H19 lncRNA alters stromal cell growth via IGF signaling in the endometrium of women with endometriosis

Abstract

Endometriosis affects approximately 15% of reproductive aged women and is associated with chronic pelvic pain and infertility. However, the molecular mechanisms by which endometriosis impacts fertility are poorly understood. The developmentally regulated, imprinted H19 long noncoding RNA (lncRNA) functions to reduce the bioavailability of microRNA let-7 by acting as a molecular sponge. Here we report that H19 expression is significantly decreased in the eutopic endometrium of women with endometriosis as compared to normal controls. We show that decreased H19 increases let-7 activity, which in turn inhibits Igf1r expression at the post-transcriptional level, thereby contributing to reduced proliferation of endometrial stromal cells. We propose that perturbation of this newly identified H19/Let-7/IGF1R regulatory pathway may contribute to impaired endometrial preparation and receptivity for pregnancy in women with endometriosis. Our finding represents the first example of a lncRNA-based mechanism in endometriosis and its associated infertility, thus holding potential in the development of novel therapeutics for women with endometriosis and infertility.

Keywords: H19; IGF; endometrium; let‐7; long noncoding RNA.

© 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Figures

Figure 1

The expressions of H19 and Igf1r are significantly decreased in the eutopic endometrium of women with endometriosis compared to those without endometriosis

1. A–F (A, B, D–F) RNA levels determined by RT–qPCR. Results are presented as mean ± SD,n = 10. P-values are indicated on the top of each plot. (C) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner. Spearman correlation coefficient, P-values, and sample numbers are marked on the top left of the plot.

Figure 2

H19 promotes Igf1r expression in endometrial stromal cells by inhibiting let-7

1. A–D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). RNA and proteins were analyzed at 48 h post-transfection. Combined results from the two patient cells are presented. Western blot gels from #166 cells are shown in (C). Quantitation of Western blots combining both patient cells are shown in (D).

2. E–H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented. Western blot gels from #98 cells are shown in (G). Quantitation of Western blots combining both patient cells are shown in (H).

Data information: Results are presented as mean ± SD, n = 6. P-values are indicated on the top of each plot.

Figure 3

H19 stimulates proliferation of endometrial stromal cells

1. A–F The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented.

2. G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT–qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.

3. H A proposed model of the H19/let-7/IGF1R-mediated regulation of endometrial stromal cell proliferation. During the proliferative phase of the endometrium, E2 stimulates the expression of H19. As its level rises, H19 acts as a sponge to sequester let-7 and prevent it from inhibiting its target gene Igf1r. Increased IGF1R protein level leads to increased IGF1 signaling with a biological endpoint of increased proliferation of endometrial stromal cells.

Data information: Results are presented as mean ± SD, n = 6. P-values are indicated on the top of each plot.