fig. 2C). Next, flow cytometry and immunofluorescence were used to determine changes in M1- and M2-polarization markers in BV-cells. Results showed that the expression of the M1 polarization marker CD68 was significantly increased while the expression of the M2 polarization marker CD206 was significantly decreased in BV-2 cells after LPS stimulation. In contrast, transfection with miR-330-5p mimics significantly downregulated CD68 expression and significantly upregulated CD206 expression (Fig. 2D–G). Real-time PCR results showed that the expression of the M1 polarization markers—CD11b, CD86, CD16, and MHCII—were significantly increased, while the expression levels of the M2 polarization markers, IL-10 and ARGINASE1, were significantly decreased after BV-2 cells were stimulated with LPS. miR-330-5p overexpression significantly inhibited M1 polarization marker expression and promoted M2 polarization marker expression (Fig. 2H, I). Collectively, miR-330-5p inhibited M1 polarization and promoted M2 polarization. Appendix File 1 shows the effects of miR-330-5p inhibitors on BV-2 polarization.
miR-330-5p targets Ehmt2 to promote CXCL14 transcription through H3K9me2 in the regulation of microglial polarization
Next, we explored the downstream mechanism of miR-330-5p regulation. Three databases (Targetscan, microRNA.ORG, and miRDB) were used for target gene prediction of miR-330-5p and overlapping data from these three databases were obtained (Fig. 3A, Appendix File 2). From these data, we selected Ehmt2—which is associated with microglia development as a candidate target gene. Real-time PCR was used to measure changes in Ehmt2 expression after miR-330-5p was upregulated. Results showed that miR-330-5p significantly inhibited Ehmt2 expression (Fig. 3B). Western blotting was used to validate the effects of miR-330-5p upregulation on Ehmt2 protein levels. Results showed that Ehmt2 expression was decreased when miR-330-5p was upregulated (Fig. 3C, D). A luciferase reporter plasmid containing either wild-type or mutant Ehmt2 was constructed (Fig. 3E) according to its predicted binding site and was co-transfected with either miR-330-5p mimics or miR-330-5p inhibitors into 293T cells. The luciferase reporter assay was used to determine the relationship between miR-330-5p and Ehmt2 (Fig. 3F).
Ehmt2 is a major methyltransferase that catalyzes mono-methylation and di-methylation of H3K9. We found that miR-330-5p overexpression induced H3K9me2 downregulation (Fig. 3G). It was reported that H3K9me2 inhibits the transcription of CXCL14, a key factor in macrophage polarization. Therefore, we further measured CXCL14 expression after transfection with miR-330-5p mimics. Real-time PCR results showed that miR-330-5p overexpression increased CXCL14 transcription (Fig. 3H). ChIP further showed that mir-330-5p inhibited the enrichment of Ehmt2 and H3K9me2 at the promoter region of CXCL14 (Fig. 3I, J).
Ehmt2 rescues the inhibition of M1 polarization and promotion of M2 polarization of microglia mediated by miR-330-5p
We stably overexpressed Ehmt2 in BV-cells, stimulated them with LPS, and transfected them with miR-330-5p mimics before measuring changes in the levels of the pro-inflammatory factors, IL-6 and TNF-α, and the anti-inflammatory factor, IL-10. Results showed that Ehmt2 reversed miR-330-5p-induced inhibition of pro-inflammatory factors, while exerting opposite effects on anti-inflammatory factors (Fig. 4A, B). Ehmt2 restored miR-330-5p-induced decreases in nitrite concentrations (Fig. 4C). Flow cytometry, immunofluorescence, and Real-time PCR showed that Ehmt2 reversed miR-330-5p-induced inhibition of M1 polarization markers and miR-330-5p-induced promotion of M2 polarization markers in BV-2 cells (Fig. 4D–I).
miR-330-5p affects microglial polarization to ameliorate traumatic brain injury in rats
In order to determine whether SHED-EXs miR-330-5p has any in vivo effects, we established a rat model of TBI and used SHED-EXs and miR-330-5p for local-injection treatments at the wound site (Fig. 5A). Motor function tests were conducted in TBI rats at 48 h, one week, two weeks, and three weeks after injury. The BBB scoring showed significant motor dysfunction in all groups at 48 h after TBI, except for the sham group. Motor functions in the SHED-EXs group and miR-330-5p-mimics group were significantly improved after one week (Fig. 5B). Sectioning of brain tissues and hematoxylin and eosin (H&E) staining were carried out in the different groups of rats at two weeks after TBI. Results showed significant tissue defects in brain tissues from the TBI and DMEM groups, whereas significant recovery in brain tissue injuries were seen in the SHED-EXs group and miR-330-5p mimics group (Fig. 5C). Inflammation status was examined in the damaged brain tissues from different groups after 48 h of treatment. Results showed that the expression of the pro-inflammatory factors IL-6 and TNF-α were significantly lower in the SHED-EXs group and miR-330-5p mimics group compared with those in the TBI group and DMEM group, whereas the expression of the anti-inflammatory factor IL-10 was significantly increased (Fig. 5D, E).
Immunofluorescent staining was performed on brain tissue sections from the different groups. Results showed that the expression of the M1 polarization marker CD68 was significantly decreased in the SHED-EXs and miR-330-5p mimics groups, while the expression of the M2 polarization factor CD206 was significantly increased compared with those in the TBI group and DMEM group (Fig. 6).
Taken together, our present study found that miR-330-5p from SHED-EXs ameliorated functional impairment caused by TBI in rats. From a mechanistic perspective, TBI decreased miR-330-5p expression in microglia, and SHED-EXs delivery of miR-330-5p promoted M2 polarization and inhibited M1 polarization, thus reducing neuro-inflammation and promoting tissue repair through the downstream Ehmt2-H3K9me2-CXCL14 signaling axis (Fig. 7).