Endometrial cancer is the most common gynecologic malignancy, whose incidence rate is on the rise. However, the underlying mechanisms of endometrial cancer are not very clear yet. miRNAs have been considered to be playing important roles in malignant behavior. Here, miR-652 was significantly upregulated in endometrial cancer, which correlated with shorter overall survival and earlier recurrence. Moreover, overexpression of miR-652 in endometrial cancer cells promoted proliferation, migration, and invasion in vitro and facilitated tumor growth and metastasis in vivo. In contrast, downregulation of miR-652 in endometrial cancer cells inhibited these processes both in vitro and in vivo. Mechanistically, miR-652 promotes proliferation and metastasis through directly targeting RORA. Both mRNA and protein level of RORA were negatively related with miR-652 and overexpression of RORA can rescue the promotion effect of miR-652. Further experiments indicated miR-652 overexpression can activate the Wnt/β-catenin pathway and RORA can downregulate β-catenin and function as a tumor suppressor in endometrial cancer. Collectively, these findings demonstrate that miR-652 functions as an oncomir in endometrial cancer.

Implications:

This study suggests that the miR-652 is a critical regulator of proliferation and metastasis in endometrial cancer and may serve as a therapeutic target.

Endometrial cancer is the fourth most common malignant tumor among women (1), and is the most common gynecologic malignancy, with an estimated 63,230 new cases and 11,350 deaths in 2018 in the United States (2). Endometrial cancer has been divided into estrogen dependent (type I) and nonestrogen dependent (type II) based on its clinicopathologic features and pathogenesis (3). However, tumors showing combined or hybrid morphologic and molecular characteristics are not uncommon; several studies defined IHC and/or mutation profiles to aid in distinguishing endometrial cancer subtypes, to further stratify risk categories (4, 5). The 5-year relative survival rate of endometrial cancer has been reported to be about 82%. Although contrasting with the declining trends in cancer mortality for many other cancers, death rates for endometrial cancer rose from 2010 to 2015 by about 2% per year (2, 6). Many factors affect its prognosis, including tumor differentiation, stage, and lymph node metastasis, while effective treatment for advanced endometrial cancer is still lacking (7). All the observations reflect that a more complete understanding of the molecular genetics of endometrial cancer is necessary.

miRNAs are small noncoding RNA molecules, approximately 22 nucleotides, which play important gene-regulatory roles in animals and plants by binding to the 3′-untranslated regions (3′-UTR) of the corresponding mRNA targets, thus leading to mRNA decay and translational repression (8). It is believed that miRNAs are involved in diverse biological processes, including proliferation, metastasis, and other features of cancer, and regulate up to one third of all human genes at posttranscriptional level (9). Although previous studies found that differentially expressed miRNAs may be associated with the carcinogenesis of endometrial cancer (10, 11), the roles and potential mechanisms of miRNAs in endometrial cancer are still largely unknown. Only few miRNAs, such as miR-145 (12), miR-181c (13), miR-200a, miR-200b (14), miR-218 (15), and miR-449a (16), have been studied for their roles in endometrial carcinogenesis. miR-652 has been reported to be an oncomir in human breast cancer (17), osteosarcoma (18), and rectal cancer (19), and a tumor suppressor gene in malignant pleural mesothelioma (20). However, little is known about the function of miR-652 in endometrial cancer.

Retinoic acid receptor–related orphan receptor A (RORA), the first member of ROR subfamily of orphan receptors, is expressed in multiple tissues and cells, including brain, muscle, colon, heart, skin, lung, spleen, leukocytes, breast, and mammary epithelial cells (21, 22). Aberrant expression of RORA can influence diverse cellular pathologies, such as autoimmune disease, proliferation, obesity, and cell metastasis (23). Furthermore, RORA is frequently inactivated in cancers and functions as a tumor suppressor, making it an attractive target for cancer therapy (24).

β-Catenin is a key molecule of the Wnt signaling pathway, and plays important roles in regulating cellular proliferation and differentiation (25). Accumulation of β-catenin is associated with the tumorigenesis of several cancers, such as prostate cancer (26, 27) and endometrial cancer (28, 29). RORA can exert inhibitory function on the expression of β-catenin, thereby providing new approaches for the therapy of human cancers (30).

In this study, we investigated the miRNA expression profiles in endometrial cancer and identified a proliferation-promoting miRNA, miR-652, which is frequently upregulated in endometrial cancer. Moreover, we identified RORA, a putative tumor suppressor in endometrial cancer, as the direct functional target of miR-652. This discovery will improve the development of treatment for patients with endometrial cancer.

Human tissues and cell lines

Endometrial cancer and benign endometrial tissues were collected from April 2015 to December 2017 at Qilu Hospital (Jinan, China). A total of 74 endometrial samples were studied, which included benign endometrium (n = 22), endometrial serous carcinoma (ESC; n = 13), and endometrial endometrioid carcinoma (EEC; n = 39). All malignant cases were diagnosed using criteria of the International Federation of Gynecology and Obstetrics. Before RNA extraction, a 5-μm frozen section was sliced and stained with H&E for review to evaluate the proportion of tumor cells (>70%; ref. 31). All the benign endometrium samples were collected from age-matched hysteromyoma patients who underwent hysterectomy. The study was conducted in accordance with the Declaration of Helsinki. Ethics committee of Shandong University approved the study and all participants signed informed consents.

AN3 CA, HEC-1-A, Ishikawa, RL95-2, and SPEC-2 were purchased from the ATCC. HEK293T was purchased from China Type Culture Collection. Cell line authentication was conducted and no cross-contamination of other human cell line was found. AN3 CA and SPEC-2 cell lines were cultured in Eagle minimum essential medium; HEC-1A, Ishikawa, and HEK293T cell lines were maintained in DMEM, RL95-2 cell line was cultured in DMEM:F12 medium. All cell culture media were supplemented with 10% FBS and culture media for AN3 CA and SPEC-2 cells were also supplemented with 100 mmol/L sodium pyruvate and nonessential amino acid. Cells used for 17-β-estradiol treatment were cultured in steroid-free medium (phenol red-free DMEM with 10% FBS, pretreated with dextran-coated charcoal) and maintained for 3 days with replacement of the same fresh medium. All cell lines were cultured and maintained in 5% CO2 at 37°C.

Hormone treatment

17β-Estradiol (E2) was purchased from Solarbio (IE0210) and dissolved in DMSO. Cells were incubated with E2 at various doses for multiple time points or with DMSO (Sigma-Aldrich; control group).

Plasmid construction and transfection

The human pre–miR-652 sequence was amplified from normal human genomic DNA and cloned into pGIPZ lentiviral vector (Open Biosystems) to generate a miR-652 expression vector. Lentivirus expressing miR-652 or negative control was produced in HEK293T cells packaged with pMD2G and psPAX2. For stable transfection, cells (1 × 105) were plated in 6-well plates without antibiotics for 1 day before incubation. Then, the medium was replaced with 1 mL of retrovirus solution generated from the step mentioned above. The solution was supplemented with 8 μg/mL polybrene. After 24 hours, fresh medium containing 2 μg/mL puromycin (Sigma-Aldrich) was added to each well. Multiple colonies were obtained after 2 weeks of puromycin selection.

Transient transfection

Mimics (GenePharma) and inhibitor (GenePharma) of miR-652 and the corresponding negative control were used to achieve the transient overexpression or knockdown of miR-652. RORA cDNA was synthesized by GeneChem. RNAi-mediated knockdown was performed with the following siRNA: siRORA-1, 5′-CGCUGCCAACACUGUCGAUUATT-3′; siRORA-2, 5′-CUAAUGGCAUUUAAAGCAATT-3′. Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from endometrial cancer cells using TRizol reagent (Ambion) following the manufacturer's instructions. The cDNA of miRNA was synthesized with One Step PrimeScript miRNA cDNA Synthesis Kit (Takara). qPCR was performed with the SYBR Green Premix Ex Taq II (Takara) with StepOne Plus Real-Time PCR System (Applied Biosystems). The expression of U6 was used as the endogenous control for detection of miRNA expression level. Primer information used in the study can be found in Supplementary Table S1.

Western blot analysis

Cells were harvested and lysed in RIPA Lysis Buffer (Beyotin) with PMSF (1%). Protein samples were incubated for 30 minutes on ice, and cell debris was removed by centrifugation. The protein concentration was determined by BCA Assay Kit (Thermo Fisher Scientific). Protein samples were separated by SDS-PAGE and electrotransferred onto PVDF membrane (Millipore). After blocking with 5% nonfat milk, the membrane was incubated overnight at 4°C with the primary antibody and then with horseradish peroxidase–coupled secondary antibody for 1 hour (Dako, 1:5,000). Signal was detected with enhanced chemiluminescence (ECL; PerkinElmer) by ImageQuant LAS 4000 (GE Healthcare Life Sciences). Antibodies used include rabbit anti-RORA (Abcam, ab60134, 1:500 dilution), rabbit anti–β-catenin (Abcam, ab16051, 1:4,000 dilution), rabbit anti-CCND1 (Abcam, ab16663, 1:200 dilution), rabbit anti-estrogen receptor α (Abcam, ab75635, 1:200 dilution), mouse anti-human β-actin antibody (CST, 3,700; 1:1,000 dilution).

Cell proliferation assay

The proliferation ability of cells was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell lines were seeded in quintuplicate into 96-well plates (1.0–2.0 × 103cells/well) for 1 to 5 days. At specified time points, 20 μL of MTT reagent at 5 mg/mL concentration (Sigma-Aldrich) was added to each well, and the cells were incubated for additional 4 hours at 37°C. Then, the supernatants were carefully removed and 100 μL of DMSO was added to each well. The absorbance values at 490 nm wavelength were evaluated with a Varioskan Flash microplate reader (Thermo Fisher Scientific).

Clonogenic assay

For the colony formation assay, 500 cells were placed in each well of 6-well plate and maintained in media containing 10% FBS for 10 to 14 days. Colonies were fixed with methanol and stained with 0.1% crystal violet. Colonies of greater than 50 cells were counted.

Apoptosis

Apoptosis was quantified using 7-amino-actinomycin D (7AAD) and PE-labeled Annexin V (BD Biosciences) following the manufacturer's protocol and flow cytometry. Cells were collected 48 hours after transfection, washed twice with cold PBS, resuspended at 1 × 106 cells/mL, mixed with 100 μL of 1× buffer and 5 μL Annexin V-PE and 7AAD, and incubated for 15 minutes in the dark; 400 μL 1× buffer was added, and the cells were subjected to flow cytometry within 1 hour.

In vivo nude mice tumorigenesis and metastasis assay

Tumor cells transfected with pGIPZ-652 plasmid vector or pGIPZ-NC plasmid vector were harvested from subconfluent cell culture plates. For tumorigenesis assay, 4 × 106 cells in 150 μL PBS were injected subcutaneously into either side of the axilla of the same 4- to 5-week-old BALB/c nu/nu female mice. Size of tumors was measured weekly, and the tumor volume was calculated using the equation a × b2 × π/6, where “a” and “b” represent the longest and shortest diameter, respectively. To produce experimental peritoneal metastasis, 2 × 106 cells in 100 μL PBS were injected into the peritoneal cavity of 4- to 5-week-old BALB/c nu/nu female mice. After 5 weeks, these mice were sacrificed and examined for the growth and metastasis of the tumors. All animal experiments were performed with the approval of Shandong University Animal Care and Use Committee.

Migration and invasion assays

Cellular invasion and migration were analyzed using Boyden chamber–style cell culture inserted with and without Matrigel, respectively (BD Falcon). Endometrial cancer cells (1–2 × 105 cells) were seeded into the upper chambers of a transwell system (24-well, 8-μm pore size, BD Falcon) with 200 μL serum-free media. The lower chambers were filled with 500 μL culture media containing 10% FBS as a chemoattractant. After 12 to 48 hours, cells in the lower surface of the membrane were fixed in methanol for 15 minutes, stained with 0.1% crystal violet for 20 minutes, and counted under a light microscope.

IHC

IHC staining was performed on 4-μm sections of paraffin-embedded human endometrial cancer tissues and benign endometrial tissues to determine the expression of RORA. Tissue slides were deparaffinized in xylene and rehydrated in a graded series of ethanol. Antigen retrieval was performed by heat-induced epitope retrieval. The slides were incubated antibody in a humid chamber overnight at 4°C. Antibodies used included anti-RORA (Abcam, ab60134, 1:100 dilution) and anti–β-catenin (Abcam, ab16051, 1:1,000 dilution). Staining was detected with I-View 3, 3′-diaminobenzidine (DAB) detection system. The staining results were scored semiquantitatively on the basis of extent and intensity.

Luciferase reporter assay

The 3′UTR of potential target genes of miR-652 was amplified and cloned into pmirGLO vector (Promega) using the SacI and XhoI sites to generate the wild-type (WT) constructs. The mutant 3′UTR of RORA was generated on the basis of the pmirGLO-RORA 3′UTR-WT plasmid by overlap-extension PCR. HEK 293T cells were cultured in 96-well plates and transfected with 50 ng of WT or MT RORA 3′UTR constructs by Lipofectamine 2000 assay. After 24 hours of transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega). Renilla luciferase activity was normalized to corresponding firefly luciferase activity and plotted as a percentage.

Statistical analysis

The SPSS version 17.0 statistical software was used for statistical analyses. Student t test and Spearman correlation test were applied to analyze the statistical differences between two groups. Moreover, P < 0.05, P < 0.01, and P < 0.001 were considered as significant, very significant, and extremely significant, respectively. All the experiments were repeated for at least three times.

Expression of miR-652 is frequently increased in human endometrial cancer tissues, and its high expression predicts poor prognosis of endometrial cancer

According to the data from microarray analysis on 23 endometrial cancer samples and 23 benign endometrial samples from TCGA, we found miRNAs were significantly upregulated in endometrial cancer. Unsupervised hierarchical clustering with these 56 significantly dysregulated miRNAs was able to distinguish the endometrial cancers from corresponding benign endometrial samples (Fig. 1A). To find new potential endometrial cancer–related miRNAs from these miRNAs, we selected several targets (miR-324, -501, -652, and -1301) to evaluate their influence on the growth and migration of endometrial cancer cells using miRNA mimics transfection assay, and found that miR-652 showed the strongest proliferation and metastasis-promoting effects (data not shown).

Figure 1.

miR-652 is frequently upregulated in endometrial cancer and correlates with prognosis of patients. A, Heatmap illustrates the upregulated miRNAs in endometrial cancer compared with benign endometrium according to the 20 pairs of tissue (data from TCGA). EEC1 to EEC20 represent the 20 endometrial cancer samples, and N1 to N20 represent the 20 benign endometrial samples. B, miR-652 expression was examined by qRT-PCR in 39 EEC tissues, 13 ESC tissues, and 22 benign endometrium samples. miR-652 expression was upregulated in endometrial cancer tissues compared with the benign samples. C, The expression level of miR-652 in benign endometrium and EEC tissues according to the menstrual cycle. No significant difference was observed between the follicular and luteal phases of benign endometrial samples, and no significance was observed in comparison between the premenopausal and menopausal groups of EEC cases. D, The expression level of miR-652 in G3 endometrial cancer tissues was higher than G2 and G1 endometrial cancer tissues. E, High expression level of miR-652 exhibited shorter OS than patients with low miR-652 expression (P = 0.03). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. G1, low grade; G2, intermediate grade; and G3, high grade.

Figure 1.

miR-652 is frequently upregulated in endometrial cancer and correlates with prognosis of patients. A, Heatmap illustrates the upregulated miRNAs in endometrial cancer compared with benign endometrium according to the 20 pairs of tissue (data from TCGA). EEC1 to EEC20 represent the 20 endometrial cancer samples, and N1 to N20 represent the 20 benign endometrial samples. B, miR-652 expression was examined by qRT-PCR in 39 EEC tissues, 13 ESC tissues, and 22 benign endometrium samples. miR-652 expression was upregulated in endometrial cancer tissues compared with the benign samples. C, The expression level of miR-652 in benign endometrium and EEC tissues according to the menstrual cycle. No significant difference was observed between the follicular and luteal phases of benign endometrial samples, and no significance was observed in comparison between the premenopausal and menopausal groups of EEC cases. D, The expression level of miR-652 in G3 endometrial cancer tissues was higher than G2 and G1 endometrial cancer tissues. E, High expression level of miR-652 exhibited shorter OS than patients with low miR-652 expression (P = 0.03). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. G1, low grade; G2, intermediate grade; and G3, high grade.

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To further determine whether miR-652 was upregulated in endometrial cancer, we examined miR-652 expression level with qRT-PCR (Fig. 1B), and found that miR-652 was significantly upregulated in EEC (n = 39) and ESC (n = 13) compared with benign endometrium (n = 22); the expression level of miR-652 in ESC was higher than in EEC. In ERα-positive Ishikawa cells (Supplementary Fig. S1A), the expression level of miR-652 was found to be slightly upregulated after estrogen (E2 10−8 mol/L) stimulation (Supplementary Fig. S1B). But there was no significance difference between its expression level in premenopausal and menopausal patients with EEC; also, we obtained similar results from the TCGA data analysis (Supplementary Fig. S1C). In the control cases, the benign endometrium showed no significant difference in the expression level of miR-652 when compared between follicular phase and luteal phase. In menopausal patients with ESC, the level of estrogen was found low; however, the expression level of miR-652 was still very high (Fig. 1C). These results indicate that the upregulation of miR-652 in endometrial cancer tissues is cancer specific. High miR-652 expression level was associated with poor differentiation (P < 0.05) both in endometrial cancer tissues and cell lines (Fig. 1D and E). However, no significant association was found between miR-652 expression and tumor size, location, and stage in endometrial cancers (P > 0.05). On the basis of the analysis of the TCGA using Cox regression test, we found that the high expression level of miR-652 exhibited shorter overall survival (OS) than patients with low miR-652 expression (P = 0.03; Fig. 1F). We further analyzed the correlation between miR-652 expression and clinicopathologic parameters; besides histologic grade, miR-652 also has correlation with tumor recurrence (Table 1).

Table 1.

Correlation between miR-652 expression and endometrial cancer clinicopathologic parameters

miR-652 levela
ParametersNLow (n = 233)High (n = 137)P
Age (years) 
 <50 35 21 14 0.685 
 ≥50 334 212 122  
Grade 
 G1 83 65 18 0.000b 
 G2 101 77 24  
 G3 186 91 95  
Stage 
 I 258 166 92 0.485 
 II 31 21 10  
 III, IV 83 48 35  
BMI 
 ≥28 234 163 71 0.275 
 <28 90 57 33  
Peritoneal washing 
 Negative 244 149 95 0.539 
 Positive 29 16 13  
Lymph nodes 
 Negative 255 257 98 0.105 
 Positive 34 16 18  
Recurrence 
 Yes 0.02c 
 No 30 27  
miR-652 levela
ParametersNLow (n = 233)High (n = 137)P
Age (years) 
 <50 35 21 14 0.685 
 ≥50 334 212 122  
Grade 
 G1 83 65 18 0.000b 
 G2 101 77 24  
 G3 186 91 95  
Stage 
 I 258 166 92 0.485 
 II 31 21 10  
 III, IV 83 48 35  
BMI 
 ≥28 234 163 71 0.275 
 <28 90 57 33  
Peritoneal washing 
 Negative 244 149 95 0.539 
 Positive 29 16 13  
Lymph nodes 
 Negative 255 257 98 0.105 
 Positive 34 16 18  
Recurrence 
 Yes 0.02c 
 No 30 27  

NOTE: The follow-up data of some patients are not complete, so the case number of some groups is smaller than the total, but there is no difference between the expression level of miR-652 in the omitted individuals compared with the listed ones.

aThe median value of miR-652 levels in 370 endometrial cancer tissues is used to divide these cases into 2 groups with low or high miR-652 level.

bP < 0.001.

cP < 0.05.

Overexpression of miR-652 promotes proliferation of endometrial cancer cells in vitro and in vivo

To investigate the possible role of miR-652 in endometrial cancer, we established three endometrial cancer cell lines with transient miR-652 upregulation (Supplementary Fig. S2A) and downregulation (Supplementary Fig. S2B), and another three cell lines with stable miR-652 overexpression (Supplementary Fig. S2C). The cell proliferation assays and colony formation assays revealed that overexpression of miR-652 can significantly promote endometrial cancer cell proliferation (P < 0.05; Fig. 2A and C), whereas RNAi-mediated silencing of miR-652 decreased cell growth ratio (P < 0.05; Supplementary Fig. S2B). Overexpression of miR-652 cannot influence the apoptosis of endometrial cancer cells (Supplementary Fig. S3). Furthermore, overexpression of miR-652 could significantly promote the tumorigenicity of endometrial cancer cells in nude mice (Fig. 2D). These findings suggest that miR-652 exerts a growth-promoting function in human endometrial cancer.

Figure 2.

miR-652 enhances endometrial cancer cell growth in vitro and in vivo. A, The effect of miR-652 on cell proliferation of miR-652–overexpressing cells AN3 CA (i) and Ishikawa (ii) compared with their individual controls. B, Downregulation of miR-652 inhibited the proliferation in endometrial cancer cells AN3 CA (i) and Ishikawa (ii). C, The effect of miR-652 on colony formation of endometrial cancer cell lines AN3 CA (i) and Ishikawa (ii). Colony formation of vector controls (left) and miR-652-transfected (right). D, The effect of miR-652 on tumor formation in a nude mouse xenograft model. pGIPZ-NC and pGIPZ-652 Ishikawa cells (4 × 106) were injected subcutaneously into either axilla of the same nude mouse (right, pGIPZ -NC and left, pGIPZ-652). The tumor weight of the miR-652 group was significantly increased compared with the control group (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

Figure 2.

miR-652 enhances endometrial cancer cell growth in vitro and in vivo. A, The effect of miR-652 on cell proliferation of miR-652–overexpressing cells AN3 CA (i) and Ishikawa (ii) compared with their individual controls. B, Downregulation of miR-652 inhibited the proliferation in endometrial cancer cells AN3 CA (i) and Ishikawa (ii). C, The effect of miR-652 on colony formation of endometrial cancer cell lines AN3 CA (i) and Ishikawa (ii). Colony formation of vector controls (left) and miR-652-transfected (right). D, The effect of miR-652 on tumor formation in a nude mouse xenograft model. pGIPZ-NC and pGIPZ-652 Ishikawa cells (4 × 106) were injected subcutaneously into either axilla of the same nude mouse (right, pGIPZ -NC and left, pGIPZ-652). The tumor weight of the miR-652 group was significantly increased compared with the control group (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

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miR-652 promotes migration and invasion of endometrial cancer cells in vitro and in vivo

To determine the impact of miR-652 on endometrial cancer cell migration and invasion, we first observed the morphologic changes in miR-652–overexpressed cells. AN3 CA and Ishikawa cells overexpressing miR-652 exhibit mesenchymal morphology (Supplementary Fig. S4). The migration and invasion effect of miR-652 was analyzed using transwell assay. miR-652 can promote migration and invasion in endometrial cancer cells, and as predicted, transfection of inhibitory miR-652 can markedly impair the migrating and invading capacity (Fig. 3A). As showed in Fig. 3B, much more metastatic nodules can be seen in the group injected with Ishikawa cells with miR-652 overexpression. These results suggest that overexpression of miR-652 promotes tumor metastasis in vitro and in vivo.

Figure 3.

Overexpression of miR-652 promotes endometrial cancer cell migration and invasion in vitro and in vivo. A, Overexpression of miR-652 promotes cell migration and invasion in AN3 CA and Ishikawa cells. Inhibition of miR-652 evidently suppresses the migratory and invasive nature in both cell lines. B, Peritoneal metastasis of nude mouse, the metastasis nodules of miR-652 group were more than the control group (*, P< 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Overexpression of miR-652 promotes endometrial cancer cell migration and invasion in vitro and in vivo. A, Overexpression of miR-652 promotes cell migration and invasion in AN3 CA and Ishikawa cells. Inhibition of miR-652 evidently suppresses the migratory and invasive nature in both cell lines. B, Peritoneal metastasis of nude mouse, the metastasis nodules of miR-652 group were more than the control group (*, P< 0.05; **, P < 0.01; ***, P < 0.001).

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RORA is a direct target of miR-652

To identify the target gene of miR-652 in endometrial cancer, we analyzed the proteins downregulated in endometrial cancers and the predicted targets of miR-652 from public available databases as TargetScan, miRNAMap, and miRDB. RORA was among the predicted target genes. To investigate whether RORA contributes to the development of endometrial cancer, we first examined the expression of RORA in endometrial cancer and benign endometrial tissues. The mRNA level of RORA showed higher expression in benign endometrial tissues than in endometrial cancer by qRT-PCR, and the expression level of RORA in EEC was higher than in ESC (Fig. 4A). These findings were in accordance with the results analyzed from TCGA data (Supplementary Fig. S5A). We also found that the expression of RORA could not be downregulated significantly after estrogen (E2 10−8 mol/L) stimulation (Supplementary Fig. S5B) in Ishikawa cell line. And there was no significant difference in RORA expression level between premenopausal and menopausal EEC cases (Supplementary Fig. S5C), and the results from TCGA data comply with our findings (Supplementary Fig. S5D). The protein level of RORA also showed higher expression in benign endometrial tissues than endometrial cancer on Western blot analysis (Fig. 4C) and IHC (Fig. 4E), although the expression level of RORA between premenopausal and menopausal EEC cases showed no significant difference.

Figure 4.

RORA is a direct target of miR-652. A, mRNA level of RORA is lower in endometrial cancer tissues than benign endometrial tissues as validated by qRT-PCR. B, mRNA level of RORA is lower in miR-652–overexpressed cells than in the control group. C, The protein level of RORA in benign endometrium is higher than endometrial cancer tissues on Western blot analysis. T1 to T13 present EEC tissue samples. T1–T3, T8, T10, and T11 were collected from premenopausal patients; the others were from menopausal patients. D, Western blot analysis reveals an inverse correlation of RORA expression with miR-652 expression in malignant cell lines. Upregulation of miR-652 can inhibit RORA expression and downregulation of miR-652 can promote the expression of RORA in AN3 CA and Ishikawa cell lines. E, IHC analysis of RORA expression. (i) Representative photomicrographs show RORA expression in normal endometrial samples and EEC tissues; (ii) RORA is expressed higher in benign endometrium than endometrial cancer tissues; (iii) the expression of RORA in benign endometrium has no significant difference between follicular phase and luteal phase; and (iv) the expression of RORA in EEC tissues has no significant difference between premenopausal and menopausal cases. F, RORA is the downstream target of miR-652. (i) Putative miR-652–binding sequence in the RORA 3′UTR. The binding sequence of miR-652 in the RORA 3′UTR sequence was deleted as mutated type, and (ii) analysis of the luciferase activity of the luciferase reporter plasmid containing either WT or mutant (MT) RORA 3′UTR in HEK 293T cell line (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

Figure 4.

RORA is a direct target of miR-652. A, mRNA level of RORA is lower in endometrial cancer tissues than benign endometrial tissues as validated by qRT-PCR. B, mRNA level of RORA is lower in miR-652–overexpressed cells than in the control group. C, The protein level of RORA in benign endometrium is higher than endometrial cancer tissues on Western blot analysis. T1 to T13 present EEC tissue samples. T1–T3, T8, T10, and T11 were collected from premenopausal patients; the others were from menopausal patients. D, Western blot analysis reveals an inverse correlation of RORA expression with miR-652 expression in malignant cell lines. Upregulation of miR-652 can inhibit RORA expression and downregulation of miR-652 can promote the expression of RORA in AN3 CA and Ishikawa cell lines. E, IHC analysis of RORA expression. (i) Representative photomicrographs show RORA expression in normal endometrial samples and EEC tissues; (ii) RORA is expressed higher in benign endometrium than endometrial cancer tissues; (iii) the expression of RORA in benign endometrium has no significant difference between follicular phase and luteal phase; and (iv) the expression of RORA in EEC tissues has no significant difference between premenopausal and menopausal cases. F, RORA is the downstream target of miR-652. (i) Putative miR-652–binding sequence in the RORA 3′UTR. The binding sequence of miR-652 in the RORA 3′UTR sequence was deleted as mutated type, and (ii) analysis of the luciferase activity of the luciferase reporter plasmid containing either WT or mutant (MT) RORA 3′UTR in HEK 293T cell line (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

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To test whether RORA expression is regulated by miR-652, we compared RORA expression with miR-652 upregulation and downregulation. The expression of RORA in endometrial cancer cell lines is shown in Supplementary Fig. S5E and S5F. And the protein level of RORA in endometrial cancer cell lines was found to be negatively correlated with the expression level of miR-652 (Supplementary Fig. S5G). As shown in Fig. 4B and D, the RNA and protein levels of RORA were both downregulated in miR-652–overexpressed cells and could be restored in miR-652 depleted cells. The expression of RORA was also inhibited when miR-652 was upregulated in the subcutaneous tumor of nude mice (Supplementary Fig. S5H). We analyzed the binding site of miR-652 in the 3′UTR of RORA, and then introduced the RORA 3′UTR and the corresponding mutant counterparts into pmirGLO vector to evaluate the influence of miR-652 on expression of target gene using a luciferase assay. We found that miR-652 overexpression reduced the luciferase activity in cells transfected with the WT 3′UTR of RORA but not in cells with mutant 3′UTR in HEK 293T cells (Fig. 4F). Taken together, these results indicate that RORA is a direct downstream target of miR-652.

miR-652 promotes tumor proliferation and metastasis via directly targeting RORA in endometrial cancer

To confirm whether the downregulation of RORA is responsible for the proliferation and metastasis of endometrial cancer, we suppressed the expression of RORA by two different siRNAs. The expression level of RORA is shown in Fig. 5A. Downregulation of RORA was able to promote proliferation, migration, and invasion (Fig. 5B and C) in endometrial cancer cells.

Figure 5.

miR-652 promotes tumor proliferation and metastasis via downregulating of RORA. A, Protein levels of RORA after downregulation using two different siRNAs. B, Promotive effect on proliferation of Ishikawa cell line after downregulation of RORA. C, Effect of RORA downregulation on migration and invasion of Ishikawa cell line. D, Colony formation showed RORA could abrogate miR-652–induced cell growth. E, MTT assay showed that RORA could significantly abrogate miR-652–induced cell growth. F, Transwell assay revealed that the promotion of migration and invasion caused by expression of miR-652 could be rescued by introduction of RORA. D, Overexpression of miR-652 and inhibition of RORA could both increase β-catenin expression in endometrial cancer cells (AN3 CA, Ishikawa; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 5.

miR-652 promotes tumor proliferation and metastasis via downregulating of RORA. A, Protein levels of RORA after downregulation using two different siRNAs. B, Promotive effect on proliferation of Ishikawa cell line after downregulation of RORA. C, Effect of RORA downregulation on migration and invasion of Ishikawa cell line. D, Colony formation showed RORA could abrogate miR-652–induced cell growth. E, MTT assay showed that RORA could significantly abrogate miR-652–induced cell growth. F, Transwell assay revealed that the promotion of migration and invasion caused by expression of miR-652 could be rescued by introduction of RORA. D, Overexpression of miR-652 and inhibition of RORA could both increase β-catenin expression in endometrial cancer cells (AN3 CA, Ishikawa; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

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To further explore whether the promotion effect of miR-652 was achieved through RORA, we performed rescue experiment of RORA overexpression in cells with ectopically expressed miR-652 and corresponding control cells. We cotransfected Ishikawa cells with RORA cDNA or negative control and mimics. We established four subgroups of cells and measured the expression level of miR-652 by qPCR and RORA by Western blot analysis. As shown in Fig. 6A, we successfully established the four subgroups for rescue experiment. The overexpression of RORA can inhibit the proliferation of Ishikawa cells, and the promotion effect of miR-652 can also be reversed (Fig. 5D and E). Also, overexpression of RORA with a cDNA without 3′UTR could partially abrogate miR-652–mediated promotion on migration and invasion of endometrial cancer cells (Fig. 5F).

Figure 6.

miR-652 promotes tumor proliferation and metastasis via miR-652/RORA/β-catenin pathway. A, Inhibition of miR-652 could promote RORA and decrease β-catenin expression in AN3 CA cell; overexpression of miR-652 could inhibit RORA and increase β-catenin expression in Ishikawa cells. B, Inhibition of RORA could increase β-catenin expression in endometrial cancer cells (AN3 CA, Ishikawa). C, Overexpression of miR-652 could still inhibit RORA and increase β-catenin expression after estrogen stimulation in Ishikawa cells (*, P < 0.05; **, P < 0.01).

Figure 6.

miR-652 promotes tumor proliferation and metastasis via miR-652/RORA/β-catenin pathway. A, Inhibition of miR-652 could promote RORA and decrease β-catenin expression in AN3 CA cell; overexpression of miR-652 could inhibit RORA and increase β-catenin expression in Ishikawa cells. B, Inhibition of RORA could increase β-catenin expression in endometrial cancer cells (AN3 CA, Ishikawa). C, Overexpression of miR-652 could still inhibit RORA and increase β-catenin expression after estrogen stimulation in Ishikawa cells (*, P < 0.05; **, P < 0.01).

Close modal

In addition, overexpression of miR-652 could increase β-catenin expression and inhibition of RORA could also increase β-catenin expression (Fig. 6A and B). The expression of β-catenin in endometrial cancer cell lines is shown in Supplementary Fig. S6B and S6C, which was found to be negatively correlated with RORA expression (Supplementary Fig. S6D). It is found that β-catenin could be upregulated by 17β-estradiol (E2) stimulation in an estrogen receptor (ESR)–dependent manner (32). To exclude the possibility that whether the observed regulatory axis of miR-652/RORA/β-catenin is menstrual cycle biased, we detected the expression level of RORA and β-catenin in miR-652 upregulated cell lines after estrogen (E2 10−8 mol/L) stimulation, which showed that overexpression of miR-652 could still downregulate RORA and enhance β-catenin expression (Fig. 6C); we also found that the expression level of β-catenin showed no difference after estrogen stimulation (E2 10−6 mol/L and E2 10−8 mol/L; Supplementary Fig. S6F), which indicated the modulation of RORA on Wnt/β-catenin signaling pathway. Taken together, these results support that RORA is one of the major targets through which miR-652 promotes proliferation and metastasis of human endometrial cancer.

Although the 5-year survival rate of early diagnosed and treated endometrial cancer can be up to 90%, the 5-year survival rate of patients with stage III and IV disease is dramatically low (33). Tumor stage and myometrial invasion are considered as important prognostic factors (34). Women with locally recurrent advanced or metastatic endometrial cancer have only a 7- to 12-month median survival (35). Knowledge of the precise molecular mechanism of proliferation and metastasis is important for developing a better therapeutic strategy for patients with endometrial cancer. Thus, it is critical to figure out the key molecular mechanism involved in the proliferation and metastasis of endometrial cancer.

miRNAs express aberrantly in cancers and have a profound effect on many processes that are usually disrupted during malignant transformation, such as cell proliferation, apoptosis, stress responses, maintenance of stem cell potency, and metabolism (36). Recent advances have suggested that dysregulation of miRNAs is a common event in endometrial cancer (37). In this study, we revealed 56 dysregulated miRNAs through which endometrial cancer tissues can be distinguished from benign endometrium. Some of these miRNAs have been reported in endometrial cancer before, such as miR-200a (14), miR-182 (37), and miR-183 (38). These data suggest that miRNAs play important roles in tumorigenesis. On the basis of our preliminary functional experiments, we focused on miR-652. miR-652 functions as an oncomir in lung cancer (39) and inhibits microenvironment-induced epithelial–mesenchymal transition in pancreatic cancer (40), but little is known about the function or molecular mechanism in endometrial cancer.

In this study, we found that miR-652 was significantly upregulated in EEC tissues compared with benign endometrium, and the expression of miR-652 was significantly associated with tumor differentiation and recurrence, indicating its role as an oncomir in EEC. There have been few studies that theorized that miR-652, RORA, and β-catenin can be regulated by estrogen (32, 41, 42), and in our study, we found that miR-652 and RORA could be slightly regulated by estrogen in endometrial cancer cell lines; however, in endometrial cancer tissues, all three showed no significant correlation with menstrual cycle. So, the upregulation of miR-652 is cancer specific. In addition, in endometrial cancer cell lines, miR-652 promoted cell proliferation and colony formation. In vivo studies also revealed that the xenograft tumors derived from Ishikawa cells with miR-652 overexpression exhibited a higher growth rate than the control groups. Overexpression of miR-652 can also improve the migration and invasion ability of endometrial cancer cells, further highlighting the oncomir ability of miR-652. Furthermore, high miR-652 expression level correlated with poor prognosis in patients with endometrial cancer. To our knowledge, our report is the first one examining the effect of ectopic miR-652 expression on malignant behaviors in endometrial cancer.

RORA is a member of nuclear receptor family. There are four isoforms of RORA (RORA1-RORA4), which differ in the N-terminal. It is demonstrated that RORA functions through binding to ROR response elements (RORE) in the promoter region of its target gene. ROREs are specific DNA sequences, which include one core motif AGGTCA or two direct AGGTCA repeats spaced by two nucleotides preceded by a six nucleotide–long AT-rich sequence (43). Although all isoforms of RORA are expressed in normal human tissues, only RORA1 and RORA4 are mainly transcribed (24). The RORA1 isoform was reported mainly expressed in the central nervous system (44). RORA, identified as a potent tumor suppressor, is downregulated in several types of cancers, such as cervical cancer, breast cancer, and colorectal cancer (23, 30). Lee and colleagues reported that RORA could function by repressing canonical Wnt/β-catenin signaling, leading to the inhibition of colon cancer growth (30) and by increasing p53 stability upon DNA damage response (45). Xiao and colleagues reported that RORA could inhibit proliferation and migration of colorectal cancer cell and chick embryo chorioallantoic membrane angiopoiesis (46). Xiong and colleagues reported RORA suppresses breast tumor invasion through inducing SEMA3F expression (47). Fu and colleagues reported downregulated RORA expression was associated with poor prognosis in hepatocellular carcinoma (48). These studies suggest that RORA acts as a tumor suppressor in many cancers. Our study confirms that RORA is a downstream target gene of miR-652, and upregulation of miR-652 can reduce RORA expression, thus promoting cell proliferation, migration, and invasion of endometrial cancer cells.

β-Catenin is a key molecular counterpart in Wnt/β-catenin signaling pathway, which participates in tissue proliferation and differentiation (29). β-Catenin is found to be mutated frequently in type I endometrial cancer (49), and plays important roles in endometrial tumorigenesis. Kurnit and colleagues demonstrated that β-catenin mutation identifies patients with low-grade, early-stage endometrial cancer at increased risk of recurrence (50). Lee and colleagues studied that RORA can repress the Wnt/β-catenin pathway through attenuating β-catenin transcriptional activity (30), and it is in accordance with our results. Our study suggests a potential regulatory mechanism of proliferation and metastasis in endometrial cancer in which abnormally upregulated miR-652 suppresses RORA, thus inducing RORA inhibition and β-catenin upregulation, thus leading to poor prognosis.

A mouse model of endometrial cancer was established to further investigate the function of miR-652 in endometrial cancer in vivo. Overexpression of miR-652 significantly promoted tumor xenograft growth and led to an increased tumor volume. IHC staining confirmed that the tumors formed by miR-652 transfected cells expressed lower levels of RORA. These results provide further confirmation that miR-652 may act as an oncomir in endometrial cancer by targeting RORA and provide new clues of gene therapy for endometrial cancer.

In conclusion, we demonstrate that miR-652 promotes cell proliferation, invasion, and metastasis in endometrial cancer by targeting RORA. High expression level of miR-652 is associated with poor prognosis of patients with endometrial cancer. This knowledge may provide the basis for further investigations to identify novel diagnostic and therapeutic methods for endometrial cancer.

No potential conflicts of interest were disclosed.

Conception and design: X. Sun, Q. Zhang, B. Kong

Development of methodology: X. Sun, B. Kong

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Sun, Q. Zhang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Sun, C. Qiu, Y. Xu, Q. Zhang

Writing, review, and/or revision of the manuscript: X. Sun, S. Dongol, C. Qiu, C. Sun, Z. Zhang, Q. Zhang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Yang, B. Kong

Study supervision: X. Yang, B. Kong

The authors would like to extend sincere thanks to all those who have helped make this thesis possible and better. This study was supported by the National Clinical Research Center for Gynecological Oncology (grant no. 2015BAI13B05) and by the Science and Technology Development Project of Shandong Province (2016GSF201164).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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