Down-Regulation of miR-29b Is Essential for the Pathogenesis of Uterine Fibroids.

Abstract

Uterine leiomyomas (ULs) are the most common neoplasm in women of reproductive age with an accumulative incidence of over 70%. They are believed to arise from the myometrium, but the etiology of this tumor remains unknown. The most notable characteristic of UL is the excess extracellular matrix (ECM), composed mainly of collagen Types I and III. It has been repeatedly reported that many collagen genes (COL1A1, 1A2, 3A1, 5A1, 5A2, and 7A1) are up-regulated in ULs. Since the production of ECM protein is one of the factors that contribute to the tumor growth, it is important to understand the mechanism of collagen over-production in ULs. In addition to collagens, mRNAs of many genes are differentially expressed in the myometrium and ULs. Recent gene expression profiling studies have revealed that many microRNAs (miRNAs) are also aberrantly expressed in ULs. Specifically, miR-29b is consistently found to be down-regulated in ULs in comparison to normal myometrium. Down-regulation of miR-29 has been correlated with an over-expression of collagen in the liver, lung, kidney and cardiac fibrosis. In vitro studies have demonstrated that miR-29b can down-regulate multiple collagen genes including COL1A1, 1A2, 5A1, and 5A2. Moreover, COL3A1 was ranked top among ~1000 potential targets of miR-29b in a target prediction algorism (miSVR) based on the Support Vector Regression model. These observations suggest that the down-regulation of miR-29b may play a role in the overproduction of ECM in ULs. To investigate the role of miR-29b in the pathogenesis of UL, we first established stable human leiomyoma cell lines that restored miR-29b expression levels to normal by lentiviral infection. The miR-29b and control cell lines were then compared in cell proliferation and the expression of potential miR-29b targets including COL1A1 and 3A1. While the growth rate was not affected by an up-regulation of miR-29b, genes for ECM protein including the collagens were significantly down-regulated. It is well known that myometrial and UL cells lose their original characteristics in vitro. Therefore, we further examined the effect of miR-29b expression levels on tumor growth in vivo by utilizing the xenograft model. When the miR-29b level was restored to the level of myometrium, UL cells did not form a solid tumor. Instead, the UL cells grew flat on the surface of the host mouse kidney. Histological analysis indicated that an up-regulation of miR-29b inhibited collagen accumulation. The experiment was repeated with primary human UL cells, and it confirmed the result of the cell line experiment. Thus, the over-production of ECM in UL is due to the down-regulation of miR-29b, which targets mRNAs of multiple collagens including two major collagen genes, COL1A1 and 3A1. Reversely, we generated myometrial cell lines in which miR-29b was permanently knocked down. While the knock-down of miR-29b increased the levels of collagen in vitro, the down-regulation of miR-29b itself was not sufficient to make myometrial cells tumorigenic in vivo. When cell lines were grafted into mice, neither the mir-29b knock-down nor the control cell lines formed tumors. In conclusion, down-regulation of miR-29b is essential for the formation of ULs, but it is not sufficient to transform myometrium into ULs. Administration of miR-29 mimics may prove to be an effective treatment for symptomatic ULs by reducing the tumor volume. This research was supported by NIH/NICHD Grant R01 HD064402.