Role of microRNA-21 and programmed cell death 4 in the pathogenesis of human uterine leiomyomas

Abstract

Objective: To determine whether programmed cell death 4 (PDCD-4) is altered in autologous leiomyoma and myometrial tissues and what microRNA-21's (miR-21) role is in PDCD-4 expression, apoptosis, and translation.

Design: Laboratory research.

Setting: Academic medical center.

Patient(s): Myometrial and leiomyoma tissues from patients with symptomatic leiomyomata.

Intervention(s): Tissue analysis and miR-21 knockdown in cultured immortalized myometrial (UtM) and leiomyoma (UtLM) cells.

Main outcome measure(s): MiR-21 and PDCD-4 mRNA and protein expression.

Result(s): Leiomyoma tissues robustly expressed the full-length 51 kd isoform of PDCD-4, but normal myometrial tissue had negligible expression. Consistent with autologous tissues, UtLM cells expressed elevated miR-21 and a similar pattern of PDCD-4 compared with UtM cells. Knockdown of miR-21 increased PDCD-4 levels in UtM cells and UtLM cells, indicating that it can regulate PDCD-4 expression. Loss of miR-21 also increased cleavage of caspase-3 (apoptosis marker) and increased phosphorylation of elongation factor-2 (marker of reduced translation) in both cell lines.

Conclusion(s): Elevated leiomyoma miR-21 levels are predicted to decrease PDCD-4 levels, thus leiomyomas differ from other tumors where loss of PDCD-4 is associated with tumor progression. Our studies indicate regulation of PDCD-4 expression is not a primary miR-21 function in leiomyomas, but instead miR-21 is able to impact cellular apoptosis and translation, through unknown targets, in a manner consistent with its involvement in the pathophysiology of uterine fibroids.

Figure 1

Differential expression of PDCD-4 and miR-21 in leiomyoma and myometrial tissues and immortalized cell lines, UtLM (leiomyoma) and UtM (myometrial). A) Western analysis of PDCD-4 isoforms in 3 representative paired leiomyoma and normal myometrial samples of a total of 11 examined are shown. B) Western analysis of PDCD-4 isoforms in UtLM and UtM cell lines. *Means ± SEM (n=3) for the 51 kDa band between cells types are different (P<0.05). C) qRT-PCR analysis of miR-21 and PDCD-4 mRNA levels in UtLM and UtM cell lines. *Means ± SEM (n=3) are different (P<0.05) between UtLM cells and UtM cells.

Figure 2

miR-21 regulation of PDCD-4 in UtM and UtLM cells. Locked nucleic acid (LNA) oligonucleotides complementary to miR-21 (LNA-miR-21) or a scrambled (LNA-scr) control were transfected into UtM cells (A) or UtLM cells (B). *Means ± SEM (n=3) PDCD-4 (51 kDa isoform) levels normalized to actin are different (P<0.05) between LNA-scramble and LNA-miR-21 treated cells.

Figure 3

Knockdown of miR-21 in UtM and UtLM cells increases cleaved caspase-3. Locked nucleic acid (LNA) oligonucleotides complementary to miR-21 (LNA-miR-21) or a scrambled (LNA-scramble) control were transfected into UtM cells (A) or UtLM cells (B). Western analysis showed a significant induction of cleaved caspase-3 in the LNA-miR-21 treated UtM and UtLM cells across 3 independent experiments.

Figure 4

Expression of phosphorylated EF-2 in UtM and UtLM cells following knockdown of miR-21. A) Basal ph-EF2 expression levels in UtM cells and UtLM cells. *Means ± SEM (n=3) ph-EF2 levels normalized to actin are different (P<0.05). B) Locked nucleic acid (LNA) oligonucleotides complementary to miR-21 (LNA-miR-21) or a scrambled (LNA-scr) control were transfected into UtM cells or UtLM cells. *Means ± SEM (n=3) ph-EF2 levels normalized to actin are different (P<0.05) between LNA-scramble and LNA-miR-21 treated cells.