Study Of Differentially Expression Profile Of Long Nocoding RNAs And MRNAs In The Eutopic Endometria Of Adenomyosis

Background and perposesAdenomyosis is a common condition that causes dysmenorrhea, abnormal uterine bleeding and may also cause infertility and miscarriages in women in their reproductive years, which severely affect their health and quality of life. The exact pathogenesis of adenomyosis is still unknown.Several theories regarding the pathogenesis of adenomyosis have been postulated. The downward extension of the endometrium from the uterine cavity to the myometrium of the uterus is mostly accepted. Previous research identified a series of alterations at the molecular level in the eutopic endometria of women with adenomyosis, which include genetic factors, hormonal factors, immunologic factors and metabolic factors. However, coding gene-related researches are fragmented, a systematic omics approach to screen the alterations of the coding gene in the eutopic endometrium of adenomyosis will be helpful for revealling the mechanism of the disease. Previous studies have demonstrated that genetic factors play pivotal roles in the pathogenesis of adenomyosis, partially through epigenetic regulation. Studies have shown that long non-coding RNA (lncRNAs) as functional regulatory elements play a very important role in the regulation of eukaryotic gene expression and participate in a variety of important signal transduction regulation. Abnormal expression of lncRNAs closely relates to a variety of benign or malignant diseases. However, to date there has been no study of lncRNAs expression profiles or their functional roles in adenomyosis.In our study, we firstly plan to construct the lncRNAs and mRNAs expression profile in eutopic endometria from adenomyosis patients and from that of normal control subjects. In addition, we conduct bioinformatics analysis and construct co-expression networks to find the potential core genes associated with adenomyosis pathogenesis. Then we measure the expression of several potential core genes and provides new foundations for further mechanistic studies in this enigmatic disorder.Materials and Methods1. The expression of lncRNAs and mRNAs in the eutopic endometrium in women with versus without adenomyosis were detected by human gene microarray (where the sample size in each groups were 4 cases) and the differently expression profiles of lncRNAs and mRNAs were conducted.2. Six differentially expressed lncRNAs in the microarray results were chosen for quantitative real-time polymerase chain reaction validation to further demonstrate the consistency with the microarray.3.Bioinformatics analysis was done for further investigation of the differentially expressed lncRNAs and mRNAs.3.1 GO-Analysis:The Gene Ontology analysis were applied to determine the significant, low false positive rate and targeted GO function that the differentially expressed genes involved.3.2 Pathway-Analysis:Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways analysis were applied to determine the significant, low false positive rate and targeted pathway that the differentially expressed genes involved.3.3 Construction of co-expression networks:To identify the interactions between gene and lncRNA, we constructed a lncRNA-mRNA co-expression network for each group. This co-expression network was built according to the normalized signal intensity of specific expression in coding gene and lncRNA. While considering different networks, core regulatory factors were determined by the degree differences between two class samples.4. Quantitative real-time polymerase chain reaction and immunohistochemistry were used to measure the expression levels of TLN1 and CCND2 in the eutopic endometria of women with adenomayosis.Results1. In total,165 lncRNAs and 612 mRNAs were identified with differential expression between endometria from patients with and without adenomyosis. Among these lncRNAs,117 were significantly downregulated and 48 were significantly upregulated. n333955 was the most significant downregulated lncRNA (Fold-change:0.0032) and n342839 was the most significant upregulated one (Fold-change:4.13). Among these mRNAs,414 were significantly downregulated and 198 were significantly upregulated. The most down-regulated and up-regulated mRNA transcripts were HBAl (Fold-change:0.02) and THBS2 (Fold-change: 5.14).2. Six chosen lncRNAs were validated by quantitative real-time polymerase chain reaction. The result showed that n333955, n337373, n338909 were decreased expression in the eutopic endometria of adenomyosis, whereas n341651, n342794, n387706 were increased expression which was consistent with the trend of microarray.3. The result of bioinformatics analysis3.1 In the GO analysis, upregulated transcripts were highly enriched for extracellular matrix organization endothelial cell differentiation、intracellular signal transduction、 positive regulation of cell migration、angiogenesis. The highest enriched GOs targeted by under-regulated transcripts were gene expression、DN A replication、cell division、Gl/S transition of mitotic cell cycle、immune response.3.2 Pathway analysis showed that 40 pathways corresponded to up-regulated transcripts and that the enriched pathways included MAPK signaling pathway、Focal adhesion、 Adherens junction、ECM-receptor interaction、PI3K-Akt signaling pathway. With respect to down-regulated transcripts, there were 39 pathways corresponded and enriched pathways included DNA replication、Cell cycle、Primary immunodeficiency、Cytokine-cytokine receptor interaction.3.3 Based on the normalized signal intensity of specific expression in coding gene and lncRNA, we respectively constructed a coding-noncoding gene co-expression network for each group. The structure of the co-expression networks of endometria samples with and without adenomyosis were significantly different. By comparing the differences between them, we have got a list of lncRNAs and mRNAs that may closely related to the pathogenesis of adenomyosis, including TLN1、n342794、 MYBL1、CCND2、ENST00000406939、n338909.4. The gene and protein expression levels of TLN1 in the eutopic endometria of women with adenomyosis were significantly higher than in the control endometria. The gene and protein expression levels of CCND2 in the eutopic endometria of women with adenomyosis were significantly lower than in the control endometria.Conclusions1. We firstly constructed the lncRNA and mRNA differently expression profile in the eutopic endometria from patients with adenomyosis and in the control endometria from subjects without adenomyosis at the microarray level.2. Our results of qRT-PCR demonstrated a strong consistency with the microarray, thus proving the reliability of our microarray data.3. Bioinformatics analysis could be used for investigation of the role of the differentially expressed lncRNAs and mRNAs in the pathogenesis of adenomyosis.4. TLN1 and CCND2, which were potential core genes, were aberrantly expressed in in the eutopic endometria of women with adenomyosis.