Effect and mechanism of miR-135a-5p/CXCL12/JAK-STAT axis on inflammatory response after myocardial infarction

To investigate the regulatory role of miR-135a-5p/CXCL12/JAK-STAT signaling axis in inflammatory response after myocardial infarction (MI).

With the construction of mouse model with MI by ligation of left descending coronary artery, modeling mice were subdivided into sh-NC group, sh-CXCL12 group, agomir-NC group, miR-135a-5p agomir group and miR-135a-5p-agomir+pcDNA-CXCL12 with intravenous injection of corresponding adenovirus, and no modeling was made for mice in the sham operation group. Simulation of MI in vivo was realized by hypoxia model in vitro, with the establishment of groups including mimic-NC group, miR-135a-5p mimic group, inhibitor-NC group, miR-135a-5p inhibitor group, sh-NC group, sh-CXCL12 group, oe-NC group, oe-CXCL12 group, mimic NC+oe-NC group, miR-135a-5p mimic+oe-NC group, and miR-135a-5p mimic+oe-CXCL12 group. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the level of miR-135a-5p, CXCL12, TNF-α, IL-1β and IL-6, and Western blot were further performed to detect the mRNA and protein expression of JAK2/p-JAK2 and STAT3/p-STAT3, respectively. Hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to evaluate MI in mice. Dual-Luciferase reporter assay was used to verify the targeting relationship between miR-135a-5p and CXCL12. Annexin V-fluorescein isothiocyanate/propidium iodide (V-FITC/PI) double staining analysis by flow cytometry was used to detect apoptosis.

After hypoxia of myocardial cell line H9c2 for 24 h, there were increased expression of CXCL12, decreased expression of miR-135a-5p, increased number of apoptotic cells, as well as upregulated levels of TNF-α, IL-1β and IL-6 (all p<0.05). Meanwhile, similar results were found in the myocardial tissues. Dual-Luciferase reporter assay indicated that miR-135a-5p could target the expression of CXCL12. Transfection of miR-135a-5p mimic or sh-CXCL12 could reduce the number of apoptotic myocardial cells and inhibit the level of TNF-α, IL-1β and IL-6 (all p<0.05). Furthermore, miR-135a-5p mimic or sh-CXCL12 could result in the suppressed expression of p-JAK2 and p-STAT3 (all p<0.05). Compared with miR-135a-5p mimic +DMSO group, the expression of JAK2 and STAT3 in miR-135a-5p mimic +RO8191 group had no significant change (p>0.05); the expression of p-JAK2 and p-STAT3 was increased (all p<0.05), suggesting that miR-135a-5p negatively regulated the expression of CXCL12 and inhibited the activation of JAK-STAT signaling pathway. In addition, for further verification, experiments carried out in sh-NC group, sh-CXCL12 group, agomir-NC group and miR-135a-5p agomir group found that sh-CXCL12 and miR-135a-5p agomir resulted in decreased area of MI decreased, the number of apoptotic cells, the expression of p-JAK2 and p-STAT3 (all p<0.05); while compared with miR-135a-5p-agomir group, miR-135a-5p-agomir+pcDNA-CXCL12 group showed increased area of MI decreased, the number of apoptotic cells, the expression of p-JAK2 and p-STAT3 (all p<0.05).

Inhibition of miR-135a-5p/CXCL12/JAK-STAT signaling axis can reduce inflammatory reaction and apoptosis after MI, and hence contribute to the improvement of the degree of myocardial injury.