miR-93/106b and their host gene, MCM7, are differentially expressed in leiomyomas and functionally target F3 and IL-8

Abstract

miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3'-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.

Fig. 1.

A, The relative expression (mean ± sem) of miR-93, miR-106b, and MCM7 in leiomyoma (LYO) and paired myometrium (MYO) from the untreated group (n = 41) with the expression of miR-93 (B) and MCM7 (C) in paired tissues representing Caucasian (W; n = 18) and African-Americans (AA; n = 20). The data were analyzed using nonparametric Student t test (*, P < 0.05 and **, P < 0.01, indicating significant difference as indicated by the corresponding lines for each paired tissues).

Fig. 2.

Lentiviral transduction of miR-106b∼25 cluster in spontaneously transformed leiomyoma cells (TF324). A, Green fluorescent protein expression (derived miR-106b∼25cluster expression) transduced after treatments with 10 μg/ml of doxycycline (DOX+) or without (Dox−) for 6 d and Western blot analysis of p21 (miR-93/106b target), p57 (miR-25 target), and F3 and β-actin used as a loading control (B). Note a significant reduction in the level of p21, p57, and F3 but not MCM7, which is coexpressed in the same pre-mRNA with miR-106b∼25cluster, in cells with gain of function of miR-106∼25. C, Treeview analysis of 226 genes differentially expressed in TF324 after miR-106∼25 transduction (+Dox 6d) and untreated control (Ctrl, −Dox) and the pattern of same genes derived from del(7q) and none-del(7q) leiomyomas (LYO-ND and LYO-DL, respectively) and myometrium (MYO). Each column represents the mean expression values from 11 matched tissues as described by Hodge et al. (16).

Fig. 3.

Relative expression (mean ± sem) of miR-93, miR-106b, F3, and IL8 in leiomyoma (LYO) and paired myometrium (MYO) from the untreated group (n = 41) and the expression of F3 (B) and IL8 (C) in paired tissues from Caucasian (W; n = 18) and African-Americans (AA; n = 20). The data were analyzed using a nonparametric Student t test (*, P < 0.05 and **, P < 0.01, indicating significant difference as shown by the corresponding lines for each paired tissues).

Fig. 4.

Western blot analysis of MCM7, F3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control) (A) with the F3 band densities shown below as well as IL8 levels determined by ELISA (B) of tissue extracts prepared from 11 leiomyoma (L) and paired myometrium (M) from the untreated group. The results are presented as mean ± sem and analyzed using nonparametric student t test (*, P < 0.05; **, P < 0.01, indicating a significant difference and shown by the corresponding lines for each paired tissues).

Fig. 5.

Relative expression of F3 and IL8 (mean ± sem) in MSMC, LSMC, and SKLM-S1 transfected with pre-miR-93, pre-miR-106b (gain of function), and preNC (NC) determined by QRT-PCR (A and B), Western blot analysis (D), and ELISA (IL-8, C). The assays were performed using three to five sets of independent cell preparation, with QRT-PCR and ELISA assayed in triplicates, and α-tubulin was used as loading control for Western blotting. The data were analyzed using a nonparametric Student t test [*, P < 0.001 (A and C); *, P < 0.01 (B); *, P < 0.05 and **, P < 0.01 (D), indicated by the corresponding lines].

Fig. 6.

Dose-dependent influence of miR-93 and miR-106b gain of function (pre-miR transfected) on F3 (A) and IL8 (B) mRNA and protein (C) expression in LSMC as compared with control (preNC). The assays were performed using four sets of independent cell preparations with QRT-PCR assayed in triplicates, and in Western blotting, α-tubulin was used as a loading control. The data are presented as mean ± sem and analyzed using a nonparametric Student t test (*, P < 0.05, **, P < 0.01, ***, P < 0.001, as indicated by the corresponding lines). D and E, Firefly luciferase assay with pZEX-MT01 and pGL3 constructs carrying a 3′UTR fragments of F3 and IL8, respectively. LSMC and SKLM-S1 were cotransfected with firefly luciferase reporters, Renilla luciferase transfection control plasmid (in case of IL8), pre-miR-93, pre-miR-106b, or preNC. The ratio of firefly to Renilla was determined and reported as relative luciferase activity as compared with empty vector. The results are presented as the average fold change from three sets of independent experiments performed in duplicates and analyzed using a nonparametric Student t test [*, P < 0.01, **, P < 0.001 (D), and *, P < 0.05, **, P < 0.01, and ***, P < 0.001 (E) and shown by the corresponding lines]. The sequence alignment of the miR-93 and miR-106b seed regions and the F3 and IL-8 mRNA target sits at their 3′UTR are shown at the top of each graph.

Fig. 7.

The expression of IL8 in LSMC after treatment with FVIIa (+FVIIa), which activates the endogenous F3 pathway (A) as compared with the untreated control (−FVlla) and LSMC transfected with pre-miR-93 and pre-miR-106b as compared with control (preNC transfected) treated with and without FVIIa (B). The assays were performed in two sets of independent experiments in triplicate and the data are reported as mean ± sem and analyzed by a nonparametric Student t test [*, P < 0.001 (A), *, P < 0.05 and **, P < 0.01 (B) as indicated by the corresponding lines]. C–F, The influence of F3 silencing (siRNA transfection) in LSMC on the expression of F3 (C) and downstream genes IL8 (D), CTGF (E), and PAI-1 (F). Western blot analysis of F3 in LSMC after transfection with F3 siRNA and scrambled siRNA (NC) is shown as an insert in C. The assays were performed in three independent cell preparations, and the results are reported as mean ± sem and analyzed by a nonparametric Student t test [*, P < 0.001 (C), *, P < 0.01 (D) *, P < 0.05 (E), and *, P < 0.001 (F) as compared with NC as indicated by the corresponding lines].

Fig. 8.

The expression of PAI-1 (A) and CTGF (B) in MSMC and LSMC after transfection with pre-miR-93, pre-miR-106b, and the corresponding controls (preNC) determined using QRT-PCR (A and B) and Western blot analysis (C). The assays were performed using four independent sets of cell preparations and the results are reported as mean ± sem and analyzed by a nonparametric Student t test [*, P < 0.001 (A) and P < 0.05 (B), and **, P < 0.001 (B) as indicated by the corresponding lines].

Fig. 9.

The influence of gain of function of miR-93 and miR-106b and corresponding controls (preNC) on the LSMC growth rate (A), viability (B), and caspase-3/7 activity (C). The cell growth and caspase-3/7 activity were determined after 4 d of incubation, and the rate of cell viability was determined using an MTT assay on the indicated days with culture media changed every 2 d. D (a-d), Photomicrographs of LSMC transfected with pre-miR-93/106b or the corresponding negative controls (preNC) for 96 h, and the LSMC migration was determined for the last 24 h after the biocompatible gels were removed. The images represent preNC-transfected cells at 0 h (a) and 24 h (b) and cells transfected with pre-miR-93 (c) or pre-miR-106b (d). The cell growth, viability, caspase activity, and migration assays were performed, at least in triplicates, using three independent cell preparations. The results are shown as mean ± sem and analyzed using a nonparametric Student t test (*, P < 0.01 as compared with preNC).

Fig. 10.

Schematic presentation of miR-106b∼25 cluster and their host gene, MCM7, expression with their posttranscriptional regulation as well as specific genes either directly (p21, F3, and IL8) or indirectly through the regulation of F3 (CTGF, PAI-1, uPA, PTX3) regulated by miR-106/93 in myometrium and leiomyoma cells. Through such regulatory functions and altered expression due to the chromosomal rearrangement of 7q22, miR-106/93 could influence the cell growth, migration, inflammation, and tissue turnover, events with central roles in leiomyoma development, growth, and associated symptoms.