MiR-211/STAT5A Signaling Modulates Migration of Mesenchymal Stem Cells to Improve its Therapeutic Efficacy

Abstract

Our previous study showed that the therapeutic effects of mesenchymal stem cells (MSCs) transplantation were improved by enhancing migration. MicroRNA-211 (miR-211) can modulate the migratory properties of some cell types by mechanisms that are not fully understood. This study was designed to investigate a possible role for miR-211 in MSC migration, and whether genetic manipulation of miR-211 in MSCs could be used to enhance its beneficial effects of cell transplantation. Transwell assays confirmed that MSCs migration of was significantly impaired by miR-211 knockdown but enhanced by miR-211 overexpression. MiR-211 overexpressing MSCs also exhibited significantly increased cell engraftment in the peri-infarct areas of female rat hearts 2 days after intravenous transplantation of male MSCs as shown by GFP tracking and SYR gene quantification. This conferred a significant decrease in infarct size and improved cardiac performance. By using a loss or gain of gene function approach, we demonstrated that miR-211 targeted STAT5A to modulate MSCs migration, possibly by interacting with MAPK signaling. Furthermore, the beneficial effects of miR-211 overexpression in MSCs were abolished by simultaneous overexpression of STAT5A whereas the negative effects of miR-211 silencing on MSC migration were rescued by simultaneous downregulation of STAT5A. Finally, using ChIP-PCR and luciferase assays, we provide novel evidence that STAT3 can directly bind to promoter elements that activate miR-211 expression. STAT3/miR-211/STAT5A signaling plays a key role in MSCs migration. Intravenous infusion of genetically modified miR-211 overexpressing MSCs conveys enhanced protection from adverse post-MI remodeling compared with unmodified MSCs. Stem Cells 2016;34:1846-1858.

Keywords: Mesenchymal stem cells; MicroRNA-211; Migration; Myocardial infarction; Retention; STAT5A.

Figure 1

MiR-211 modulated mesenchymal stem cell (MSC) migration. Migrated MSCs assessed by transwell assay (A, scale bar=100 μm), and cell number was quantified for each group (B, n>3 per group). Fluorescent image showed green fluorescent protein (GFP) positive MSCs in the peri-infarct area (C, scale bar=50 μm), and number of GFP positive cells was quantified (D, n≥3 heart samples per group). TnT and Hoechst costaining were performed as shown in red and purple, respectively. SRY gene copies were quantified by real time polymerase chain reaction, the migrated MSCs cells were determined as the absolute number of SRY gene copies per mg myocardium tissue (E, n=4 per group). MSCs^miR-211-over was abbreviated as over, MSCs^{miR-211-shRNA} as shRNA, and their controls, MSCs^{miR-211-control} and MSCs^{miR-211-scramble}, are abbreviated as CV and NC, respectively. Abbreviations: GFP, green fluorescent protein; TnT, Troponin T.

Figure 2

MiR-211 overexpressing mesenchymal stem cells (MSCs) improve post-myocardial infarction (MI) remodeling. Echocardiography examination was performed for each group rats at 28 days after MI (A), and fractional shortening was quantified (B). Left ventricle (LV) pressure recording was also obtained, and LV-end-diastolic pressure (C), and ±dP/dt_max (D, E) then measured. Masson trichrome staining was performed (F) to evaluate the infarct size (G). Immunofluorescence microscopy with antibody targeting CD31 was performed, and CD31 positive structure was quantified in the peri-infarct area using the heart tissue obtained at day 28 after infarction, where troponin I and Hoechst were costained for identifying myocardium and nuclei (H, I, scale bar=75 μm). Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) staining was also done and positive cells quantified (K) in the peri-infarct area using the heart tissue obtained at day 3 after infarction (J, scale bar=100 μm). All the abbreviations are the same as in Figure 1. Abbreviations: HRF, high resolution field; LVFS, left ventricular fractional shortening; PBS, phosphate buffer solution; shRNA, short hairpin ribonucleic acid; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 3

Identification of STAT5A as a target of miR-211. Schematic of the miR-211 putative binding site alignment with rat 3′-UTR mRNA of STAT5A, the mutated STAT5A 3′-UTR binding site for miR-211 are illustrated with the mutated nucleotides underlined (A). Using Hela cells, dual luciferase report assay was performed. Renilla and Firefly Luciferase activities were measured 48 hours after transfection with Renilla used as baseline control (B, n=3). Using real time polymerase chain reaction, the effect of miR-211 on mRNA level of STAT5A was tested in mesenchymal stem cells after infected with miR-211-shRNA or miR-211-over (in grey bars and their respective controls shown in black bars, respectively. C). Western blot was also done (representative bands in D) to quantify the effect of miR-211 on STAT5A protein expression in MSCs (E, the layout is the same as in (C), n=3). All the abbreviations are the same as above. Abbreviations: shRNA, short hairpin ribonucleic acid; UTR, untranslated region; WT, wild type.

Figure 4

Role of STAT5A in miR-211-mediated mesenchymal stem cell (MSC) migration. Transwell assay was performed for MSCs^{miR-211 over}, MSCs infected with STAT5 over alone or MSCs simultaneously infected with miR-211-over and STAT5A-over (double over), representative image was shown in (A) (scale bar=200 μm) and cell number quantified in (B) (n=3). MSCs^{miR-211-shRNA}, MSCs infected with STAT5 shRNA, or MSCs simultaneously infected with miR-211-shRNA and STAT5A-shRNA (double shRNA) were used for transwell assay, with representative image shown in (C) (scale bar=200 μm) and quantified in (D) (n=3). MSCs obtained from male rats that were infected with miR-211-over alone or Double over, miR-211-shRNA alone or double shRNA were delivered in female MI rats, the number of engrafted MSCs was measured by either counting GFP positive cells in the peri-infarct area (representative image in (E) (scale bar=50 μm) and quantified in (F), n≥3 per group) or quantifying the copy of SRY gene per mg heart tissue (G, n≥3 per group). Western blot showed phosphorylation levels of ERK1/2, p38MAPK and JNK, and MMP9 protein expression respectively for MSCs when infected with miR-211-over (abbreviated as over, CV for empty vector, and WT as control) or miR-211 shRNA (denoted as shRNA, NC for miR-211 scramble and WT as control) with representative blots shown in (H) where β-Actin was used as loading controls (data repeated for three times). Abbreviations: GFP, green fluorescent protein; shRNA, short hairpin ribonucleic acid; WT, wild type.

Figure 5

MiR-211 overexpressing mesenchymal stem cells (MSCs) improved post-myocardial infarction (MI) remodeling via STAT5A. Male MSCs infected with different lentivirus as described in Figure 4E were delivered for female MI rats, another group MI rats received PBS served as controls. At 28 days after MI, before the rat were sacrificed, echocardiography was performed for each group rats (A, n≥5) and fractional shortening quantified (B). With LV pressure recording, LVEDP (C),+dP/dt_max (D) and −dP/dt_max (E) were analyzed for each group rats. After rats were sacrificed, heart tissue slices were obtained for Masson’s trichrome staining was then performed (F) and the infarct size was quantified (G). Abbreviations: LVEDP, left ventricular end-diastolic pressure; PBS, phosphate buffer solution; shRNA, short hairpin ribonucleic acid.

Figure 6

STAT3 directly mediated hypoxia-induced miR-211 expression. MiR-211 expression levels were analyzed by polymerase chain reaction (PCR) for both normoxia-treated MSCs (N-MSCs, in black bars) and HP-treated MSCs (HP-MSCs, in white bars) when they were infected with STAT3-over (over, CV as control, shown in A) or STAT3-shRNA (shRNA, NC as control, shown in B), respectively (three separate experiments). The promoter region of miR-211 was divided into seven consecutive segments (C), and ChIP with STAT3 specific antibody was carried out and the pull-down precipitates were analyzed with quantitative PCR, PCR products were run in the ethidium bromide agarose gel, and the image was captured with the grey-scale inverted for better illustration (D), the relative fold of enrichment was normalized to the input (E). Luciferase assay showed that STAT3 can positively regulate the transcription activities specific for the different promoter regions 3, 4, and 5 of miR-211 (F). The experiments were repeated for three times. **Denotes p less than .01 vs. groups that were not infected with vectors containing both STAT3 and specific promoter regions of 3–5 of miR-211. Abbreviations: HP, hypoxic preconditioning; MSCs, mesenchymal stem cells; shRNA, short hairpin ribonucleic acid.

Figure 7

Low miR-211 expression impairs migration of aged hMSCs ability. Downregulated miR-211 expression levels were detected by quantitative real time polymerase chain reaction in aged hMSCs (hMSCs^old, n=3) compared with young hMSCs (hMSCs^young, n=3. A). Migration ability of hMSCs was assessed by transwell assay when both young and old hMSCs were infected with miR-211-over (denoted as hMSCs^{young+miR-211} and hMSCs^old+miR-211, respectively) compared with hMSCs^young and hMSCs^old controls, respectively, with representative images shown in (B) (scale bar=200 μm) and the fold changes in number of migrated cells (mean value from three wells and total number of five fields counted for each well, C). Scale bar=200 μm. Abbreviation: hMSCs, human mesenchymal stem cells.