Circulating microRNA Profile throughout the Menstrual Cycle

<div><p>Normal physiological variables, such as age and gender, contribute to alterations in circulating microRNA (miRNA) expression levels. The changes in the female body during the menstrual cycle can also be reflected in plasma miRNA expression levels. Therefore, this study aimed to determine the plasma miRNA profile of healthy women during the menstrual cycle and to assess which circulating miRNAs are derived from blood cells. The plasma miRNA expression profiles in nine healthy women were determined by quantitative real time PCR using Exiqon Human Panel I assays from four time-points of the menstrual cycle. This platform was also used for studying miRNAs from pooled whole blood RNA samples at the same four time-points. Our results indicated that circulating miRNA expression levels in healthy women were not significantly altered by the processes occurring during the menstrual cycle. No significant differences in plasma miRNA expression levels were observed between the menstrual cycle time-points, but the number of detected miRNAs showed considerable variation among the studied individuals. miRNA analysis from whole blood samples revealed that majority of miRNAs in plasma are derived from blood cells. The most abundant miRNA in plasma and blood was hsa-miR-451a, but a number of miRNAs were only detected in one or the other sample type. In conclusion, our data suggest that the changes in the female body during the menstrual cycle do not affect the expression of circulating miRNAs at measurable levels.</p> </div

RNA-Seq statistics of E2, P4, TAM, and RU486 treated and non-treated Ishikawa cells.

*<p>Significant genes (5% FDR) are counted from known genes and compared to non-treated cells.</p>**<p>E2 and P4 significant genes present in human endometrium during the time of embryo implantation.</p

Selection of genes with FPKM >1000 after hormone/modulator treatments.

*<p>FPKM abundance >1000.</p

Selection of biomarkers related to reproductive system diseases among E2 and P4 significant genes in Ishikawa cell line.

<p>Expression changes are provided in logarithmic scale calculated as following: log (Expression treated/Expression non-treated).</p

Top 1 network with TAM 12 h significant genes related to DNA replication, recombination and repair, cell cycle, cellular assembly and organization.

<p>Red molecules represent up-regulated and green down-regulated genes among TAM 12 h significant genes in Ishikawa cells. The networks were generated through the use of IPA (Ingenuity® Systems, <a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>).</p

Changes in the Transcriptome of the Human Endometrial Ishikawa Cancer Cell Line Induced by Estrogen, Progesterone, Tamoxifen, and Mifepristone (RU486) as Detected by RNA-Sequencing

<div><p>Background</p><p>Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.</p><p>Methodology/Principal findings</p><p>Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including <i>PSAP, ATP5G2, ATP5H</i>, and <i>GNB2L1</i> following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: <i>EZH2, MDK, MUC1, SLIT2,</i> and <i>IL6ST</i>, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (<i>CCND1</i>) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as <i>CTNND1, JUP, CDH2, IQGAP1,</i> and <i>COL2A1.</i></p><p>Conclusions</p><p>Significant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.</p></div

Venn diagram showing significant gene expression changes 12 h E2, TAM, P4 or RU486 treatment, relative to non-treated Ishikawa cells.

<p>A Unique and common genes after 12 h E2 and TAM treatment. B Unique and common genes after 12 h P4 and RU486 treatment. The numbers given within each of the circles represent the number of significantly changed genes unique to treatment, and arrows show the manner they are regulated (up- or down-regulation compared to non-treated Ishikawa cells). Overlaps indicate the number of commonly changed genes.</p

Selection of endometrial specific biomarkers found in 12 h E2 (left) and P4 (right) treated Ishikawa cells and their relative abundance in human endometrial biopsy samples at the time of embryo implantation (n = 2).

<p>Red genes up-regulated in E2 and P4 treated Ishikawa cells compared to non-treated cells; green genes down-regulated. Genes situated on the left side of the diagonal line show higher relative abundance (FPKM) in human endometrial biopsy sample compared to non-treated Ishikawa cells. Genes situated on the right side of the diagonal line show lower relative abundance (FPKM) in human endometrial biopsy sample compared to non-treated Ishikawa cells.</p

Selection of TAM and RU486 regulated gene products in Ishikawa cells related to reproductive system diseases.

<p>Selection of TAM and RU486 regulated gene products in Ishikawa cells related to reproductive system diseases.</p

Additional file 1: Table S1. of Somatic mosaicism for copy-neutral loss of heterozygosity and DNA copy number variations in the human genome

General characteristics of the four subjects examined in the study. Table S2. Studied tissues and sample naming. Table S3. Summary of the non-mosaic (germ-line) CNV regions identified in all tissues of the body from four individuals studied. Table S4. Summary of tissue-specific CNVs observed in one of the four individuals studied (KT538). Table S5. Summary of tissue-specific cn-LOH events (>5 Mb) observed in three out of the four individuals studied. Table S6. DNA concentrations (ng/μl) and quality parameters (260/280 and 260/230 nm ratios) for each tissue type and subject. (PDF 548 kb