Diagnostic potential of differentially regulated microRNAs among endometriosis, endometrioid ovarian cancer, and endometrial cancer

Endometriosis is a most frequent estrogen-dependent gynecological disease, in which ectopic endometrium develops outside the uterine cavity with an array of symptoms such as dysmenorrhea, dyspareunia, pelvic pain, and infertility.[] Endometriosis shows similarity to malignancies in certain ways such as metastasizing tendency, escalating, invasive, and estrogen-dependent growth with chances of reappearance.[] Exact mechanism and etio-pathology of endometriosis is still ambiguous. Risk of developing ovarian cancer increases in women having endometriosis.[] Around 15%–50% of clear-cell and endometrioid type of ovarian tumors were associated with endometriosis and there are two-to-three-fold risk of developing ovarian carcinoma in patients having endometriosis in their early age.[] However, endometrioid ovarian cancers share many molecular features with endometrioid endometrial cancers as well;[] therefore, it is plausible that endometriosis may also increase the risk of developing endometrial cancer at later age. MicroRNAs (miRNAs) are small approximately 22–23 nucleotide long endogenous, noncoding RNAs which posttranscriptionally regulates gene expression through either inhibition of mRNA translation or less frequently degradation of mRNA.[] Numerous recent studies showed that differential miRNA expression might be one of the important factors in the pathogenesis and progression of endometriosis as well as endometrioid subtypes of endometrial and ovarian carcinomas[] and altered miRNA expression are involved in initiation, progression, and transformation of endometriosis as well as ovarian and endometrial cancer. There are several studies which states specific miRNAs may act as promising noninvasive biomarkers in the molecular diagnosis of these three diseases.[] From the previous published studies, we selected six miRNAs (miR-16, miR-20a, miR-99b, miR-125a, miR-143, and miR-145) which besides targeting the genes of interest also play key role in the pathogenesis of endometriosis, endometrioid ovarian cancer, and endometrioid endometrial cancer separately.[] We hypothesized that these miRNAs would be involved in regulation of common processes such as hypoxia, inflammation, angiogenesis, and cell proliferation which are common in pathophysiology of endometriosis, endometrioid carcinoma of the ovary, and endometrioid endometrial cancer and may act as important diagnostic tools and potential link in associating these three diseases with other genetic as well as epigenetic factors. Therefore, in the present study, we compared the differential expression of these six miRNAs and some of the common target genes of these miRNAs and tried to find the diagnostic potential of these miRNAs in discriminating these three debilitating diseases.

Sample collection and ethical clearance The present study was carried out from 2016 to 2019 for the period of 3 years. Ethical approval of the study has been obtained from the “Institutional Ethics Committee” of the Institutes. Histopathologically, confirmed cases (n = 40) were enrolled in the study of which Group I comprises; Group-Ia: Subjects with endometriosis (n = 10), Group-Ib: Subjects with endometrioid type of ovarian cancer (n = 7 [fresh] +3 Formalin-fixed paraffin-embedded [FFPE] tissues) Group-Ic: Subjects with endometrioid endometrial cancer (n = 10), and Group II comprises: Controls with healthy endometrium (n = 10), with no records of tumor complication. After taking written informed consents from the patients, tissue samples were collected in vials containing RNA later at PGIMER Gynae Operation Theater at the time of laparoscopy/laparotomy/hysterectomy and stored at −80°C till further experiments. FFPE tissue blocks of endometrioid carcinoma of ovary (n = 3) were also obtained. Baseline characteristics and clinical data of all the study groups are shown in Table 1. MicroRNAs rich RNA isolation and cDNA synthesis miRNA rich RNA isolated from the tissue samples by using commercially available miRNeasy® Mini Kit (Cat # 217004, Qiagen India Pvt., Ltd., New Delhi, India) according to the manufacturer's instructions. From the FFPE tissue samples, RNA was extracted by using recover all total nucleic acid isolation kit for FFPE (Cat # AM1975, Invitrogen, USA) using manufacturer's instructions. Total 1 μg/μl of RNA (for fresh tissue) and 200 ng/μl RNA (for FFPE tissue) was reverse transcribed by miScript II RT Kit (Cat # 21816, Qiagen India Pvt., Ltd., New Delhi) for cDNA synthesis. Quantitative reverse transcription-polymerase chain reaction to check microRNAs expressions and mRNA levels of target genes The expression patterns of all selected miRNAs (miR-16, miR-20a, miR-99b, miR-125a, miR-143, and miR-145) were analyzed by real-time polymerase chain reaction (PCR) (Light Cycler LC96 Real-Time PCR system, Roche applied science, USA) by using miScript SYBR Green PCR Kit, (Cat # 218073, Qiagen, India Pvt., Ltd., New Delhi) with specific miRNA primer assays for each miRNA (miScript Primer assays, Qiagen, India Pvt., Ltd., New Delhi). U6 snRNA was used as an internal reference control. Furthermore, the expression pattern of some of the target genes of previously selected miRNAs promoting angiogenesis and inflammation, i.e., vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1A (HIF1A), cyclooxygenase 2 (COX2), and tumor necrosis factor (TNF) were quantified by using same method. Glyceraldehyde 3-phosphate dehydrogenase and β-actin housekeeping genes were used as reference controls. Melting curve and cycle threshold values were obtained, and the relative quantification of all miRNAs and target mRNA expression was determined by 2−ΔΔCt method.[] Primers used for individual miRNA/gene and their annealing temperature are summarized in Table 2. Statistical analysis Statistical software IBM SPSS Statistical Package 22.0 (IBM, Armonk, New York, US) was used to perform all the statistical analysis. Data were represented as mean ± standard deviation (SD) or SD from standard error of the mean as appropriate. Kolmogorov–Smirnov test was used to check the normality of the data. The expression levels of miRNAs and mRNA levels of their target genes between control group and three disease groups were compared using Kruskal–Wallis test and within groups by Mann–Whitney test as appropriate. To check the diagnostic potential of all differentially expressed miRNAs, receiver operating characteristic (ROC) curve analysis was performed on all miRNAs to investigate the diagnostic accuracy of these miRNAs in three diseases and also for each individual disease.

Expression of miR-16, miR-20a, miR-99b, miR-125a, miR-143, and miR-145 in all study groups We have observed statistically significant downregulated expression of miR-16 and miR-20 P=0.000 or P < 0.001 in endometriosis, endometrioid type of endometrial cancer, and ovarian cancer study participants as compared to controls. Expression of miR-99b, miR-125a, and miR-143 were found to be statistically upregulated in all three diseased study participants as compared to controls P=0.000 or P < 0.001 as shown in Figure 1. miR-145 was significantly upregulated in endometriosis and endometrioid carcinoma of the ovary P=0.000 or P < 0.001, but in endometrioid endometrial cancer, its levels were significantly downregulated P=0.000 or P < 0.001. Evaluation of target gene prediction for all selected microRNAs Using miRWalk 3.0 and Target Scan online tools we have predict the target genes for selected miRNAs, i.e., miR-16, miR-20a, miR-99b, miR-125a, miR-143, and miR-145. Each micro RNA targets thousands of genes, but we mainly focus on the genes promoting angiogenesis and inflammation which are the main regulatory processes in the pathogenesis of endometriosis and endometrioid type of ovarian and endometrial cancer. miR-16 and miR-20a targets many genes operational in hypoxia such as HIF1A, inflammation (COX2 and TNF), and angiogenesis (VEGF). Whereas, miR-99b, miR-125a, miR-143, and miR-145 mainly targets the genes involved in cell proliferation, tumor suppression, and tissue remodeling pathways. Relative mRNA levels of target genes of selected microRNAs in all study subjects

of the present study revealed that mRNA levels of VEGF, HIF1A, COX2, and TNF were statistically significantly increased in endometriosis, endometrioid carcinoma of the ovary and endometrioid endometrial cancer P=0.000 or P < 0.001 as compared to controls as shown in Figure 2. Assessment of diagnostic potential of selected microRNAs using receiver operating characteristics curve analysis To check the diagnostic potential of these six miRNAs expression for endometriosis, endometrioid ovarian, and endometrial cancer, ROC analysis was done [Figure 3]. For endometriosis disease, miR-99b and miR-125a showed the highest area under curve (AUC) 0.950 (95% confidence interval [CI], 0.857–1.000, P = 0.000), and 0.733 (95% CI, 0.551–0.914, P = 0.041), respectively. We have calculated sensitivity and specificity for miR-99b and miR-125a expression levels. miR-99b displayed sensitivity and specificity of 90% and 95%, respectively, at the cutoff value of 2.945 and miR-125a displayed sensitivity and specificity of 90% and 60%, respectively, at the cutoff value of 2.15 in discriminating endometriosis from endometrioid ovarian carcinoma and endometrioid endometrial cancer. For endometrioid carcinoma of the ovary, miR-143 showed the highest AUC of 0.933 (95% CI, 0.842–1.000, P = 0.000). It displayed sensitivity and specificity of 90% and 80%, respectively, at the cutoff value of 3.89 in discriminating endometrioid carcinoma of the ovary from the other two diseases. For endometrioid endometrial cancer, miR-16, miR-99b, and miR-145 gives the highest AUC values of 0.815 (95% CI, 0.620–1.000, P = 0.006), 0.920 (95% CI, 0.820–1.00, P = 0.000) and 0.985 (95% CI, 0.950–1.00, P = 0.000), respectively. Here, miR-16 displayed sensitivity and specificity of 80% and 90%, respectively, at the cut-off value of 0.255. Sensitivity and specificity for miR-99b were calculated 80% and 90%, respectively, at the cut-off value of 1.93 and for miR-145, sensitivity and specificity showed values 90% and 85%, at the cut-off value of 1.00 in discriminating endometrioid endometrial cancers from other two diseases. Tables 3–5 represent the AUC for miR-16, miR-20a, miR-99b, miR-125a, miR-143, and miR-145 expression levels in all three diseases.

In the present study, we evaluated the expression of miR-16, miR-20a, miR-99b, miR-125a, miR-143, and miR-145 in the tissue samples of endometriosis, endometrioid ovarian cancer, and endometrioid endometrial cancer along with the expression of inflammatory and angiogenesis promoting genes (VEGF, HIF1A, COX2, and TNF). We observed significant differential expression of miRNAs under consideration along with the significant upregulated expression of genes. Further, ROC curve analysis was also done to check the diagnostic potential of study miRNAs in differentiating these diseases for their early diagnosis and finding possible molecular mechanisms associating with these three diseases. Since their discovery, miRNAs have been shown to play an important role in various physiological processes and their role in cancer development is also widely known. Several papers have been published on the possible role of miRNAs in pathogenesis endometriosis, endometrioid ovarian carcinoma, and endometrioid endometrial carcinoma,[] but there is lack of much research in finding miRNAs which can actually discriminate these three diseases and helps in better diagnosis and deciding treatment strategies. For the first time, the role of miRNAs differential regulation in human cancer development was shown in a type of leukemia (B-cell chronic lymphocytic leukemia), where miR-15/16 expression was downregulated.[] Downregulation of miR-16 was also observed in the present study in endometriosis and endometrioid type of ovarian and endometrial cancers as compared to healthy controls. ROC curve analysis showed that miR-16 was a good predictor for endometrioid endometrial cancer with AUC of 0.815 (P = 0.006). It displayed a sensitivity and specificity of 80% and 90%, respectively, at the cutoff value of 0.255. We also predicted targets of miR-16 by miRwalk and Target scan online tools, and VEGF, HIF1A, and TNF genes were found to be targets of miR-16. Furthermore, the mRNA levels of these target genes were found to be upregulated in endometriosis, endometrioid ovarian cancer, and endometrioid endometrial cancer (P = 0.000). Thus, we can say that the downregulation of miR-16 might play an important role in inducing hypoxia, inflammation, and angiogenesis in these three diseases and miR-16 might act as good diagnostic biomarkers for endometrioid endometrial cancer. In the present study, expression of miR-20a was found to be significantly decreased in endometriosis, endometrioid ovarian cancer, and endometrioid endometrial cancer (P = 0.000) as compared to control samples. Downregulation of miR-20a was previously confirmed by many studies in endometriosis.[] We also predicted the targets of miR-20a by miRwalk and Target Scan online tools and HIF1A, TNF, and VEGF genes were found to be the targets of miR-20a. Furthermore, the mRNA levels of these target genes of miR-20a were significantly upregulated in endometriosis, endometrioid ovarian cancer, and endometrioid endometrial cancer as compared to controls. Thus, we can say that miR-20a downregulation might be the promoter of hypoxia, inflammation, and angiogenesis in these three diseases and plays an important role in the pathogenesis of these three debilitating diseases. Further, in this study, miR-99b was found to be significantly upregulated in endometriosis as well as endometrioid ovarian and endometrial cancer study participants (P = 0.000) as compared to controls. According to studies of Ohlsson Teague et al., 2009, upregulated expression miR-99b was found in ectopic endometriotic tissue as compared to control healthy endometrium and surfaced as an important player in the pathogenesis of endometriosis by targeting angiogenic pathways.[] miR-99b belongs to the miR-99 family, which consists of three members, miR-99a, miR-99b, and miR-100. Several studies have shown that the miR-99 family regulates cell survival, cell stress response, proliferation, angiogenesis, DNA damage, and wound healing process.[] In the present study, ROC curve analysis revealed miR-99b as a good predictor for endometriosis showing AUC of 0.950 (95% CI, 0.857–1.000, P =0.000) and displayed sensitivity and specificity of 90% and 95% respectively, at the cut-off value of 2.945. Based on our observation and evidence from the literature, miR-99b seems to be important in the pathogenesis of endometriosis with good diagnostic potential in discriminating endometriosis from the rest of the two cancerous diseases. However, the role of overexpression of miR-99b in endometrioid carcinoma of the ovary and endometrioid endometrial cancer is still not fully understood and more research in this field would definitely help in better understanding of this miRNA family in the pathogenesis and progression of these two cancers. In the previous studies, miR-125a was upregulated in endometriosis which would repress Erythroblastic leukemia viral oncogene homologue (ERBB) signaling as well as control cell migration and invasion.[] In the present study, miR-125a was observed to be upregulated in endometriosis along with AUC of 0.733 (95% CI, 0.551–0.914, P = 0.041) and thus differentiate endometriosis from endometrioid carcinoma of the ovary and endometrioid endometrial cancer. We also observed upregulated expression of miR-125a in endometrioid subtypes of ovarian and endometrial cancer which are in contrast with some previous studies with downregulated expression in the same disease.[] Since the endometrioid histological subtype of ovarian and endometrial cancer share many similarities with endometriosis; hence, miR-125a upregulation suggests some common molecular pathogenesis of these three diseases. However, more directed research is required to find out the exact role of miR-125a in the pathogenesis and progression of endometrioid subtype of ovarian and endometrial cancer. Further, miR-143 and mir-145 belong to the miR143/miR145 cluster which usually coexpressed in many types of cancers.[] In the present study, miR-143 was significantly upregulated in endometriosis, endometrioid ovarian carcinoma and endometrioid endometrial carcinoma (P = 0.000), but miR-145 was significantly upregulated in endometriosis and endometrioid carcinoma of the ovary (P = 0.000) and significantly downregulated in endometrioid endometrial cancer (P = 0.000). Both miR-143 and miR-145 were upregulated in endometriosis[] with a predicted role in suppression of cell proliferation by repressing growth-promoting proteins mitogen-activated protein kinase 7[] and Kirsten rat sarcoma viral oncogene homologue (KRAS),[] thereby, helps in cell survival of endometriotic cells. In endometriosis, increased expression of miR-145 may regulate several important processes such as the growth of the endometriotic lesion, invasiveness, and long-term establishment of the lesion by downregulation of its target genes such as JAM-A, PAI-1 etc.[] Thus, we conclude that miR-143 and 145 might play a significant role in the pathogenesis and progression of endometriosis. In addition, ROC curve analysis revealed miR-143 as a good predictor of endometrioid carcinoma of ovary with AUC of 0.933 (P = 0.000, 95% CI, 0.842–1.000) and displayed sensitivity and specificity of 90% and 80%, respectively, at the cutoff value of 3.89 in discriminating endometrioid ovarian cancer, from endometriosis and endometrioid endometrial cancer. Zhang et al. concluded that, upregulated expression of miR-143 results posttranscriptional suppression of its target gene (fibronectin type III domain-containing 3B) and results in enhanced cell invasion and migration.[] Corroborating to this, we can also speculate aberrant miR-143 as an imperial player in progression and pathogenesis of endometrioid type of ovarian cancer and may act as an important noninvasive biomarker for this particular disease. Future studies in this direction would be helpful in finding the possible role of miR-143 in the pathogenesis of these three associated diseases. ROC curve analysis revealed that miR-145 is a good predictor for endometrioid endometrial cancer with AUC of 0.985 (95% CI, 0.950–1.000, P = 0.000) in discriminating endometrioid endometrial cancer from rest of two diseases. Downregulation of miR-145 has been reported in endometrial cancer with possible role in enhanced expression of its target genes such as SOX11 which is tumorigenic in nature.[] Hence, current findings enhance the possibility of the pivotal role of miR-145 in the pathogenesis and progression of endometrial cancer and its potential use as a biomarker for this specific disease. However, the validation of target genes of these differentially expressed miRNAs is necessary to estimate the role of these miRNAs in posttranscriptional regulation of hypoxia, angiogenesis, and inflammation in endometriosis and endometrioid subtypes of ovarian and endometrial cancer. The sample size of the present study was also small to give concluding statements but future studies on the large population will further reveal the role of these miRNAs as diagnostic and therapeutic targets. Although thousands of miRNAs are involved in the complex pathogenesis of endometriosis, endometrioid ovarian and endometrial cancer, definitely there might be some common molecular denominators that may associate these three complex disorders. It seems very necessary to accurately diagnose them in deciding treatment strategies.

In summary, we can conclude that, studied miRNAs can act as potential biomarkers for early detection and discrimination of these three ambiguous diseases. Observations of present study pointed significant operational leads, which can be further targeted and help in deciding accurate biomarkers for these three debilitating diseases in age specific manner. More importantly, a precise strategy should be set up for better prevention, early detection, specific diagnosis, and treatment targeting molecular pathogenesis to understand the mechanism of endometriosis and associated cancer subtypes. Financial support and sponsorship DST-SERB (ECR/2016/000359), UGC JRF (22/06/2014(i) EU-V) New Delhi, India and UGC CAS-II (F.4-28/2015 [CAS-II] [SAP-II]) New Delhi, India. Conflicts of interest There are no conflicts of interest.

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