Cross-talk between miR-29c and transforming growth factor-β3 is mediated by an epigenetic mechanism in leiomyoma

Objective: To determine the expression of miR-29c and its target gene transforming growth factor-beta 3 (TGF-beta 3) in leiomyoma and the mechanisms of their reciprocal regulation. Design: Experimental study. Setting: Academic research laboratory. Patient(s): Women undergoing hysterectomy for leiomyoma. Intervention(s): Overexpression and underexpression of miR-29c; blockade of DNA methyltransferase 1 (DNMT1). Main Outcome Measure(s): The miR-29c and its target gene TGF-beta 3 in leiomyoma and the effects of TGF-beta 3 and blockade of DNMT1 on miR-29c expression. Result(s): Leiomyoma expressed significantly lower levels of miR-29c, but higher expression of TGF-beta 3 compared with matched myometrium. The expression of TGF-beta 3 and miR-29c were independent of race/ethnicity. Using 3' untranslated region luciferase reporter assay we confirmed that TGF-beta 3 is a direct target of miR-29c in leiomyoma smooth muscle cells (LSMCs). Gain-of-function of miR-29c in LSMCs inhibited the expression of TGF-beta 3 at protein and messenger RNA levels, whereas loss-of-function of miR-29c had the opposite effect. Treatment of LSMCs with TGF-beta 3 inhibited the expression of miR-29c, whereas it stimulated DNMT1 expression. Knockdown of DNMT1 through transfection with small interfering RNA significantly decreased the expression of TGF-beta 3, and induced miR-29c expression. Knockdown of DNMT1 also attenuated the inhibitory effect of TGF-beta 3 on miR-29c expression. Furthermore, we demonstrated that TGF-beta 3 increased the methylation level of miR-29c promoter in LSMCs. Conclusion(s): There is an inverse relationship in the expression of TGF-beta 3 and miR-29c in leiomyoma. The TGF-beta 3 is a direct target of miR-29c and inhibits the expression of miR-29c through an epigenetic mechanism. The cross-talk between miR-29c and TGF-b3 provides a feed forward mechanism of fibrosis in leiomyoma. ((C) 2019 by American Society for Reproductive Medicine.)