MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease
Seung-Ok Lee, Tatyana Masyuk, Patrick Splinter, Jesús M. Banales, Anatoliy Masyuk, Angela Stroope, Nicholas LaRusso
Seung-Ok Lee, Tatyana Masyuk, Patrick Splinter, Jesús M. Banales, Anatoliy Masyuk, Angela Stroope, Nicholas LaRusso
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Research Article

MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease

Abstract

Hyperproliferation of bile duct epithelial cells due to cell-cycle dysregulation is a key feature of cystogenesis in polycystic liver diseases (PCLDs). Recent evidence suggests a regulatory role for microRNAs (miRNAs) in a variety of biological processes, including cell proliferation. We therefore hypothesized that miRNAs may be involved in the regulation of selected components of the cell cycle and might contribute to hepatic cystogenesis. We found that the cholangiocyte cell line PCK-CCL, which is derived from the PCK rat, a model of autosomal recessive polycystic kidney disease (ARPKD), displayed global changes in miRNA expression compared with normal rat cholangiocytes (NRCs). More specific analysis revealed decreased levels of 1 miRNA, miR15a, both in PCK-CCL cells and in liver tissue from PCK rats and patients with a PCLD. The decrease in miR15a expression was associated with upregulation of its target, the cell-cycle regulator cell division cycle 25A (Cdc25A). Overexpression of miR15a in PCK-CCL cells decreased Cdc25A levels, inhibited cell proliferation, and reduced cyst growth. In contrast, suppression of miR15a in NRCs accelerated cell proliferation, increased Cdc25A expression, and promoted cyst growth. Taken together, these results suggest that suppression of miR15a contributes to hepatic cystogenesis through dysregulation of Cdc25A.

Authors

Seung-Ok Lee, Tatyana Masyuk, Patrick Splinter, Jesús M. Banales, Anatoliy Masyuk, Angela Stroope, Nicholas LaRusso

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Figure 9

Effect of miR15a suppression in normal cholangiocytes on miR15a levels, Cdc25A expression, cell proliferation, and cyst growth.

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Effect of miR15a suppression in normal cholangiocytes on miR15a levels, ...
(A) Representative light (top panels) and fluorescent (bottom panels) images show that both scrambled miRNA and anti-miR15a were present in cultured cells in contrast with cholangiocytes treated with vehicle. Original magnification, ×20. (B) Quantitative PCR demonstrates that levels of miR15a in cholangiocytes treated with anti-miR15a (anti) were decreased compared with those in cholangiocytes treated with vehicle or scrambled miRNA. Data are representative of 3 independent experiments. (C) Representative Western blot and quantitative analysis based on densitometry measurements (D) of 5 independent experiments show that in anti-miR15a cholangiocytes, Cdc25A protein levels were increased by 38%. Thin white lines on the gel indicate that the lanes were run on the same gel but were noncontiguous. (E) Rates of cell proliferation were assessed in cholangiocytes treated with scrambled miRNA (n = 5) or anti-miR15a (n = 6) and expressed as a percentage of changes compared with rates of proliferation in nontreated cholangiocytes (considered to be equal to 100%). Cholangiocytes exposed to miR15a antisense display higher rates of proliferation than cholangiocytes exposed to scrambled miRNA. (F) Representative light microscopic images of cystic structures formed in 3D culture by normal cholangiocytes and quantitative assessment of changes in cystic areas over time (G) reveal that in the presence of anti-miR15a, cysts grow more rapidly compared with those in the presence of scrambled miRNA. Changes shown in G are in comparison with day 0. *P < 0.05. Scale bars: 250 μm.