Gene expression profiling of multiple leiomyomata uteri and matched normal tissue from a single patient

Abstract

Objective: To identify differentially expressed genes between fibroid and adjacent normal myometrium in an identical hormonal and genetic background.

Design: Array analysis of three leiomyomata and matched adjacent normal myometrium in a single patient.

Setting: University of Colorado Hospital.

Patient(s): A single female undergoing medically indicated hysterectomy for symptomatic fibroids.

Interventions(s): mRNA isolation and microarray analysis, reverse-transcriptase polymerase chain reaction, Western blotting, and immunohistochemistry.

Main outcome measure(s): Changes in mRNA and protein levels in leiomyomata and matched normal myometrium.

Result(s): Expression of 197 genes was increased and 619 decreased significantly by at least twofold, in leiomyomata relative to normal myometrium. Expression profiles between tumors were similar and normal myometrial samples showed minimal variation. Changes in, and variation of, expression of selected genes were confirmed in additional normal and leiomyoma samples from multiple patients.

Conclusion(s): Analysis of multiple tumors from a single patient confirmed changes in expression of genes described in previous, apparently disparate, studies, and identified novel targets. Gene expression profiles in leiomyomata are consistent with increased activation of mitogenic pathways and inhibition of apoptosis. Down-regulation of genes implicated in invasion and metastasis, of cancers, was observed in fibroids. This expression pattern may underlie the benign nature of uterine leiomyomata and may aid in the differential diagnosis of leiomyosarcoma.

Figure 1

A. The statistically significant differentially expressed genes that exhibited consistent changes in all 3 tumors relative to normal myometrium of a magnitude of at least 2 fold. B. Heat map showing cluster analysis of the differentially expressed 816 genes in 3 uterine leiomyoma and 3 samples of matched adjacent normal myometrium. From left to right the first 3 columns represent the 3 tumors followed by the corresponding normal tissue. Rows represent individual genes. Red, increased gene expression; green, decreased gene expression. The color intensity is proportional to the hybridization intensity of a gene from its median level across all samples.

Figure 2

Semiquantitative RT-PCR analysis. RNA from paired myometrium/leiomyoma from 5 different patients was analyzed using specific primer pairs, as indicated and described in Materials and Methods and electrophoresed on agarose gels. Bands were quantitated by scanning densitometry and expressed as relative intensity normalized to GAPDH. B2, B3, B4, B9, B11 – patient designations. N1-5 normal myometrium; L1-5 corresponding leiomyoma samples.

Figure 3

A. Semiquantitative RT-PCR analysis of MMP11 (Stromelysin 3) expression. RNA from paired myometrium/leiomyoma from 5 different patients was analyzed using specific primer pairs, as indicated and described in Materials and Methods and electrophoresed on agarose gels. Bands were quantitated by scanning densitometry and expressed as relative intensity normalized to GAPDH. B2, B3, B4, B9, B11 – patient designations. N1-5 normal myometrium; L1-5 corresponding leiomyoma samples. B. Immunohistochemical staining for matrix metalloproteinase 11 (MMP-11) in normal myometrial tissue (top panels) and leiomyomata (bottom panels). Paraffin embedded sections were stained with anti-MMP-11 antibody as described in Materials and Methods. Representative 20X fields are depicted. B2, B3, B4, B9, B11 patient designations. N: normal myometrium, L matched leiomyoma.

Figure 4

Western blots analysis of leiomyoma and myometrium samples from 5 patients using antibodies to A. PKC β1 and B. MST-4. Blots were reprobed for GAPDH as a loading control. Chemiluminescence was quantitated using a BioRad Chemdoc XRS imager and relative levels expressed as a ratio of leiomyoma to normal myometrium, normalized to GAPDH. B2, B4, B5, B8 and B9; patient designations, numbers indicate independent tumor/normal paired samples derived from that patient. N: myometrium, L: leiomyoma.