Preliminary Screening Of Differentially Expression Profile Of Long Non-coding RNA In The Eutopic Endometrial Tissues Of Ovarian Endometriosis

?Objective?our study firstly aims to explore the expression of long non-coding RNAs?lncRNAs? in eutopic endometrial tissues of ovarian endometriosis by human gene microarray, constructing lncRNA expression profile and screening out specific lncRNAs.Simultaneously,we plan to predict the potential biological function of dysregulated lncRNAs by bioinfromatics techniques.In addition,in order to provide foundation for further study on the possible molecular mechanisms of ovarian endometriosis,We chose specific lncRNAs to verify their expression in eutopic endometrial tissues and cell lines by quantitative real-time polymerase chain reaction.?Methods?1.The eutopic endometrial tissues specimens from patients with ovarian endometriosis and normal endometrium specimens from patients with uterine fibroids were collected in Zhongda Hospital of Southeast University,which were confirmed by pathology.Study was approved by Ethical Committee of Zhongda Hospital of Southeast University.2.Human LncRNA Array v4,4x180 K was used to detect the expression of lncRNAs and mRNAs of three patients with ovarian endometriosis and three patients with uterine myoma,establishing the differential expression profiles of lncRNAs and mRNAs related to ovarian endometriosis.3.6 significantly differentially expressed lncRNAs?3 up-regulated and 3 down-regulated?in microarray results were chose randomly.QRT-PCR was performed in eutopic endometrial tissues from 12 patients with ovarian endometriosis and control endometrium from 12 patients with uterine fibroids to verify the validate of the microarray analysis results.4.Bioinformatics analysis,including:?1?Kycrto Encyclopedia of Genes and Genomes?KEGG? Pathway analysis and Gene Ontology?GO?analysis were used to analyze aberrant expressed mRNAs;?2?lncRNA-mRNA correlation analysis;?3?In order to explore the possible biological function involving in the differential expression of 1ncRNAs,the possible target genes of lncRNAs were forecasted.5.According to the results of bioinformatics analysis,We chose 3 abnormally expressed lncRNAs with potential biological significance and detected their expression both in tissue and cell lines.?Results?1.In total,845 lncRNAs and 1,793 mRNAs were found to differentially express between eutopic endometrial tissues from patients with ovarian endometriosis and normal endometrium from patients with uterine fibroids.In additional,312 lncRNAs were up-regulated and 533 lncRNAs were down-regulated.The most up-regulated was ENSG00000234665.3?fold-change:15.166?.The most down-regulated lncRNA was ENSG00000253978.1?fold-change:26.482?.1,433 mRNAs were up-regulated and 360 mRNAs were down-regulated.The most up-regulated was SEMA3E?fold-change:25.025?.The most down-regulated mRNAs was STEAP4?fold-change:32.799?.2.The results of qRT-PCR were consistent with the trend of microarray data.ENSG00000270346.1?ENSG00000259327.1 and ENSG00000231312.2 were up-regulated.XLOC₀11334?ENSG00000204054.7 and ENSG00000271738.1 were down-regulated.3.The results of bioinformatics analysis?1?The results of GO analysis showed that aberrantly expressed mRNAs were associated with many biologic processes,such as metabolic process,cell process,regulation and localization etc.Aberrantly expressed mRNAs were mainly located in some cell components including cell,cell membrane,cytoplasm and nucleus etc.Aberrantly expressed mRNAs were associated with many molecular functions,such as binding, protein binding,catalytic activity and ion binding etc.?2?Pathway analysis showed that 87 pathways corresponded to up-regulated transcripts and that enriched pathways included metabolic pathways,pathway in cancer,PI3K-Akt signaling pathway and focal adhesion etc.With regard to down-regulated transcripts,there were 38 pathways corresponded and enriched pathways included Tyrosine metabolism,Ras signaling pathway,complement and coagulation cascades and TNF signaling pathway etc.?3?lncRNA-mRNA correlation analysis:483 pairs of lncRNAs and mRNAs were identified to be coexpressed,with 253 pairs presented as positive correlation and 230 pairs presented as negative correlation.LncRNA ENST00000538219.1-mRNA CYCS was the highest negative correlated lncRNA-mRNA pair.LncRNA ENST00000558838.1-mRNA PCSK6 was the highest positive correlated lncRNA-mRNA pair.?4?The results of GO analysis indicated that target genes of aberrantly expressed lncRNAs were associated with many biologic processes,such as metabolic process,signal transduction,regulation,immune system process,response to stimulus and cell response etc.Aberrantly expressed mRNAs were mainly located in some cell components including extracellular matrix,organelle,cell junction and membrane etc.Aberrantly expressed mRNAs were associated with many molecular functions including catalytic activity, enzyme regulator activity,binding and antioxidant activity etc.The results of pathway analysis indicated that target genes of aberrantly expressed lncRNAs were associated with many signaling pathways,including Mucin type O-Glycan biosynthesis,Thyroid hormone signaling pathway,metabolic pathway,pathways in cancer,p53 signaling pathway,mTOR signaling pathway and VEGF signaling pathway etc.4.The results of qRT-PCR showed that 3 up-regulated lncRNAs?RP11-363G2.4 ?RP11-548O1.3 and WAC-AS1?significantly up-regulated both in eutopic endometrial tissues from patients with ovarian endometriosis and human eutopic endometrial stromal cells.RP11-363G2.4?RP11-548O1.3 and WAC-AS1 were involved in a series of biologic processes and signal transduction pathway related to ovarian endometriosis.?Conclusion?1.We successfully constructed ovarian endometriosis related lncRNA expression profile and screened a lot of aberrantly expressed lncRNAs.These lncRNAs may play an important role in the occurrence and development of ovarian endometriosis by regulating a series of signaling pathways and biological processes.2.RP11-363G2.4?RP11-548O1.3 and WAC-AS1 might be involved in the occurrence anddevelopment of ovarian endometriosis.These dysregulated lncRNAs deserved further study on their functions and related mechanisms in the eutopic endometrial stromal cells of ovarian endometriosis.