Differential expression of microRNAs in myometrium and leiomyomas and regulation by ovarian steroids

Abstract

Given the emerging roles of microRNAs (miRNAs) as key regulator of mRNA stability we assessed their expression profile in paired myometrium and leiomyoma, their isolated smooth muscle cells (MSMC and LSMC), a spontaneously transformed leiomyoma smooth muscle cells (T-LSMC) and SK-LMS-1, a leiomyosarcoma cell line using microarray and real time PCR. Based on global normalization of expression values of 385 miRNAs and statistical analysis (anova), 91 miRNAs were expressed above the threshold levels in myometrium, with a progressive decline in numbers in leiomyomas, MSMC, LSMC, T-LSMC and SK-LMS-1 (P<0.05). We selected and validated the expression of miR-20a, miR-21, miR-26a, miR-18a, miR-206, miR-181a and miR-142-5p and found their differential expression in tissue and cell-specific manners (P<0.05). Treatments of MSMC and LSMC with 17beta estradiol and medroxyprogesterone acetate (10(-8)M), or ICI-182780 and RU-486 (10(-6)M) resulted in differential regulation of these miRNAs (P<0.05). In conclusion, the expression of a number of miRNAs in myometrium and leiomyoma with their progressive aberrant from normal MSMC into LSMC, transformed and cancerous stage, suggests that miRNAs and their regulation by ovarian steroids play a key role in pathogenesis of leiomyoma through gene expression stability.

1

Hierarchical cluster and tree-view analysis of differentially expressed miRNAs profile in paired myometrium (MY1, MY2, MY3 and MY4) and leiomyomas (LY1, LY2, LY3 and LY4) (Fig. 1A) and in paired myometrial smooth muscle cells (MSMC1 and MSMC2) and leiomyoma smooth muscle cells (LSMC1 and LSMC2) isolated and cultured from the above tissues, transformed leiomyoma cells (TLSMC-1 and T-LSMC-2) and leiomyosarcoma cell line (SLKMS1 and SKLMS2). Each column represents data from a single cohort with shades of red and green indicating up- or down-regulated miRNA according to the colour scheme shown below.miRNAs represented by rows were clustered according to their similarities in pattern of expression in each tissue. The dendrogram at the top of the image displays similarity of expression among these cohorts (see Table 1 and Table 2 for the list of differentially expressed miRNAs).

2

Bar graphs show real time PCR expression of miR-20a, miR-21 and miR-26a, miR-18a, miR-206, miR-181a and miR-142-5p in paired myometrium (Myo) and leiomyomas (LYOM) form African-Americans (AA) and Caucasians (C). The data is presented as relative expression following normalization and setting the expression of each miRNA independently in Caucasian myometrium arbitrarily as 1.Data represent mean ±standard error of three paired tissues from each ethnic group with asterisks *, ** and *** significantly different from C-Myo. P<0.05 was considered significant. Arrows indicates significant difference between the expression of these miRNAs in AA-Myo and AA-LYMO.

3

Bar graphs show real time PCR expression of miR-20a, miR-21 and miR-26a in myometrial and leiomyoma smooth muscle cells (MSMC and LSMC), spontaneously transformed LSMC (tLSMC) and leiomyosarcoma cell line, SKLM-1. The cells (1_10⁶/well in 6-well plates) were cultured as described in materials and methods and their total RNA was isolated and subjected to real time PCR. The data is presented as relative expression following normalization and setting their expression level in MSMC arbitrarily as 1. Data represent mean ± standard error from three separate experiments, with asterisks ** and *** displaying significant difference with * with P<0.05 considered significant. Arrow indicates significant difference in the expression of these miRNAs between the cell types.

4

Bar graphs show the expression of miR-20a, miR-21 and miR-26a in myometrial and leiomyoma smooth muscle cells (MSMC and LSMC).The cells (1_10⁶/well in 6-well plates) were cultured as described in materials and methods and following 24 hrs of treatment with 17b estradiol (E₂), ICI-182780 (ICI), E2+ICI, medroxyprog-esterone acetate (MPA), RU-486 (RU) and MPA+RU, their total RNA was isolated and subjected to real time PCR. The data is presented as relative expression following normalization and setting their expression level in untreated MSMC arbitrarily as 1. Data represent mean ± standard error from three separate experiments. The asterisks * indicate statistical difference between the expression of these miRNAs in treated as compared to untreated controls (Ctrl), with arrows pointing out the difference in their expression between MSMC and LSMC. A probability level of P<0.05 was considered significant.