PLIN2 Functions As a Novel Link Between Progesterone Signaling and Metabolism in Uterine Leiomyoma Cells

Abstract

Context: Uterine leiomyoma (fibroids) are the most common tumors in women. Recently, perilipin-2 (PLIN2) was identified as a critical target gene of the progesterone receptor; however, its function in the pathogenesis of fibroids is unknown.

Objective: To determine the function of PLIN2 in leiomyoma cells.

Design: Tissue and primary cells from leiomyoma and myometrium were analyzed. PLIN2 function in leiomyoma was assessed using small interfering RNA. RNA-sequencing was performed to identify genome-wide effects of PLIN2 depletion. Metabolic activity was measured using the Seahorse XF96 analyzer. Real-time quantitative PCR and immunoblotting were also performed.

Setting: Laboratory.

Patients or other participants: Forty-one premenopausal women undergoing surgery for fibroids.

Main outcome measures: Gene expression, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and cell proliferation.

Results: PLIN2 gene expression was 2.4-fold lower in leiomyoma compared with adjacent myometrium, suggesting a link between PLIN2 deficiency and fibroids. A total of 3877 genes were differentially expressed after PLIN2 knockdown. Gene ontology analysis identified metabolism as the second-highest biological process affected by PLIN2 depletion. OCR (mitochondrial respiration) and ECAR (glycolysis) were significantly upregulated after PLIN2 knockdown; PLIN2-depleted cells had a greater basal metabolic activity and higher metabolic stress response. Cell proliferation was also significantly increased after PLIN2 knockdown.

Conclusions: PLIN2 depletion increases mitochondrial respiration and glycolysis, suggesting that PLIN2 is a critical regulator of metabolic function in leiomyoma cells. PLIN2 deficiency also reprograms leiomyoma cells to a proproliferative phenotype. These findings introduce metabolomics as an area to explore to better understand leiomyoma tumorigenesis.

Figure 1.

Relative PLIN2 mRNA levels in human leiomyoma tissue and matched adjacent myometrium. A total of 48 samples from 24 women were analyzed. To standardize comparisons between samples, PLIN2 expression in leiomyoma tissue was normalized to one. PLIN2 expression was 2.4-fold lower in leiomyoma compared with matched myometrium tissue (2.4 ± 0.51, mean ± SEM; ***P = 0.007 comparing myometrium with leiomyoma).

Figure 2.

Effect of UPA treatment on the expression of PLIN2 in leiomyoma cells. Serum-starved leiomyoma cells were treated with varying concentrations of UPA (ranging from 10⁻⁹ to 10⁻⁵ M) or vehicle for 24 h. PLIN2 mRNA levels were normalized to GAPDH. Results are reported as fold change compared with vehicle and represent the mean ± SEM of experiments performed in five independent patient samples (**P = 0.003 compared with vehicle; 10⁻⁹ vs 10⁻⁵: P = 0.004; 10⁻⁸ vs 10⁻⁵: P = 0.004; 10⁻⁷ vs 10⁻⁵: P = 0.02; 10⁻⁶ vs 10⁻⁵: P = 0.5).

Figure 3.

PLIN2 knockdown significantly decreases its expression in leiomyoma cells. Leiomyoma cells were transfected with control siRNA or PLIN2 siRNA for 72 h. PLIN2 mRNA expression was detected using RT qPCR, which was downregulated ∼0.17-fold (0.166 ± 0.04). Results are reported as fold change compared with control siRNA. The mean ± SEM for four patient samples is reported. ***P = 0.0004.

Figure 4.

Metabolic activity of leiomyoma cells after PLIN2 knockdown. Leiomyoma cells were transfected with control siRNA or PLIN2 siRNA for 72 h. Metabolic activity was determined by the measurement of extracellular flux using the Seahorse XF 96 Analyzer. The OCR reflects mitochondrial respiration, whereas the ECAR reflects glycolysis. Basal activity represents metabolic activity under basal conditions. Stress response represents metabolic activity after injection of FCCP and oligomycin. (A and B) Representative kinetics of increased (A) OCR and (B) ECAR over time in control or PLIN2-depleted cells. (C and D) Quantification of data from five patient samples demonstrates that PLIN2 depletion is associated with increased (C) mitochondrial respiration (mean ± SEM, ***P < 0.0001) and (D) glycolysis (mean ± SEM, ***P < 0.0001) under both basal and stressed conditions. (E and F) Percent change in OCR and ECAR transitioning from the basal state to the stress state. PLIN2 depletion causes leiomyoma cells to rely more heavily on (F) glycolysis (73 ± 21.3% vs 64 ± 21.3%, n = 5, ***P < 0.0001) over (E) mitochondrial respiration (115% vs 117%, n = 5, P = 0.63) to meet increased energy demand.

Figure 5.

Effects of siRNA knockdown of PLIN2 on leiomyoma cell proliferation. Cell proliferation was determined by the measurement of PCNA protein levels using immunoblot. Blots were reprobed with a β-actin antibody as a loading control. (A) Representative immunoblot showing PLIN2 depletion led to increased PCNA protein expression. (B) Quantification of immunoblots from three independent patient samples demonstrating a 4.8-fold increase in PCNA protein levels (**P = 0.021; 1.63 ± 0.08 vs 0.34 ± 0.06, means ± SEM). (C) Leiomyoma cells were transfected with control siRNA or PLIN2 siRNA for 72 h. RT qPCR assay demonstrated a 0.46-fold decrease in CDKN1A mRNA levels in PLIN2-depleted vs control cells (***P = 0.043; 0.46 ± 0.08 vs 1.0 ± 0.23, mean ± SEM).