Downregulated miR-129-5p expression inhibits rat pulmonary fibrosis by upregulating STAT1 gene expression in macrophages

Objective: This study investigated the mechanism by which microRNA-129-5p (miR-129-5p) in macrophages affects pulmonary fibrosis in rats by regulating the expression of the signal transducer and activator of transcription 1 (STAT1) gene. Methods: After the establishment of a pulmonary fibrosis rat model, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the expression of miR-129-5p in the sham group and model group. The binding sites between miR-129-5p and STAT1 were predicted online and verified by using a dual luciferase reporter system. qRT-PCR and Western blot analyses were used to test the effect of miR-129-5p on STAT1 gene expression. M2 macrophages were isolated and induced, and exosomes were extracted. Cell proliferation was detected by EdU. Furthermore, qRT-PCR was performed to detect the expression of STAT1, collagen type I A2 (COL1A2), collagen type III A1 (COL3A1), fibronectin, and alpha-SMA in cells and tissues followed by the detection of CD9, CD63, CD81, CD31 and STAT1 protein expression using a Western blot analysis. The pulmonary fibrosis area was detected by Masson staining followed by the immunohistochemical detection of alpha-smooth muscle actin (alpha-SMA) and type I collagen (COL-I) expression in pulmonary fibroblasts. Results: Compared with the sham group, the expression level of miR-129-5p in the model group was significantly increased (P < 0.05). miR-129-5p was observed to negatively regulate the expression of STAT1 (P < 0.05). The in vitro cell transfection experiments showed that after inhibiting the expression of miR-129-5p, the expression of STAT1 was increased, and the proliferation of fibroblasts and pulmonary fibrosis were inhibited (all P < 0.05). Furthermore, compared with the fibroblasts without coculture, the proliferation of the fibroblasts cocultured with M2 macrophage-secreted exosomes was clearly increased, and the expression levels of COL1A2, COL3A1, fibronectin and alpha-SMA were significantly increased (all P < 0.05). Compared with the mimic NC-exo group, the miR-129-5p-exo group had significantly increased proliferation of fibroblasts, decreased expression of STAT1, and significantly increased expression of COL1A2, COL3A1, fibronectin and alpha-SMA, and M2 macrophagesecreted exosomes could carry miR-129-5p to fibroblasts. Furthermore, the in vivo experiment confirmed that the exosomes of M2 macrophages could carry miR-129-5p, which could regulate M2 macrophages with pulmonary fibrosis in vivo. Conclusion: M2 macrophages can carry miR-129-5p to pulmonary interstitial fibroblasts and inhibit STAT1 gene expression, which may lead to the proliferation of fibroblasts and promote pulmonary fibrosis. The downregulation of miR-129-5p can significantly promote STAT1 gene expression in macrophages to inhibit pulmonary fibrosis in rats.

Automated summary

The study revealed that miR-129-5p promotes pulmonary fibrosis in rats by inhibiting the expression of STAT1 gene, which can be carried by M2 macrophages to inhibit the expression of STAT1 and suppress the development of pulmonary fibrosis.