Endometriosis is a common disease in women, which impairs the quality of life in patients. Recently, accumulating evidences reported that miRNAs play an essential role in diagnosis and treatment of endometriosis. However, the regulatory mechanism of miRNAs has not been fully explored. The expression of miR-17-5p and VEGFA was detected using qRT-PCR. The protein level of VEGFA was measured via Western blot. Cell proliferation was determined by CCK-8 assay. Cell migration and invasion were measured via transwell assay. The relationship of miR-17-5p and VEGFA were verified via luciferase reporter assay. Then miR-17-5p was remarkably down-regulated in endometriosis tissues, serums and cells, and overexpression of miR-17-5p inhibited cell proliferation, migration and invasion in endometriosis. Results showed that VEGFA was significantly up-regulated in endometriosis tissues and cells and acted as a target of miR-17-5p. Moreover, miR-17-5p negatively regulated VEGFA expression in endometriosis. Otherwise, up-regulation of VEGFA improved cell proliferative, migrated and invasive ability in ECSCs with transfection of miR-17-5p mimics group. Our data showed miR-17-5p inhibits cell proliferation, migration and invasion in endometriosis by directly repressing VEGFA expression.
Endometriosis is defined that the endometrial tissue runs backward through the uterus and proliferates at the ectopic location, which is a polygenic gynecological disease in women (Linden 2014; Nyholt et al. 2012). Meanwhile, endometriosis pathogenesis may be related to chronic pelvic inflammatory disease, uterine bleeding and even ovarian cancer (Bulun 2009; Dinulescu et al. 2005). However, the mechanism of miRNA has not been fully understood in endometriosis.
MicroRNAs (miRNAs) are short, non-coding, single-stranded RNA which are 18–23 nucleotides in length. Mechanistically, miRNA may bind to the 3′ untranslated region (3′-UTR) of transcribed mRNA, then suppress the target mRNA expression by either hindering mRNA translation or mediating mRNA degradation. miRNAs may play an essential role in cell progression and are associated with various types of diseases as a regulatory molecule. (Bartel 2004; Chen 2007). In recent years, the dysfunction of microRNAs has been extensively studied in gynecological diseases, including endometriosis (Hawkins et al. 2011). In our study, we found that miR-17-5p expression was decreased in endometriosis tissues and cells, which may be related to the pathogenesis of endometriosis. Previous studies showed that miR-17-5p was associated with cell progression in various diseases, containing breast cancer (Hossain et al. 2006) and ovarian cancer (Li et al. 2015). Besides, miR-17-5p was down-regulated in endometriosis in Jia et al. study consistent with our results (Jia et al. 2013). Since the pathogenesis of endometriosis is complex and the regulation mechanism of miR-17-5p has not been fully demonstrated, more functional studies are needed to reveal the molecular mechanism of microRNAs in endometriosis.
It is well known that miRNAs usually act together with their target mRNAs to affect the occurrence and development of cancer through degradation of mRNA. So, we predicted that vascular endothelial growth factor A (VEGFA) may be the target gene of miR-17-5p together with miR-17-5p affecting the characteristics of endometriosis. Vascular endothelial growth factor A (VEGFA) belongs to the platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) family which exists as a disulfide-linked homodimer (Mattei et al. 1996). Moreover, VEGFA may induce angiogenesis, promote endothelia cell growth and metastasis, inhibit apoptosis and is implicated in both physiological and pathological biological processes. (Banerjee et al. 2008; Fiedler et al. 2005; Ma et al. 2015; Terme et al. 2013; Xu et al. 2014; Zhou et al. 2013). Meanwhile this protein is up-regulated in many known diseases including tumor. More than that, VEGFA was elevated in peritoneal fluid of endometriosis women, which was involved in cell progression in endometriosis (Mclaren et al. 1996; Young et al. 2015).
In this study, we demonstrated that miR-17-5p could inhibit cell proliferation, migration and invasion in endometriosis by directly targeting VEGFA. Our finding revealed a novel role of miR-17-5p in endometriosis which maybe a potential target in therapy.
Materials and methods
Tissues and blood samples
Normal tissues (n = 10) was collected from 10 female patients who were diagnosed with uterine fibroids and no endometriosis occurred. Ectopic endometriotic (n = 20) and eutopic endometrial tissues (n = 20) were obtained from 20 patients who were diagnosed with endometrial. All patients were informed consent at Zhou Pu Hospital. All blood samples were collected from patients in EDTA tubes (BD Biosciences, San Jose, CA, USA) and centrifuged at 1800g for 10 min at room temperature. All samples were stored at − 80°C. This research was approved by the Institutional Review Board of Zhou Pu Hospital.
Cell culture and transfection
Endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) were isolated from ovarian ectopic endometriotic tissues and the eutopic endometrial tissues, respectively (Nishida et al. 2004). Then cultured in PMI-1640 medium (GIBCO, Grand Island, NE, USA) containing 10% fetal bovine serum (FBS), 100U/ml penicillin and 100mg/ml streptomycin at 37°C with 5% of CO2. Medium was changed every 3 days.
pcDNA3.1-miR-17-5p (MiR-17-5p mimics), pcDNA3.1-VEGFA and their corresponding negative control (pcDNA3.1-NC, pcDNA3.1-vector) were synthesized and purchased from Ribobio (Guangzhou, China). And a negative control siRNA (Silencer R, Life Technologies) and siRNAs targeting miR-17-5p (anti-miR-17-5p) were also were synthesized and purchased from Ribobio (Guangzhou, China). On reaching up to 70% confluence, all plasmids and oligos were transfected into ECSCs cells with Lipofectamine 3000 (ThermoFisher Scientific, San Jose, CA, USA) according to the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from tissues and cells with the Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was reverse transcribed with TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) for miRNA and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) for mRNA reverse transcription. U6 snRNA and Glyceraldehyde phosphate dehydrogenase (GAPDH) were employed as reference genes for miR-17-5p and VEGFA, respectively. Quantitative RT-PCR was performed using SYBR Green Real-time PCR kit (Invitrogen), according to the manufacturer’s protocol. Then each sample was run in three wells in iQTM5 Multicolor Real-Time PCR Detection System (Bio-Rad). The data were analyzed using the 2−ΔΔCt method. The primers used in qRT-PCR reactions were, respectively, as follows: the primers for U6: forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′
The primers for miR-17-5p, forward, 5′-TGCGCCAAAGTGCTTACAGTGCA-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGTATT-3′,
The primers for VEGFA: forward 5′-TATTCAGCGGACTCACCAGC-3′ and reverse, 5′-AACCAACCTCCTCAAACCGT-3′
The primers for GAPDH: forward 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.
Cells were washed with ice-cold Tris-buffered saline (TBS) and added the RIPA buffer (ThermoFisher Scientific, 1 ml per 100 mm dish) and PMSF (10 mg/ml) maintained constant agitation for 30 min at 4°C. Then the supernatant was transferred to a new tube. The protein concentrations were measured by Nanodrop2000 (Thermo Fisher Scientific, USA). Took 20 μg of each sample and added an equal volume of 4x NuPAGETM LDS sample buffer (ThermoFisher Scientific), then boiled at 95°C for 3 min. Then the equal amounts of protein were added into the wells of SDS-PAGE gel. Ran the gel at 60V for 5 min and increased the voltage to 120 V to finish about 1 h. The proteins in the gel were transferred to PVDF membranes (Millipore) for 0.5 h. Membranes were blocked into 3% BSA in TBST for 0.5 h and then incubated with primary rabbit anti-human VEGFA antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-human GAPDH antibody (1:2000 dilution, Cell Signaling Technology) for 1 h at room temperature. The bolt was washed 4 times for 15 min with TBST. After incubation with HRP-conjugated secondary antibody solution (goat anti-mouse IgG, 1:2000 dilution, Cell Signaling Technology), the blots were detected using PierceTM ECL western blotting substrate (ThermoFisher Scientific).
Cell migration and invasion assays
Cell migration and invasion were measured by transwell migration and invasion assay. Transfected-cells were incubated with PMI-1640 medium media containing 10% FBS for 48 h and then prepared the transwell compartment (Sigma-Aldrich, CLS3421; 24-well format with 8 μm pore size insert). 1 × 105 cells were added to the upper chamber with transwell compartment for the migration experiment. And 1 × 105 cells were seeded on the upper chamber with Matrigel (BD Biosciences) for the invasion experiment. Then took out the upper chamber, and the migrated cells in the lower chamber were stained with crystal violet in methanol for 30 min. The migrated and invasive cells were counted using a Countess automatic cell counter (Invitrogen).
Cell counting Kit-8 proliferation assay
Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan). Cells were seeded to 96-well plates at a density of 2 × 103 cells per well and incubated at 37°C with 5% CO2 overnight. Then discarded the media and added 100 μl serum-free media containing 10ul CCK-8 solution without bubbles. The plates were incubated at 37°C with 5% CO2 for 2 h. The optical density (OD) was determined with a microplate reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 490 nm.
Luciferase reporter assay
Bioinformatics analysis showed that VEGFA was a target of miR-17-5p by TargetScan (http://www.targetscan.org/). The complete 3′-UTR sequences of VEGFA and the mutated 3′-UTR were amplified by PCR and inserted into the pGL3-control luciferase reporter vectors (Promega, Madison, WI, USA) respectively to construct the plasmids of VEGFA-WT and VEGFA-MUT. Then VEGFA-WT or VEGFA-MUT was co-transfected with NC or miR-17-5p into ECSCs respectively by Lipofectamine 3000. After transfection for 48 h, cells were collected, and the relative luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega).
All statistical analyses were evaluated by GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). All data are displayed as means ± standard deviation (SD) from three independent experiments. All comparisons were performed using the Student t-test. Differences were considered to be significant when P < 0.05.
miR-17-5p expression was decreased in endometriosis tissues, serums and cells
To verify miR-17-5p expression pattern in endometriosis, we investigated 10 normal tissues from normal women, 20 eutopic tissues and 20 ectopic tissues from normal women and endometriosis patients. These tissues were divided into three groups: normal group, eutopic group and ectopic group. The qRT-PCR results showed that miR-17-5p expression was significantly decreased about 25% and 80% in eutopic and ectopic groups respectively compared with normal group (figure 1A). Also, serums were collected from normal women and endometriosis patients. The expression of miR-17-5p was down-regulated in serums of endometriosis patients and its expression in serums was similar to that in tissue in endometriosis (figure 1B). Next, we compared the expression of miR-17-5p in endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) (figure 1C). The results displayed that miR-17-5p expression was remarkably lower in ECSCs than that in NESCs. Consequently, miR-17-5p may be related to the pathogenesis of endometriosis.
Overexpression of miR-17-5p inhibited cell proliferation, migration and invasion in endometriosis
Since miR-17-5p expression was down-regulated in ECSCs, we transfected ECSCs with a miR-17-5p mimics fragment to determine the functions of miR-17-5p in endometriosis. figure 2A showed that miR-17-5p expression was obviously enhanced in transfected miR-17-5p group compared with control and NC groups. CCK-8 assay was applied to measure whether miR-17-5p influences cell proliferative ability in ECSCs. High miR-17-5p expression remarkably inhibited cell proliferation in ECSCs (figure 2B). Moreover, PCNA was a key cell proliferation marker, which could reflect cell proliferation at the protein level. As shown in figure 2C, the protein expression of PCNA was inhibited by miR-17-5p transfection (figure 2C). Following transwell experiments demonstrated that migratory and invasive cells were sharply decreased in miR-17-5p group compared with control and NC groups (figure 2D and E). Thus, overexpression of miR-17-5p attenuated cell proliferative ability, migrated and invasive capacity.
miR-17-5p negatively regulated VEGFA by binding its 3′-UTR
To further explore the underlying mechanism of miR-17-5p in endometriosis, we used TargetScan to predict the downstream candidate target mRNA of miR-17-5p (figure 3A). Then luciferase reporter assay was used to confirm the relationship between miR-17-5p and VEGFA. The plasmids of VEGFA-wt and VEGFA-mut were constructed and co-transfected respectively with miR-17-5p mimics into 293T cells. As shown in figure 3B, the luciferase activity was decreased when miR-17-5p binding to VEGFA-wt, but not VEGFA-mut. So miR-17-5p may mediate the degradation of VEGFA. Subsequently, to determine whether VEGFA expression is affected by miR-17-5p, we transfected HEK293T (because of their reliable growth and propensity for transfection) with NC, miR-17-5p, anti-NC, anti-miR-17-5p groups and detect VEGFA expression via qRT-PCR and western blot. As shown in figure 3C and D, VEGFA mRNA and protein expression was significantly decreased after exogenous overexpression of miR-17-5p comparing to control and slightly up-regulated when using the miR-17-5p inhibitor. Based on the results, miR-17-5p negatively regulated VEGFA by binding its 3′-UTR.
VEGFA expression was up-regulated in endometriosis tissues and cells
Previous study reported that VEGFA was increased in endometriosis. In our study, VEGFA expression was significantly increased in ECSCs compared with that in NESCs (figure 4A). To further verify the protein level of VEGFA, western blot analysis showed that the VEGFA protein level was higher in ECSCs than that in NESCs (figure 4B). We also measured the protein level of VEGFA in normal, eutopic and ectopic tissues. In ectopic and eutopic groups, VEGFA protein expression was remarkably elevated (figure 4C). In conclusion, VEGFA was increased in patient samples and ECSCs and negatively correlated with miR-17-5p.
Overexpression of VEGFA mitigated the suppressive effects of miR-17-5p in endometriosis
To understand whether miR-17-5p affects cell growth through VEGFA, the plasmids of control (vector) or VEGFA overexpression (VEGFA) was co-transfected with miR-17-5p into ECSCs respectively. As shown in figure 5A and B, VEGFA expression was significantly increased in miR-17-5p+VEGFA group compared with that in miR-17-5p+vector group by qRT-PCR and western blot. Moreover, cell proliferation was remarkably increased in miR-17-5p+VEGFA group compared with that in miR-17-5p+vector group (figure 5C). Moreover, PCNA protein expression was sharply higher in miR-17-5p+VEGFA group than that in miR-17-5p+vector group (figure 5D). Meanwhile, cell migration and invasion were also obviously enhanced in miR-17-5p+VEGFA group (figure 5E and F). Therefore, overexpression of VEGFA abolished the suppressive effect of miR-17-5p overexpression on cell proliferation, migration and invasion in endometriosis.
Endometriosis is a common gynecological disease, which can lead to dysmenorrhea and infertility in women. Nearly half of the patients have chronic pelvic pain of which 70% occurs during menstruation and nearly half of the female patients are sterile (Holoch and Lessey 2010). Accumulating evidence suggested that miRNAs regulated cellular and biological processes in various diseases, such as non-small cell lung cancer (Zhang et al. 2010), gastric cancer (Huang and Lu 2017), ovarian cancer (Yang et al. 2016) and endometriosis (Chen et al. 2017).
Recently, miRNAs act as potential biomarkers for endometriosis and are involved in cell progression of endometriosis. For example, the expression of miR-202 was up-regulated in eutopic and ectopic endometrial tissues compared with that in normal endometrial tissues, which promoted cell proliferation and metastasis by down-regulating sex determining region Y-box 6 (SOX6) (Zhang et al. 2015). Okamoto et al. reported that up-regulated miR-210 expression increased cell proliferative ability and inhibited apoptosis through targeting signal transducer and activator of transcription 3 (STAT3) (Okamoto et al. 2015). Thus, miRNAs/mRNAs axis widely takes part in cellular processes of endometriosis. Meanwhile, through microRNA microarray expression profiling, Jia et al. showed that miR-17-5p, miR-20a and miR-22 were down-regulated miRNAs in endometriosis (Jia et al. 2013). In our study, miR-17-5p expression was lower in ectopic and eutopic tissues than that in normal tissues. Thus, we focused on and speculated that miR-17-5p was involved in endometriosis pathogenesis.
Accumulating evidence determined that miR-17-5p is an important regulatory molecule in many diseases. For example, in breast cancer, miR-17-5p was up-regulated which regulated cell proliferation, migration and invasion by inhibiting amplified in breast cancer 1 (AIB1) and HMG box-containing protein 1 (HBP1) respectively (Hossain et al. 2006; Li et al. 2011). Similarly, miR-17-5p was highly expressed in pancreatic cancer (Yu et al. 2010), colorectal cancer (Ma et al. 2012), ovarian cancer (Ying et al. 2015) and hepatocellular carcinoma (Zheng et al. 2013), associated with a poor prognosis, cellular processes and drug resistance. Inversely, miR-17-5p was down-regulated in chronic lymphocytic leukemia (CLL) (Mraz et al. 2009) and endometriosis (Jia et al. 2013), which may be due to disease differences. Based on these reports, we further confirmed the important regulatory role of miR-17-5p in cancers. However, the function of miR-17-5 was barely verified in endometriosis. Therefore, we improved the expression of miR-17-5p in ECSCs to investigate its function. According to our data, overexpression of miR-17-5p not only suppressed cell proliferation, but also decreased migratory and invasive cells in endometriosis.
To further explore the regulatory mechanism of miR-17-5p, we used TargetScan to select the target mRNA of miR-17-5p and found that VEGFA was a candidate which may bind miR-17-5p via its 3′-UTR. Besides miR-17-5p, VEGFA was also a target of various miRNAs, including miR-185, miR-361-5p, miR-29a, miR-203 and miR-199a-5p, which regulated the cellular processes of breast cancer (Wang et al. 2016), hepatocellular carcinoma (Cui et al. 2016), gastric cancer (Ling et al. 2014), cervical cancer (Zhu et al. 2013) and endometriosis (Hsu et al. 2014). Previous studies reported that VEGFA, a potent angiogenic factor, was known to be up-regulated and relevant to cell progression and blood vessel formation in endometriosis (Donnez et al. 1998). For example, improved level of miR-199a-5p inhibited cells proliferation, motility and angiogenesis by down-regulating VEGFA (Hsu et al. 2014). Meanwhile, VEGFA and Matrix metalloproteinase 3 (MMP3) acted as target genes of miR-93 and were highly expressed in endometriosis. Low expression of miR-93 contributed to cell growth in endometriosis by up-regulating MMP3 and VEGFA (Lv et al. 2015). However, in this study, miR-17-5p targeted VEGFA was first put forward in endometriosis, which may further clarify the regulatory mechanism of endometriosis induction and become a new target for endometriosis treatment.
Consistently, we observed that VEGFA expression was up-regulated in endometriosis tissues and ECSCs compared with normal tissues and NESCs. More than that, VEGFA was identified as a target gene of miR-17-5p by luciferase reporter assay, which was down-regulated in ECSCs with transfection of miR-17-5p mimics and up-regulated in ECSCs with transfection of anti-miR-17-5p. Therefore, miR-17-5p negatively regulates the expression of VEGFA in endometriosis. Subsequent functional studies showed that overexpression of VEGFA could reverse the suppressive effect in cell proliferation, migration and invasion caused by miR-17-5p overexpression in ECSCs.
In conclusion, our results demonstrated that miR-17-5p was down-regulated in endometriosis and overexpression miR-17-5p could mitigate endometriosis via regulating VEGFA expression, provided that miR-17-5p/VEGFA axis might become a biomarker in endometriosis diagnosis and a therapeutic target in endometriosis treatment.
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Corresponding editor: Tapas Kumar Kundu
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Pang, QX., Liu, Z. miR-17-5p mitigates endometriosis by directly regulating VEGFA. J Biosci 45, 78 (2020). https://doi.org/10.1007/s12038-020-00049-y
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