MicroRNA-323a-3p Promotes Pressure Overload-Induced Cardiac Fibrosis by Targeting TIMP3

Cardiac fibrosis is a major cause of diverse cardiovascular diseases. MicroRNAs have recently been proven a novel class of regulators of cardiac fibrosis. In this study, we sought to investigate the role of miR-323a-3p and its mechanisms in regulating cardiac fibrosis.

The transverse aortic constriction (TAC) mice model was induced and neonatal cardiac fibroblasts (CFs) were cultured. MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay was used to detect the cell viability. Echocardiography was used to evaluate cardiac function. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site. Real-time PCR and Western blot were used to evaluate the level of mRNA and protein.

MiR-323a-3p was found up-regulated in myocardial tissues subjected to TAC and in CFs cultured with Angiotensin Ⅱ (Ang Ⅱ). Overexpression of miR-323a-3p significantly increased the mRNA levels of collagen Ⅰ, collagen Ⅲ, MMP2 and MMP9, while inhibition of miR-323a-3p prevented the proliferation, collagen production and the protein level of transforming growth factor (TGF-β) in rat neonatal CFs. Strikingly, injection of antagomiR-323a-3p elevated cardiac function and inhibited the expression of TGF-β in the TAC mice. TIMP3 was a direct target of miR-323a-3p, as the overexpression of miR-323a-3p decreased the protein and mRNA levels of TIMP3. In the CFs with pre-treatment of Ang Ⅱ, siRNA-TIMP abolished the effects of AMO-323a-3p on the inhibition of the proliferation of CFs, the down-regulation of collagen Ⅰ and collagen Ⅲ, and the expression of TGF-β.

Our findings provide evidence that miR-323a-3p promotes cardiac fibrosis via miR-323a-3p-TIMP3-TGF-β pathway. miR-323a-3p may be a new marker for cardiac fibrosis progression and that inhibition of miR-323a-3p may be a promising therapeutic target for the treatment of cardiac fibrosis.