Exosomal miRNAs as novel potential biomarkers for endometriosis

Background: Endometriosis (EMS) is a chronic gynaecological disease. Exosomal miRNAs appear to be associated with the progression of EMS, which suggest potential circulating biomarkers in EMS. Methods: Differential centrifugation, illumina small RNA sequencing, bioinformatics analysis and qRT-PCR are used to compare the pelvic uid of patients with a clinical diagnosis of EMS and matched controls. Results: Six miRNAs (miR-135a-5p, miR-196a-5p, miR-449b-5p, miR-4454, miR-4286, and miR-5100) showed signicant differences in the EMS group in initial screening (log2FC > 2). Receiver-operator curve (ROC) analysis further indicated miR-4454 (area under the curve (AUC) = 0.956, P < 0.05) and miR-5100(area under the curve (AUC) = 0.900, P < 0.05) were highly correlated with the pathogenesis of EMS. Validation on the samples from 10 patients and 10 healthy people. Conclusion: Our miRNA expression data provide altered expression and potential prospects for biomarkers in EMS. miR-5100) Q-PCR another samples. The results show that the levels of miR-196a-5p, and miR-449b-5p decreased signicantly (p < 0.05),while miR-4454 and miR-5100 increased signicantly (p < 0.05) in cyst uid exosomes with endometriosis patients. ROC curve analysis that AUC CI, and CI, Thses results


Introduction
Endometriosis (EMS), the presence of endometrial-like tissue outside the uterus, is a chronic gynaecological disease which affects approximately 10% of reproductive-aged women 1 .EMS morbidity has displayed a marked increasing trend in recent years 2 .It has been recognized as a precursor lesion of several types of malignancies and endometriosis-associated carcinoma, such as clear cell carcinoma, endometrioid carcinoma and seromucinous borderline tumor 3 .Patients suffering from EMS typically experience chronic pelvic pain, infertility, or both, but can also be asymptomatic. Unfortunately, the pathogenesis of EMS remains unclear. Laparoscopic visualization of lesions con rmed by histology 4 is the go-to-method for EMS diagnosis, and clinically de nitive diagnosis could take up to 11 years. Although CA125 and HE4 are widely used to monitor the occurrence of ovarian cancer, they are not speci c for the detection of EMS 5,6 . There is a clear need for noninvasive and sensitive methods for early and speci c diagnosis of EMS.
The recent discovery of exosomes presents a much-needed opportunity in this regard. Isolated from a variety of biological uid, including blood, saliva, urine, nasal secretions, breast milk, and cerebrospinal uid 7 , these nanosized vesicles function as intercellular communicators with the inclusion of active proteins, mRNA, microRNA (miRNAs), and DNA 8,9 , of which miRNAs are of particular interest in this capacity as they regulate gene expression at the post-transcriptional level by binding to 3'-untranslated regions (3'-UTRs) of the target mRNAs 10 . Recent studies have indicated that the level of exosomal miRNAs is strongly related to progression of certain diseases. For example, serum exosomal miR-126 and miR-125a-3p have been used as biomarkers for the diagnosis of non-small-cell lung cancer and earlystage colon cancer, respectively 11,12 .
In search for miRNAs as potential biomarkers of EMS, we are encouraged by the increasing amount of evidence that points to the important role of miRNAs in EMS progression 13 . For example, Zhang and coworkers reported that exosomal miRNA-138 could promote EMS through in ammation and apoptosis via the vascular endothelial growth factor (VEGF)/nuclear factor-kB signaling pathway 14 . Harp and coworkers demonstrated that exosomal miRNAs could contribute to EMS angiogenesis 15 , while Sun and coworkers revealed that EMS exosomes could promote neuroangiogenesis 16 . Most directly related to the development of biomarkers of EMS is the recent work by Zhang and coworkers. They found that the level of serum exosomal miR-22-3p and miR-320a is signi cantly higher in EMS patients than in the control group. Their potential uses as circulating biomarkers for EMS 17 is thus suggested.
Progress notwithstanding, we note that in these studies the exosomes used were from serum, most of which being platelet-and/or megakaryocyte-derived 18 . As such, information so obtained does not directly re ect on the microenvironment in the endometriotic lesion. However, studies have shown that the local microenvironment plays a pivotal role in the onset and progression of an endometriotic lesion.
We believe that the state of EMS could be more directly correlated with certain miRNAs isolated from exosomes extracted from pelvic uid (PF) due to its source. PF derive from macrophage secretions, ovarian exudate, re uxed tubal uid, serum transudate, and re uxed endometrial material via retrograde menstruation,which can re ect better the changes in the pelvic environment 13 . EMS exosmoses will appear in PF via some of the above uid to promote pathological processes. Analyzing exosomes extracted from PF may be better to reveal the EMS pathological processes.The results presented here are from our initial efforts to identify exosomal miRNAs from the pelvic uid or cyst uid of EMS patients. We found that miR-4454 and miR-5100 are strongly expressed in the EMS patients studied, which portends their potential use as novel biomarkers of EMS.

Patient and sample design
Two EMS patients were recruited for the present study from the Department of Gynaecology of Shenzhen Maternity & Child Healthcare Hospital in the period from July to December 2018. They are respectively30-50 years old, nonsmoker, and had not under any hormone therapy for at least 3 months prior to the present study. Their EMS state was detected by laparoscopy and further con rmed by histopathologic examination. Two infertile and EMS-free patients served as the negative control. Pelvic uid and cyst uid were collected in the surgical process, from which exosomes used in this study were extracted. The requirements of ethics of this study were approved by the Ethics Committee of Shenzhen Maternity & Child Healthcare Hospital. All participants were informed of the details of the project, signed and dated the Consent Form.
Isolation of exosomes from pelvic uid (PF) and cyst uid (CF) The exosomes, including those (Ctr1 and Ctr 2) from the control group and those of the EMS patients (CF1 and CF2 from the cyst uid; PF1 and PF2 from the pelvic uid) were obtained in the following manner with the temperature maintained at 4 °C at all stages of the sample preparation and handling: 6 mL of pelvic uid were collected from each of the four participants. The samples were stored in sterile centrifuge tube and subject to low-speed centrifugation. The resulting supernatant was collected, followed by centrifugation at 2000 × g (Eppendorf; 5418R) for 10 min. The supernatant thus obtained was collected and centrifuged at 10,000 × g for 20 min, and this last procedure was repeated once. The supernatant was then collected and diluted with an equal volume of 4% PEG6000 (Sangon biotech, China), and allowed to react for 5 min before centrifugation at 10,000 × g for 4 min.
Exosomes for the subsequent analyses were extracted by differential ultracentrifugation. Speci cally, the supernatant was rst thawed and then subject to sequential centrifugation at 13,000 g for 30 min, followed by centrifugation at 100,000 g for 70 min (Himac CS150GXII, HITACHI, Japan). The pellets were then suspended in 100 µL of phosphate-buffered saline and stored at − 80 °C for further analyses. Transmission electron micrographs (TEM) were obtained by using Hitachi Electron Microscope H-600 (Japan). The size of the serum exosomes was measured by using Nanosight analysis (Malver NanoSight 300 instrument, UK), while the protein markers of exosomes were detected by Western blotting.
Exosome RNA isolation and Small RNA sequencing Isolation of uid exosomal RNA was performed using QIAGEN exoRNeasy Serum/Serum Maxi Kit(Cat number: 77044). The quality and quantity of RNA were checked by using Agilent 2100 Bioanalyzer. The small RNA sequencing library was constructed with QIAseq miRNA Library Kit Cat number: 331505 following Manufacture's instruction. Small RNA sequencing was performed by using SE75 on mode Illumina NextSeq 500 sequencing platform.

Bioinformatics analysis of miRNA-seq data
The miRNA-seq data were quanti ed and tested for differential expression with eRNA published in 2014 19 . After raw data cleaning, sequences with a length more than 18 nt were aligned against miRBase (v22). The miRNA pro ling was normalized using reads per million (RPM) mappable miRNA sequences.
Differential expression analysis of the miRNAs was performed for EMS patients and health controls using DESeq2 R-package. Only miRNAs with fold-change ≧ 2 (p < 0.05 ) were considered to be differentially expressed miRNAs.The prediction of miRNAs targets was performed by using the online bioinformatics tool. Miranda and RNA hybrids were used to carry out complementary pairs of miRNAs and target genes.Software parameters are as follows: Free energy of miranda≤-20, score of miranda > 150, free energy of_RNA hybird≤-25, p value of RNA hybrid < 0.05. After obtaining the target genes of miRNAs, we performed GO function analysis (http://www.geneontology.org/) and KEGG (http://www.genome.jp/kegg/) pathway analysis. The GO terms with corrected p value ≤ 0.05 and the pathway with Q value ≤ 0.05 were considered as signi cant enrichment.
Validation of miRNA by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) In order to validate the miRNA-sequencing data, transcript levels obtained from miRNA-seq for six selected different miRNAs(miR-135a-5p, miR-196a-5p, miR-449b-5p, miR-4454, miR-4286, and miR-5100) were con rmed by qRT-PCR. The expression levels of target miRNAs were normalized to that of U6. The primers used for qRT-PCR are provided in supporting Table S4. According to the Manufacturer's instructions, all reactions were carried out using a SYBR Green Master Mix (SYBR Premix EX Taq, TaKaRa). PCR ampli cation was conducted in a total volume of 20 µL with the following procedures: 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. All reactions were performed in triplicate. Relative transcription levels were calculated using the 2-ΔΔCt method. Each measurement was performed in three biological replicates.

Statistical analysis
Statistical analysis was performed using SPSS 18.0. All qRT-PCR ampli cations were performed in triplicate. Data are expressed as the mean ± standard deviation. Differences in the miRNA expression pro le were analyzed using Student's t-test. P < 0.05 was considered statistically signi cant. We applied the area under the curve (AUC) to assess the diagnostic power of the predictors. AUC can be used as an accurate measurement of the diagnostic marker; the larger the AUC, the better the prediction model.AUC = 0.5 indicates no predictive power, whereas AUC = 1 represents a perfect predictive performance.

Characterization of exosomes
The morphological feature of exosomes was observed by transmission electron microscope. As shown in Fig. 1A, most of the photo-captured round-shaped microvesicles are of a size of about 100 nm in diameter with the membrane bounded. The size distribution of the serum exosomes is shown in Fig. 1B. The protein markers of exosomes (TSG101) are shown in Fig. 1C. The results indicated that exosomes can be successfully isolated from serum using this protocol with quality adequate for subsequent experiments.
Sequence analysis of small RNAs Thirteen sRNA libraries were generated from serum exosomes via high-throughput sequencing. We used Fast-QC software (http://www.bioinformatics. babraham.ac.uk/projects/fastqc/)for the overall evaluation of the quality of sequencing data and used BWA algorithm for small RNA mapping. There are 15548736, 13804825, 12383415, 16332234, 15327209, and 16970328 total reads in Ctr1, Ctr 2, CF1, CF2, PF1, and PF2 sRNA libraries, respectively ( Table 2). The corresponding clean reads of 2654371(17.07%), 2911044(21.09%), 643916(5.2%), 5843973(35.8%), 2587012(16.9%) were respectively obtained (Table 2) after the removal of the low-quality reads, including 3'adapter null, insert null, 5'adapter contaminants, reads smaller than 18nt, and reads containing ploy A. The miRNA rates in total RNA were 16.03%, 2.01%, 1.66%, 2.17%, 7.17%, and 2.09%, respectively. Differentially expressed miRNAs We applied DESeq2 algorithm to lter the differentially expressed genes. An miRNA candidate is considered to be differentially expressed if the level between the two experiment groups shows a statistically signi cant difference (p < 0.05) of at least two folds. Compared with CF group, 54 types of miRNAs are down-regulated, and 154 are up-regulated in PF group. (Fig. 2a). The differently expressed miRNA pro les (Fig. 2B) were obtained from comparative analyses of the exosomes in the three groups (control. CF, and PF) according to the value of log2FC > 2 miRNA whose expression level was respectively highest or lowest. As shown in Table S3, the top ve most down-regulated miRNAs in the patient group were miR-135a-5p, miR-196a-5p, miR-34b-3p, miR-100-5p, and miR-125b-5p, while the top ve most upregulated miRNAs were miR-4454, miR-4286, miR-5100, miR-7977, and miR-624-5p. Validation miRNA expression by q-PCR To further validate our miRNA pro ling results, we screened 10 patients and 10 healthy people for validation of miRNA expression levels.We found that the expression trends of all miRNAs determined by q-PCR were identical to those obtained from the RNA sequencing (Fig. 3). Compared with the control group, the expression levels of miR-196a-5p and miR-449b-5p decreased, whereas those of miR-4454 and miR-5100 increased, both to a signi cant degree (p < 0.05). These observations suggest that miR-196a-5p, miR-449b-5p, miR-4454, and miR-5100 were involved in the pathogenesis of EMS and that they may serve as potential biomarkers of EMS. ROC curve analysis of exosomal miRNA as diagnostic biomarkers for EMS We subsequently used ROC curve analysis to assess the potential of the 6 miRNAs (Table 3) as EMS biomarkers. The AUC of miR-135a-5p, miR-196a-5p, miR-449b-5p, miR-4454, miR-4286, and miR-5100 were 0.53 (95% CI, 0.400-0.800), 0.570 (95% CI, 0.700 − 0.600), 0.680 (95% CI 0.600-1.000), 0.956 (95% CI, 0.800-1.000), 0.520 (95% CI, 1.000-0.200), 0.900 (95% CI, 0.800-0.900), respectively, suggesting that miR-4454 and miR-5100 from serum exosomes hold a high potential as biomarkers of EMS. Discussions EMS has been described as a cancer-like process, involving cell migration and invasion. Its pathogenesis remains poorly understand, and related research ndings and conclusions are controversial. The gold standard of EMS diagnosis is direct visualization of lesions at surgery, preferably coupled with histologic con rmation of endometrial glands and stroma in biopsies of suspected lesions. Compared with a minimally invasive diagnostic procedure, the surgical diagnosis has multiple drawbacks, including possible organ damage, hemorrhage, infection, adhesion formation, in addition to common anesthetic complications 20 . EMS can be asymptomatic or misdiagnosed in the general population, and diagnosis is always delayed, leading to di culties in medical and surgical treatments 21 . At the endometriosis deteriorating stages, the severity of pelvic pain may lead to hysterectomy that is often with oophorectomy 22 . Furthermore, increasing evidence exists for the malignant transformation of ovarian endometriomas to ovarian cancer, particularly the clear cell and endometrioid subtypes 23 . Earlier detection and timely therapeutic follow-up with noninvasive biological markers with good speci city for endometriosis is thus considered to be ultimately necessary.
With the hope of identifying a feasible biomarker for early diagnosis of EMS, we extracted miRNAs from the pelvic uid and cyst uid of EMS patients and analyze their pro les using miRNA sequencing. To our knowledge, this is the rst report of changes in pelvic uid and cyst uid miRNA expression in EMS. Moreover, we selected six (miR-135a-5p, miR-196a-5p, miR-449b-5, miR-4454,miR-4286, and miR-5100) of the above miRNAs for Q-PCR veri cation in another endometriosis samples. The results show that the levels of miR-196a-5p, and miR-449b-5p decreased signi cantly (p < 0.05),while miR-4454 and miR-5100 increased signi cantly (p < 0.05) in cyst uid exosomes with endometriosis patients. ROC curve analysis showed that the AUC of miR-4454 and miR-5100 were 0.956 (95% CI, 0.800-1.000) and 0.900 (95% CI, 0.800-0.900). Thses results suggest that miR-4454 and miR-5100 from serum exosomes hold great potential as biomarkers for EMS diagnosis.
Our study rst identi ed a number of miRNAs from the exosomes isolated from the cyst uid of EMS patients and then veri ed them in the serum exosomes of a second group of EMS patients. Different from Zhang 14 studies in serum, we believe that the miRNAs are more directly correlated with the development of EMS for the following two reasons: First is the the complexity of miRNA biogenesis, miRNA secretion, and variation in miRNA expression pro les according to variation epigenetic and environmental factors and the other the expression of lesion exosomal 24 . RNA was at high and stable levels as compared with Page 9/16 that of the plasma samples, possibly due to RNA degradation associated with the ribonucleases present in plasma 25,26 .
Furthermore, our study has several limitations. First, only a small number of miRNAs were veri ed by Q-PCR, limitations due to small sample size and suboptimal characterization of specimens. Second, this study lacked functional experiments at the cellular and molecular levels to identify the relationship between these miRNAs and endometriosis, further studies are needed to develop more clinical tests. Speci cally, a using miRNAs identi ed as speci c biomarkers will application in early detection of EMS

Conclusion
Our results demonstrated that miR-196a-5p, and miR-449b-5p were decreased signi cantly, miR-4454 and miR-5100 were increased signi cantly in endometriosis patients. Serum miR-4454 and miR-5100 may play a regulatory role in endometriosis processionand thus hold great potential as biomarkers for the early diagnosis of EMS. Ethics approval and consent to participate Approval for the study was given by the ethical committee of Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences, SIAT-IRB-170315-H0157. Written consent for participation and data presentation was received from the patients who volunteered to take part in the study.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysis during this study are included in this published article and its Additional les.