Endometrial cancer (EC) is a common malignant gynecological tumor where few biomarkers are useful for early and accurate diagnosis. Serum miRNAs, considering their stability, maybe an effective and minimally invasive diagnostic method. Circulating miRNAs have been reported to have diagnostic significance in cases of EC. Jia W. and his colleagues were the first study using a genome wide serum miRNA expression profiling analysis who found a four-miRNA signature, including miR-222, -23, -186, and − 204 that might serve as a non-invasive approach for EC diagnosis. [34]
In the current study, relative expression of miRNA 27a and miRNA150-5p were both significantly relatively overexpressed in serum of endometrial cancer patients than serum of control. This suggests the potential role for both micro RNAs in distinguishing between endometrial cancer and healthy females. (p < 0.001).
Upregulation of miRNA 27a in endometrial cancer patients is in accordance with previous studies. Mozos et al., observed upregulation of miRNA 27a expression in invasive endometrioid adenocarcinoma tissue [35]. Our findings further complete this work as we confirm the upregulation of miRNA 27a in the serum of these patients which proposes it as a strong non-invasive biomarker. Also, we note that miRNA 27a was significantly associated with the same type of endometrial cancer (type I endometroid). [35] reported that miRNA 27a is one of three miRNAs miR-15b, -27a, and − 22 that might have their unique pathway to regulate the development of EC having diagnostic role [36]
On the other hand, in cervical cancer, decrease in expression of miRNA 27a was documented. Using miRNA array and small RNA sequencing, host miRNAs were specifically regulated by viruses, HPV16 and HPV18 in organotypic raft cultures vaginal keratinocytes. Viral oncoprotein E6, E7 decreased miRNA 27a expression [37]
In a trial to understand the role of miRNA 27a as an oncogenic miRNA, it was demonstrated that activated estrogen receptor can suppress the expression of proapoptotic protein BAX through upregulating miRNA 27a. Thereby increased BCL2/BAX ratio may promote survival and proliferation leading to precancerous lesions and type I endometrial adenocarcinoma.[38] Moreover, miRNA 27a target the expression of FOXO1 (apoptosis factor) resulting in tumor cells survival by apoptosis inhibition. [35]
The current study finding can be explained also by the function of miRNA 27a as a tumor promoter in various other cancers through targeting MAP2K4 as in osteosarcoma(Pan et al. 2014), AGGF1 in bladder carcinoma [39], prohibitin in gastric carcinoma [40], and other genes that control protein transcription factors at G2-M checkpoint demonstrated in breast cancer cells.[41] Genistein anticancer agent studied in colon cancer targeted miRNA 27a also. [42]
Serum miRNA150-5p was also significantly upregulated in patients in comparison to controls. This was in accordance with previous miRNA-based on TCGA-UCEC project [43] focused on miRNA sequences downloaded from The Cancer Genome Atlas Project. Difference of miRNA profile between metastatic and nonmetastatic ECs was studied using bioinformatics technique and miRNA150-5p was differently expressed. It was demonstrated to regulate multiple pathways of cancer, including the Wnt, NOTCH, and TGF-β signaling by functional enrichment analysis.
In cancer cervix also miRNA150-5p played an important role. In invasive cervical squamous cell carcinomas 68 up-regulated miRNAs were identified including miRNA150-5p.[44] Li et al. (2015) [45] demonstrated that the level of miRNA150-5p expression was higher in the advanced stage of cervical cancer and in cervical intraepithelial neoplasia which is a well-defined precursor stages of squamous cell carcinomas.[46] In serum samples from cervical cancer patients expression of miRNA150-5p patients was also increased. MiRNA150-5p promoted the proliferation, migration and invasion of human cervical cancer cells HeLa and SiHa cells. [47]
These findings can be explained by the action of miRNA150-5p as it has multiple targets involved in the cell proliferation, apoptosis, and metastasis including p53, P2X purinoceptor 7 (P2X7 mucins 4 (MUC4), BRI1associated receptor kinase 1 (BAK1), C-Myb. zincfinger Ebox binding homeobox 1 (ZEB1), EGR2, and SRC kinase signaling inhibitor 1 (SRCIN1). Significant downregulation of FOXO4 in C-33A cells expressing miR-150 mimics and the upregulation of FOXO4 (apoptosis factor) in the cells expressing miRNA150-5p inhibitors was also found.[45]The same family of FOX protein was targeted by miRNA27-a as demonstrated by Mozos et al. (2014).[35] MiRNA150 induces the arrest of FOXO4 transcription by binding to 3′-UTR of its mRNA, therefore reduces p27 and pRb activation, and increases CyclinD1, leading to cell cycle progression and survival.MiRNA150-5p enhances the cell cycle progression from the G1/G0 toS phase due to decrease of p27 and the increase of CyclinD1(Zhang et al. 2018).[43] Cell cycle protein transcription factors act also as a common target also for miRNA 27a [41]. MiRNA150-5p targets PDCD4 gene which is a direct suppressor of NF-кB. It can also suppress AKT pathway as well as the expression of matrix metallopeptidase 9 (MMP-9) which facilitates cancer cell migration.[48] Allgayer et al. have demonstrated that PDCD4 could inhibit the invasion of cells through also regulating the expression of urokinase receptor (u-PAR) which is one of the major invasion-related genes in various cancers. [49]
We found a significant association between miRNA 27a and endometrial cell type; it was overexpressed in endometrial cancer type I than type II. Endometrial adenocarcinoma (EC) has two basic clinicopathologic forms, type I and type II. Type I EC is usually a well to moderately differentiated cancer and accounts for 80–85% of all ECs and includes tumors of endometrioid histology.[50] Type II is poorly differentiated, usually of a nonendometrioid histological subtype, frequently lack steroid receptors. Type I tumors generally arise on a background of endometrial hyperplasia and have a good prognosis survival. It develops in a steroid environment, associated with high levels of hormone receptors and usually responds to hormonal therapy.[51] In a trial to explain the current study finding miRNA 27a expression in breast cancer was demonstrated as an example.[52] A previous study showed that PR + versus PR- breast tumors had higher expression of miRNA 27a.[53] This can indicate that miRNA 27a may be regulated by the ovarian steroids estrogen and progesterone in endometrial epithelium.
Moreover, miRNAs are differentially expressed in noninvasive (stage IA) and myoinvasive adenocarcinomas (stage IB and IC), miRNA 27a was overexpressed in invasive adenocarcinomas, and its expression increased linearly according to tumor stage. Results were validated by RT-PCR in an independent series of EC. The expression of FOXO1 (miRNA 27a main target) was down-regulated in invasive compared with noninvasive tumors. Nonmutated adenocarcinomas showed miRNA 27a overexpression. It was concluded that the miRNA 27-FOXO1 tandem inhibits apoptosis and enhances tumor cell survival in-nonmutated EC. [35]
The current study also demonstrated that miRNA150-5p was overexpressed in postmenopausal more than premenopausal endometrial cancer patients. Since endometrial cancer predominantes in postmenopausal women reflecting the contribution of several extraovarian tissues to circulating estrogens pool by the production of estrogen from adrenal steroids in absence of ovarian synthesis. Estrogen can also be formed from conjugate estrogens such as E1-S. The sulfatase enzyme, which produces E1 from high circulating E1-S metabolite, also contributes significantly to estrogen synthesis in malignant endometrium.[54, 55] In vitro studies have demonstrated the modulation of uterine miRNAs by estrogen using isolated endometrial epithelial and stromal cell cultures through their respective receptor-mediated pathways. In addition to modulating miRNA expression at the level of transcription, steroids may also influence the expression of the miRNA biogenesis components necessary for their processing to the mature cytoplasmic form. Among miRNA biogenesis components, Exportin-5 and Dicer1 expressed in the mouse uterus and appear to be the major steroid regulated components in the miRNA biogenesis pathway.[56] In postmenopausal women the expression of p53, which is targeted by miRNA150-5p, was the highest in adenocarcinoma samples when compared to endometrial polyp and atrophic endometrium. [57, 46]
In the current study miRNA 27a showed 100% sensitivity and specificity, positive and negative predictive values of 100%. MiRNA150-5p showed 88.89% sensitivity and 100% specificity, 100% positive and 78.9 % negative predictive values. Areas under the curve were 1.0 for miRNA 27a, 0.982 for miRNA150-5p which were higher than serum Ca 125. A similar study showed that combination of miR27-a and Ca125 had an AUC of 0.894 (95% CI, 0.807, 0.980; sensitivity = 0.774, specificity = 0.970), which makes miRNA 27a an optimal non-invasive biomarker to diagnose EC.[36] A recent study identified a series of miRNA/mRNA pairs (miR-497/EMX1, miR-23c/DMBX1, and miR-670/KCNS1) to be associated with survival in EC.[58] In light of the above we suggest a similar diagnostic panel including miRNA 27a and miRNA150-5p to be considered for larger scale studies and further evaluation.