Long Noncoding RNA MIAT Modulates the Extracellular Matrix Deposition in Leiomyomas by Sponging MiR-29 Family

Abstract

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.

Keywords: MED12 mutation; MIAT; TGF-β3; leiomyoma; miR-29.

Figure 1.

(A) The relative expression of MIAT in 66 paired myometrium (Myo) and leiomyoma (Lyo). The results in this and subsequent figures are presented as mean ± SEM. *P < .05, **P < .01, and ***P < .001. (B) Relative MIAT levels expressed as fold change (Lyo/paired Myo) based on race/ethnicity in Caucasian (n = 12), African American (n = 24), Hispanics (n = 23), and Asians (n = 7); (C) menstrual cycle phase in proliferative phase (n = 31) and secretory phase (n = 16); (D) MED12 mutation status in MED12 wild type (n = 23) and MED12 mutated (n = 43). (E,F) Relative levels of miR-29 family (E), COL1A1 and COL3A1 (F) expressed as fold change (Lyo/ paired Myo) in MED12 wild type (n = 23) and MED12 mutations (n = 43).

Figure 2.

(A) Sequence alignment with the coordinated positions of MIAT with miR-29 family. (B) Shows the relative luciferase activity in isolated LSMCs transfected with Renilla and firefly luciferase reporter pEZX-MT01 (Control) or pEZX-MT01(MIAT). Cells were also cotransfected with pre-miR-29 family (miR-29a, -b, -c; 50 nM) or scramble oligonucleotides (NC). Ratio of firefly to Renilla was determined after 48 hours and reported as relative luciferase activity compared with NC, which was independently set as 1. The results are presented as mean ± SEM and analyzed using 1-way analysis of variance. **P < .01 and ***P < .001. (C,D) Show the results of RNA immunoprecipitation assay with Ago2 antibody which was conducted to confirm the association between MIAT and miR-29 family using lysates from fresh specimens of leiomyoma (Lyo) and matched myometrium (Myo). The expression of MIAT (C) and miR-29 family (D) from purified RNA was determined by qRT-PCR. The results are presented as mean ± SEM and analyzed using unpaired t test. *P < .05, **P < .01, and ***P < .001 vs IgG, #P < .05, ###P < .001 vs Ago2 of the Myo group.

Figure 3.

The effect of MIAT knockdown through transfection of LSMC spheroids with siRNA scrambled oligonucleotides (siNC) or siRNA against MIAT (siMIAT) for 96 hours on MIAT expression (A), miR-29 family expression (B), RNA levels of COL1A1, COL3A1 and TGF-β3 (C) and their protein levels determined by western blot (D) along with the relative band density analysis (E). The results were obtained from 4 experiments using cells isolated from different patients in each set and presented as mean ± SEM. P values are analyzed using unpaired t test and indicated as *P < .05, **P < .01, and ***P < .001.

Figure 4.

(A-C) Representative of Western blot analysis of COL1A1 and COL3A1 proteins with relative band densities shown below (A); qRT-PCR analysis of MIAT (B) and miR-29 family (C) in LSMC spheroids following treatment with DMSO or SB431542 (5 μM; TGF-β type I receptor inhibitor) with TGF-β3 (10 ng/mL) for 48 hours. (D) LSMC spheroids were transfected with siRNA scrambled oligonucleotides (siNC) or MIAT siRNA oligonucleotides (siMIAT) for 96 hours with TGF-β3 (10 ng/mL) treatment for the last 48 hours and the levels of COL1A1 and COL3A1 proteins were determined with the relative density histograms shown below. The results were obtained from 4 experiments using cells isolated from different patients in each set and analyzed using unpaired t test with presentation as mean ± SEM. *P < .05, **P < .01, and ***P < .001 vs DMSO group (A and C) or siNC group (D); ^#P < .05, ^##P < .01, and ^###P < .001 vs TGF-β3 group (C) or siNC group of TGF-β3 treatment (D).

Figure 5.

Schematic diagram representing our working model in which leiomyoma are characterized by reduced miR-29 family and elevated expression of TGF-β3 and the lncRNA MIAT, which causes an increase in extracellular matrix accumulation.