Transcriptome Analysis Of Human Endometrial Stromal Cells Reveals CNTN1 As A Potential Biomarker In Endometriosis

BackgroundEndometriosis is a common chronic inflammatory disease in gynecology,which ischaracterized by the appearance of endometria-like tissue outside the uterine cavity and similar periodic growth,proliferation and destruction of the endometrium^[1].The disease varies from superficial peritoneal and serous lesions to ovarian endometriosis cysts and deep nodules larger than 5mm（deep endometriosis）,often accompanied by tissue adhesions and fibrosis.The growth of ectopic endometrium is estrogen-dependent,so the disease mainly occurs during the child-bearing years.It has occasionally been reported in pre-menarche girls^[2],and post-menopausal women may also relapse.Although endometriosis is a benign disease,it has similar adhesion,invasion andmetastasis characteristics with malignant tumors^[3].Endometriosis can be divided into three subtypes according to its histopathology and anatomic location:superficial endometriosis,deep infiltrating endometriosis and endometriosis cysts（chocolate cysts/cisterns/endometriosis）.Among them,ovarian endometriosis is the most common.According to statistics,the incidence of endometriosis accounts for about 5%～10%of women of childbearing age,and about 176 million women in the world are affected by this disease^[5].The onset age of endometriosis is mainly between 25 and 45 years old,and the proportion of endometriosis in patients with chronic pelvic pain and dysmenorrhea is 20%to 90%,and the proportion of infertility can be 25%to 35%^[6].In 1%of cases of ovarian endometriosis,the risk of malignant degeneration is 2-4times higher than that of women without endometriosis^[7-10].This has brought great pain to the majority of women both physically and psychologically,and has become a heavy economic burden to the society.The etiology and pathophysiological mechanism of endometriosis are still not fullyunderstood.No theory,including the widely accepted theory of"meridional reflux",can explain all events of endometriosis.Multiple susceptibility factors such as genetics,hormones and environment can work together with immune inflammatory changes to promote the survival of endometrial cells outside the uterine cavity^[12].In recent years,some scholars have proposed that ectopic endometrial tissue may develop through three steps（adhesion,invasion and angiogenesis）,namely"3A theoretical model"^[18].According to this theory,the adhesion of the upstream endometrial cells to the extracellular matrix is the"primary stage"or"initial stage"of implantation in the theoretical model,which is consistent with the research viewpoints of Nisolle and Defrere et al.^[19,20].After the ectopic endometrium attached to the pelvic ectopic,it invaded the extracellular matrix,and then the new blood vessels provided necessary nutrients for the growth of the lesion^[21].Subsequently,a number of studies have confirmed the correlation between changes in adhesion function and the incidence of endometriosis.Studies on gene and function changes in ectopic endometrial tissue and in situ endometrial tissue have found that ectopic endometrial stromal cells undergo extensive metabolic reprogramming changes,increased invasiveness and adhesion,decreased apoptosis potential,and changes in immune function^[14].Local pelvic microenvironment can also change the gene expression of ectopic endometrium cells through epigenetic changes（DNA methylation,histone modification,mi RNA）,thus affecting the growth and differentiation ability of cells^[15,16].Therefore,it may be that the changes in the characteristics of the ectopic endometrium itself interact with the microenvironment to promote the occurrence and development of the disease.To understand how adverse currents of endometrium debris cause disease and what changes occur in the ectopic endometrium cells themselves,it is of great significance to compare gene expression and functional regulation between ectopic endometrium and normal endometrium tissue,and to explore the process of lesion formation.In this study,we selected ectopic endometrium from patients with ovarianendometriosis tissue and normal endometrium tissues from patients without endometriosis,with the help of a genome-wide expression profile analysis and comparison the transcriptional differences between the two group,in the hope of a better understanding of the pathogenesis of ovarian within different disease,for more accurate treatment strategy.Objective（1）To identify the difference between ectopic endometrial stromal cells and normalendometrial stromal cells;（2）To search for potential biomarkers and therapeutic targets for endometriosis.Methods（1）HE,immunohistochemistry and immunofluorescence staining are used to compare the tissue structure and stromal cell characteristics of ectopic endometrium and normal endometrium;（2）Flow cytometry are used to detect the proportion of ectopic endometrium and normal endometrium CD10-positive stromal cells;（3）MACS are used to sort CD10-positive stromal cells,and microarray probes are used to detect gene expression profiles of the two groups of cells;（4）GO and KEGG function enrichment analysis are used to annotate the differential gene functions of the two groups of cells;（5）Heat map cluster analysis and Wayne map analysis are used to screen for keydifferential genes;（6）RT-q PCR,q PCR and immunohistochemical staining are used to confirm the high expression of CNTN1 in ectopic endometrial tissues;（7）Protein interaction network analysis are used to screen for proteins that interactwith CNTN1.Results（1）Ectopic endometrial stromal cells and normal endometrial stromal cells have similar tissue structure and CD10 positive staining;（2）Gene expression profiles of CD10-positive stromal cells in ectopic endometrium and normal endometrium are similar;（3）Cell adhesion function changes most significantly in ectopic endometrial stromal cells;（4）The up-regulation of cell adhesion molecule CNTN1 in ectopic endometrial stromal cells is the most critical;（5）Multiple experiments have confirmed that CNTN1 molecules are significantly up-regulated in ectopic endometrial tissue.Conclusion（1）Compared with normal endometrium,the adhesion function in ectopicendometrium is significantly changed,which is closely related to the disease;（2）CNTN1 is the most important adhesion change molecule and a potential biomarker and therapeutic target for ovarian endometriosis.