MicroRNA-29a inhibits collagen expression and induces apoptosis in human fetal scleral fibroblasts by targeting the Hsp47/Smad3 signaling pathway.

Abstract

Members of the microRNA-29 (miR-29) gene family have been implicated as suppressors of collagen in several human diseases. The present study aimed to explore the function of miR-29a in human fetal scleral fibroblasts (HFSFs) and to investigate potential mechanisms by which the molecule regulates cellular functioning. First, HFSFs were transfected with miR-29a mimic, miR-29a inhibitor, or their corresponding controls. Then, cell proliferation and apoptosis were assessed using a CCK-8 assay and flow cytometry, respectively. Further, using real-time PCR, western blotting, and immunofluorescence staining, levels of miR-29a, heat shock protein 47 (Hsp47), COL1A1, Smad3, P-Smad3, Bax, and Bcl-2 were investigated. Next, empty vectors and SERPINH1-overexpressing vectors were used to transfect HFSFs. Western blotting and flow cytometry were performed to assess changes in levels of HFSF protein expression and apoptosis, respectively. Results indicated that the miR-29a mimic significantly inhibited Hsp47, Smad3, P-Smad3, and COL1A1 expression. Conversely, the miR-29a inhibitor enhanced the expression of the same genes. Furthermore, miR-29a overexpression inhibited HFSFs proliferation and enhanced the rate of HFSFs apoptosis. Consistent with this finding, miR-29a overexpression led to the downregulation of Bcl-2 and upregulation of Bax. In contrast, miR-29a suppression led to the upregulation of Bcl-2 and downregulation of Bax expression and reduced the rate of apoptosis. Additional research revealed that overexpression of Hsp47 prevented HFSFs apoptosis and enhanced collagen production. Findings that miR-29a overexpression reduces collagen expression levels, slows proliferation, and promotes apoptosis in HFSFs highlight the key role of miR-29a in scleral remodeling. The effects of miR-29a on scleral remodeling might mediate by targeting Hsp47 and repressing the Smad3 pathway.