MicroRNA-15b regulates reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression in human uterine leiomyoma

Abstract

Background: Human uterine leiomyoma (fibroids; LYO) are the most common benign neoplasms in reproductive-aged women. Dysregulated extracellular matrix and irregular LYO reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression are thought to be mediated by aberrant microRNA (miR) expression. The relationship of miR-15b and RECK expression in LYO has not been studied.

Methods: The expression levels of miR-15b and RECK were determined by quantitative RT-PCR, Western blot, and immunohistochemistry in cultures derived from commercial primary leiomyoma (cpLYO) and myometrial (cpMYO) cell lines and leiomyoma (pLYO) and myometrium (pMYO) tissue from surgical samples respectively. The relationship between miR-15b and RECK expression in cpLYO and pLYO (compared to their respective myometrial controls) was evaluated following transfection of cell cultures with either miR-15b mimic or inhibitor.

Results: Elevated levels of miR-15b were observed in cpLYO (2.82-fold; p = 0.04) and pLYO cell (1.30-fold; p = 0.0001) cultures respectively compared to corresponding MYO cell controls. Following transfection with miR-15b mimic, cpLYO cells (0.62-fold; p < 0.0001) and pLYO cells (0.68-fold; p < 0.0001) demonstrated reduced RECK protein expression. Following transfection with miR-15b inhibitor, cpLYO cells (1.20-fold; p < 0.0001) and pLYO cells (1.31-fold; p = 0.0007) demonstrated elevated RECK protein expression. RECK protein expression was reduced in pLYO tissues (0.73-fold; p < 0.0001) and pLYO (0.47-fold; p = 0.047) cells when compared to the corresponding MYO tissue controls.

Conclusion: Our findings suggest that miR-15b negatively regulates RECK expression in LYO, and increased miR-15b and decreased RECK expression may contribute to the pathobiology of LYO. The functional significance of miR-15b and RECK expression warrants further investigation as potential therapeutic targets for the treatment of human LYO.

Keywords: Fibroids; Myometrium; RECK; Uterine leiomyoma; miR-15b.

Fig. 1

miR-15b expression is up-regulated in uterine leiomyoma tissue and cultured cells. a Comparison of miR-15b expression level between isolated pLYO tissue and paired pMYO tissue. b Expression level of miR-15b in cpLYO cells as well as cpMYO cells. c Comparison of miR-15b level between pLYO cells and pMYO cells. Quantitative RT-PCR assay was performed as described in the methods section. All data were presented as mean ± SD and analyzed using Student’s t-test (two tail, unequal variance) and Mann–Whitney U test applicable. The experiments were performed in triplicate. Statistical significance was noted as * p < 0.05, ** p < 0.005, *** p < 0.001

Fig. 2

miR-15b mimic and inhibitor delivery into LYO and MYO cells via transient transfection. a Relative miR-15b expression in miR-15b mimic transfected cells 24 h post transfection. b Relative expression level of miR-15b in cells 48 h after miR-15b mimic transfection. c Influence of miR-15b inhibitor on miR-15b expression in cells 24 h after transfection. d Relative miR-15b expression in miR-15b inhibitor transfected cells 48 h post transfection. Cells without miR-15b mimic or miR-15b inhibitor transfection were used as control group, which was abbreviated as cell blank. Quantitative RT-PCR experiment was performed as described in methods. The data were presented as mean ± SD and analyzed using Student’s t-test (two tail, unequal variance) and Mann–Whitney U test appropriately. The experiments were conducted in triplicate. Statistical significance was noted as *** p < 0.001

Fig. 3

Inhibition of RECK mRNA expression by transfection miR-15b mimic in different type of cells. a qRT-PCR demonstrating decreased expression of RECK mRNA in cpLYO and cpMYO cells at 24 h post transfection with miR-15b mimic. b Reduction of RECK mRNA observed in miRNA-15b mimic transfected cpLYO, cpMYO and pLYO cells 48 h post transfection. Cells without miR-15b mimic transfection were used as control group, which was abbreviated as cell blank. Quantitative RT-PCR assay was performed as described in methods section. All data were presented as mean ± SD and analyzed using Student’s t-test (two tail, unequal variance) and Mann–Whitney U test when applicable. The experiments were performed in triplicate. Statistical significance was noted as* p < 0.05, ** p < 0.005, *** p < 0.001

Fig. 4

Differential expression level of RECK in pLYO and pMYO paired tissues and isolated cells. a RECK expression level in matched pLYO and pMYO tissues as determined by Western blot analysis. b Western blot analysis of RECK in pLYO and pMYO cell cultures from patient matched samples. c Representative matched pLYO and pMYO immunostained for RECK. The distribution of RECK immunohistochemical staining of paraffin sections from matched pLYO and pMYO tissue. Magnification: 10 × (left panel) and 40 × (right panel); Arrows indicated positive expression of RECK. Distribution of RECK immunohistochemical staining scores in matched LYO and MYO tissues were also showed. Data represented as Mean ± SD and analyzed using Student’s t-test (two tail, unequal variance), Mann–Whitney U test and chi-square (χ ²) test appropriately. Statistical significance was noted as * p < 0.05, ** p < 0.005, *** p < 0.001

Fig. 5

miR-15b is predicted to bind to the 3’-UTR of RECK and regulates RECK expression. a Scheme of the potential binding sites of miR-15b in the 3’-UTR of RECK. RECK mRNA has the potential complimentary binding site with miR-15b within its 3’-UTR. b miR-15b regulation of RECK protein expression in cpLYO cells. Proteins from cpLYO cells transfected with either miR-15b mimic or inhibitor at 10 nM. After 48 h, the lysates were analyzed by Western blot. c miR-15b regulation of RECK protein expression in cpMYO cells. d miR-15b regulation of RECK protein expression in pLYO cells. e miR-15b regulation of RECK protein expression in pMYO cells. Data were presented as means ± SD. Statistical comparisons between miR-15b mimic/inhibitor transfected cells and mimic/inhibitor negative control cells was analyzed using Student’s t-test (two tail, unequal variance) and Mann–Whitney U test appropriately. Statistical significance was noted as *p < 0.05, **p < 0.005, ***p < 0.001