Dataset Information

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.

SUBMITTER: Fitzgerald JB

PROVIDER: S-EPMC3432744 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Fitzgerald J Browning JB Chennathukuzhi Vargheese V Koohestani Faezeh F Nowak Romana A RA Christenson Lane K LK

Fertility and sterility 20120622 3

<h4>Objective</h4>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.<h4>Design</h4>Laboratory research.<h4>Setting</h4>Academic medical center.<h4>Patient(s)</h4>Myometrial and leiomyoma tissues from patients with symptomatic leiomyomata.<h4>Intervention(s)</h4>Tissue analysis and miR-21 knockdown in cultured immortalized myometrial (UtM) and leio ...[more]

PMID: 22728051

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Project description:Study questionWhat are the cellular composition and single-cell transcriptomic differences between myometrium and leiomyomas as defined by single-cell RNA sequencing?Summary answerWe discovered cellular heterogeneity in smooth muscle cells (SMCs), fibroblast and endothelial cell populations in both myometrium and leiomyoma tissues.What is known alreadyPrevious studies have shown the presence of SMCs, fibroblasts, endothelial cells and immune cells in myometrium and leiomyomas. However, there is no information on the cellular heterogeneity in these tissues and the transcriptomic differences at the single-cell level between these tissues.Study design, size, durationWe collected five leiomyoma and five myometrium samples from a total of eight patients undergoing hysterectomy. We then performed single-cell RNA sequencing to generate a cell atlas for both tissues. We utilized our single-cell sequencing data to define cell types, compare cell types by tissue type (leiomyoma versus myometrium) and determine the transcriptional changes at a single-cell resolution between leiomyomas and myometrium. Additionally, we performed MED12-variant analysis at the single-cell level to determine the genotype heterogeneity within leiomyomas.Participants/materials, setting, methodsWe collected five MED12-variant positive leiomyomas and five myometrium samples from a total of eight patients. We then performed single-cell RNA sequencing on freshly isolated single-cell preparations. Histopathological assessment confirmed the identity of the samples. Sanger sequencing was performed to confirm the presence of the MED12 variant in leiomyomas.Main results and role of chanceOur data revealed previously unknown heterogeneity in the SMC, fibroblast cell and endothelial cell populations of myometrium and leiomyomas. We discovered the presence of two different lymphatic endothelial cell populations specific to uterine leiomyomas. We showed that both myometrium and MED12-variant leiomyomas are relatively similar in cellular composition but differ in cellular transcriptomic profiles. We found that fibroblasts influence the leiomyoma microenvironment through their interactions with endothelial cells, immune cells and SMCs. Variant analysis at the single-cell level revealed the presence of both MED12 variants as well as the wild-type MED12 allele in SMCs of leiomyomatous tissue. These results indicate genotype heterogeneity of cellular composition within leiomyomas.Large scale dataThe datasets are available in the NCBI Gene Expression Omnibus (GEO) using GSE162122.Limitations, reasons for cautionOur study focused on MED12-variant positive leiomyomas for single-cell RNA sequencing analyses. Leiomyomas carrying other genetic rearrangements may differ in their cellular composition and transcriptomic profiles.Wider implications for the findingsOur study provides a cellular atlas for myometrium and MED12-variant positive leiomyomas as defined by single-cell RNA sequencing. Our analysis provides significant insight into the differences between myometrium and leiomyomas at the single-cell level and reveals hitherto unknown genetic heterogeneity in multiple cell types within human leiomyomas. Our results will be important for future studies into the origin and growth of human leiomyomas.Study funding/competing interest(s)This work was supported by funding from the National Institute of Child Health and Human Development (HD098580 and HD088629). The authors declare no competing interests.

Project description:The microbial loop is the conventional model by which nutrients and minerals are recycled in aquatic eco-systems. Biochemical pathways in different organisms become metabolically inter-connected such that nutrients are utilized, processed, released and re-utilized by others. The result is that unrelated individuals end up impacting each others’ fitness directly through their metabolic activities. This study focused on the impact of programmed cell death (PCD) on a population’s growth as well as its role in the exchange of carbon between two naturally co-occurring halophilic organisms. Flow cytometric, biochemical, 14C radioisotope tracing assays, and global transcriptomic analyses show that organic algal photosynthate released by Dunalliela salina cells undergoing PCD complements the nutritional needs of other non-PCD D. salina cells. This occurs in vitro in a carbon limited environment and enhances the growth of the population. In addition, a co-occurring heterotroph Halobacterium salinarum re-mineralizes the carbon providing elemental nutrients for the mixoheterotrophic chlorophyte. The significance of this is uncertain and the archaeon can also subsist entirely on the lysate of apoptotic algae. PCD is now well established in unicellular organisms; however its ecological relevance has been difficult to decipher. In this study we found that PCD in D. salina causes the release of organic nutrients such as glycerol, which can be used by others in the population as well as a co-occurring halophilic archaeon. H. salinarum also re-mineralizes the dissolved material promoting algal growth. PCD in D. salina was the mechanism for the flow of dissolved photosynthate between unrelated organisms. Ironically, programmed death plays a central role in an organism’s own population growth and in the exchange of nutrients in the microbial loop. Halobacterium NRC-1 was grown to mid-logarithmic phase (OD600 ~0.4 – 0.8) in MM2. Cells were harvested and washed in MM1 and incubated in MM1 (control) or in Dunaliella harvested supernatant. Cells were harvested at time zero, 5, 10, 20, 40, 80, 160 minutes. RNA from two biological replicate time courses were prepared, averages of these replicates are reported in the published study. The zero time point was harvested immediately after incubation Halobacteria-NRC1 in MM1 or in Dunaliella salina's supernatant.

Dataset Information

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

Objective

Design

Setting

Patient(s)

Intervention(s)

Main outcome measure(s)

Result(s)

Conclusion(s)

Publications

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

Similar Datasets

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