Novel Direct Targets and Functional Roles for MicroRNA-21 in Granulosa Cells and Human Uterine Leiomyomas

Abstract

MicroRNA-21 (miR-21) is important for maintaining optimal ovulation rates and granulosa cell viability. It is also upregulated in human uterine leiomyomas (ULMs), a disease characterized by the presence of benign tumors on the myometrium. The primary objective of this thesis was to identify miR-21 direct targets in granulosa cells that mediate its important ovarian functions. The secondary objective was to elucidate the role of miR-21 in ULMs. Gene expression and bioinformatic analysis performed on granulosa cells after miR-21 inhibition identified many potential miR-21 direct targets. Luciferase assays revealed that miR-21 regulates the 3fUntranslated Region (3fUTR) of apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (Apobec3), intestinal-specific homeobox (ISX) and ubiquitin-specific protease 30 (USP30). Further research of ISX revealed that miR-21 binds to its 3fUTR and over expression of miR-21 causes inhibition of ISX protein levels. Quantitative RT-PCR and western analysis revealed that ISX regulates scavenger receptor class B type 1 (SRB1) fÀ,fÀ-carotene 15,15Oe-monooxygenase 1 (BCMO1) and retinoic acid receptor f¿ (RARf¿) in granulosa cells, genes known to be involved in steriodogenesis, embryogenesis and meiotic resumption in the ovary, respectively. Therefore, miR-21 may be regulating these functions through directly targeting ISX in granulosa cells. Investigation of miR-21 in UtM and UtLM cell lines (cell lines derived from myometrial and leiomyoma tissue, respectively) showed that miR-21 inhibits phosphorylation of elongation factor 2 and prevents expression of cleaved caspase 3 in both cell lines; findings which suggest that miR-21 is important for preventing cell death and maintaining cellular translation in these cell lines. Further research showed that miR-21 inhibits expression of programmed cell death 4 (PDCD-4). Expression analysis of PDCD-4 showed that it is highly expressed in ULM tissue when compared to paired healthy myometrial tissue. Since PDCD-4 is a known tumor suppressor that is suppressed in tumors, this finding indicates that PDCD-4 is playing an alternative role in ULMs compared to other tumorigenic tissue. Since miR-21 is also overexpressed in ULMs, miR-21 is excluded as a means of post-transcriptional gene control that gives rise to PDCD-4 induction in ULMs. Together these studies have identified novel direct targets for miR-21 in granulosa cells and implicated one of miR-21fs most well-studied direct targets, PDCD-4 in a novel functional role in ULMs.