European Review for Medical and Pharmacological Sciences 2017; 21: 2816-2822 Over-expression of miR-1271 inhibits endometrial cancer cells proliferation and induces cell apoptosis by targeting CDK1

L. LI1, Y.-W. QU2, Y.-P. LI1

1Maternal and Child Care Service Centre of Kuiwen , , , 2Department of Gynecology Oncology, Cancer Hospital, Qingdao, Shandong, China

Abstract. – OBJECTIVE: Endometrial carci- Introduction noma (EC) is one of the most common female malignancies worldwide. Growing evidence Endometrial carcinoma (EC) is one of the showed that microRNAs (miRNAs) are involved three most common types of gynecologic can- in the EC progression. The present study aimed cer and its global incidence has increased in to investigate the role of miR-1271 in the devel- 1 opment and progression of EC. recent years . In 2016, there were 60,050 new PATIENTS AND METHODS: The EC tissues expected cases of EC with an estimated 10,470 2 and adjacent normal tissues were obtained from deaths in USA . Radical surgery might be the 42 EC patients. The expression of miR-1271 only hope for curing EC in the stage of pre- in EC tissues and cells was examined using cursor lesions and the patients are diagnosed Real-time RT-PCR. Western blot was used to at an early stage with a favorable prognosis3. quantify the level of cyclin-dependent kinase 1 However, the expected survival of patients (CDK1) in EC tissues and cells lines. Cell pro- with recurrent disease is only 12-15 months at liferation, colony formation and flow cytome- 4,5 try were done to examine effects on cancer cell the time of diagnosis . Therefore, elucidat- proliferation and apoptosis in vitro. Bioinfor- ing the potential mechanism that mediate the matics software was used to predict some po- initiation and progression of EC is urgent and tential target genes of miR-1271. Besides, the of great interest. MicroRNAs (miRNAs) are a dual luciferase reporter gene assay was used class of small (19-24 nucleotides), non-coding to determine the direct targeting relationship RNAs that are involved in post-transcriptional between miR-1271 and CDK1. gene regulation and/or degradation6,7. It has RESULTS: MiR-1271 was significantly down- regulated in human EC tissues and cells while been demonstrated that miRNAs regulate the CDK1 was strongly upregulated. Bioinformatics expression of specific target genes by bind- analysis indicated that CDK1 was a potential tar- ing to the 3’-untranslated regions (3’-UTRs) get of miR-1271. Then, luciferase reporter assay of messenger RNA8. Growing evidence shows confirmed that CDK1 was a direct target gene that miRNAs play important role in various bi- of miR-1271. In vitro studies showed that miR- ological processes, such as tumor angiogenesis, 1271 overexpression reduced EC cell prolifera- proliferation, cell differentiation, apoptosis, tion and promoted apoptosis, while restoration metastasis9,10. Indeed, miRNAs serve as onco- of CDK1 attenuated these effects of miR-1271 on EC cells. Moreover, we found that knockdown of genes or tumor suppressors depending on the 11 miR-1271 significantly promoted EC cell growth types of tumors . These information highlight and suppressed apoptosis. the critical effect of miRNAs in tumor progres- CONCLUSIONS: Our findings showed that sion and provide new insight into the molecular miR-1271 served as a tumor suppressor in EC mechanisms underlying carcinogenesis. MiR- via targeting CDK1, suggesting miR-1271 as a 1271 is a member that was newly discovered in new potential target for therapy strategy in EC. the miRNA family. Previous studies indicated that miR-1271 functions as tumor suppressors Key Words: in various tumors, including gastric cancer12, miR-1271, Endometrial carcinoma, Proliferation, 13 Apoptosis, CDK1. oral squamous cell carcinoma , and pancreatic cancer14. However, to our best knowledge, the

2816 Corresponding Author: Yan-Ping Li, MD; e-mail: [email protected] miR-1271 and endometrial cancer effect of miR-1271 in EC remains unknown. In primers for PCR were designed and purchased the present study, we aimed to explore the role from Invitrogen (Carlsbad, CA, USA). The 2-ΔΔCt of miR-1271 in progression of EC and try to method was used to calculate the relevant expres- confirm a targeting gene of this miRNA. sion values of miR-1271 in EC and corresponding normal tissues.

Patients and Methods Protein Extraction and Western Blot Total proteins were extracted with a Total Tissue Samples Extraction Kit (Solarbio, Beijing, China). Cyto- This study was approved by the Research plasmic and nuclear proteins were extracted with Ethics Committee of Maternal and Child Care a nuclear and cytoplasmic protein extraction kit Service Centre of Kuiwen District. Written in- (Beyotime, Shanghai, China). Western blotting formed consents were obtained from all the in- was performed to determine CDK1 protein ex- dividuals involved in the trial. Human endome- pression. The standard procedure performed as trial cancer tissue and matched adjacent normal described previously15. tissues from 42 patients with EC were collected in 20014 and 2015. None of the patients received Luciferase Assays radiotherapy or chemotherapy before surgery. To confirm that miR-1271 can bind to the All samples were evaluated by two pathologists. predicted site CDK1, we conducted a luciferase The collected sample was snap frozen in liquid reporter assay in the EC cell line. Reporter con- (-70°C) until use. structs containing pGL3-wt-CDK1 and pGL3- mut-CDK1 (with a mutated target seed sequence) Cell Culture and Transfection were obtained from Bio-Asia (Haiding, Beijing, Human EC cells lines, including ECC-1, China). Then, the recombinant CDK1 3’UTR- RL95-2 and AN3 CA, and endometrial fi- wt plasmid or CDK1 3’UTR-mut plasmid, and broblast cell T-HESC were purchased from miR-448 mimics were co-transfected to EC cells American Type Culture Collection. The above with Lipofectamine 3000 (Invitrogen, San Diego, cells were maintained in Dulbecco’s modi- CA, USA). Following transfection at 37°C for 48 fied Eagle’s medium (DMEM) (Invitrogen, h, the luciferase activities were determined on Carlsbad, CA, USA). The medium was supple- an LD400 luminometer (Beckman Coulter, Inc., mented with 10% fetal bovine serum (FBS), Brea, CA, USA). 100 IU/ml penicillin and 100 mg/ml strep- tomycin at 37°C in a humidified atmosphere Methylthiazolyl Tetrazolium (MTT) Assay containing 5% CO2. MiR-1271 mimics (miR- for Cell Proliferation 1271), miR-1271 inhibitors and their negative Twenty-hours after transfection, a total of 2×103 controls were purchased from GenePharma cells were seeded into 96-well plates and cultured Corporation. Overexpression CDK1 plasmid in medium with 10% FBS. MTT assay was used pCDNA3.1-CDK1 was granted from Doctor to detect cell viability at 0, 12, 24, and 48 h after Ming Wang. The empty pCDNA3.1 Vector seeding. Optical density (OD) was detected at a without any insert was used as a control of wavelength of 490 nm using an enzyme-labeled plasmid transfection. Transfection was carried analyzer. Each experiment was repeated three out using Lipofectamine 2000 (Invitrogen, times. Carlsbad, CA, USA) method. Clonogenic Assay miRNA Real-time RT-PCR At 12 h post-transfection, 3-5×103 EC cells Total RNA was extracted from surgical spec- were seeded into 60-mm Petri dishes in trip- imens or cell lines using the Trizol reagent (In- licate and maintained in RPMI 1640 (Gibco, vitrogen, Carlsbad, CA, USA) according to the Rockville, MD, USA) with 10% fetal bovine manufacturer’s instructions. For analysis of miR- serum (FBS). When there was visible colony by 1271 expression, qRT-PCR was performed using naked eye, the number of colonies containing Hairpin-it miRNAs qPCR Quantitation Kit (cat- more than 50 cells was counted manually. Col- alog number QPM-010, GenePharma, Shanghai, onies were counted and calculated in relation to China). All samples were carried out in triplicate. the values obtained from the mock and scram- U6 was used as an endogenous control. The ble-treated controls.

2817 L. Li, Y.-W. Qu, Y.-P. Li

Apoptosis Assay Results Apoptosis was assessed using Annexin V-FITC/PI double staining kit (Beyotime, Pud- miR-1271 Expression is Reduced While ong, Shanghai, China). Cells were harvested and CDK1 Expression is Increased in EC stained with Annexin V-FITC and PI according Tissues and Cell Lines to the manufacturer’s instructions. Cells were To investigate the possible role of miR-1271 then examined by flow cytometry (FACScan; BD in EC development, we first examined the ex- Biosciences, Jiangsu, Zhejiang, China) on in in- pression of miR-1271 in specimens and cells strument equipped with CellQuest software (BD in EC by RT-PCR. As shown in Figure 1A, the Biosciences, Jiangsu, Zhejiang, China). results showed that expression of miR-1271 was decreased in EC samples (p < 0.01), compared Statistical Analysis to normal matched tissues. Similarly, Figure 1B Statistical analysis was performed using SPSS revealed that miR-1271 was also significantly de- software (version 19.0, IBM, Chicago, IL, USA). Da- creased in three EC cell lines compared with that ta were collected from at least three separate exper- of T-HESC (all p < 0.01). On the other hand, we iments and were expressed as the mean ± standard further detected the expression levels of CDK1 in error. Paired t-test was used to analyze comparisons EC tissues and cell lines. The results of Western between the groups and paired data. Statistical sig- blot indicated that CDK1 was significantly upreg- nificance was considered when p < 0.05. ulated in EC tissues or cell lines compared to the

Figure 1. Analysis of miR-1271 and CDK1 expression in human EC tissues and cell lines. (A) qPCR was performed to determine the relative expression of miR-1271 in EC tissues and matched noncancerous tissues. (B) qPCR was performed to determine the relative expression of miR-1271 in EC cell lines (ECC-1, RL95-2, and AN3 CA) and normal T-HESC cells. (C) Western blot was performed to determine the relative expression of CDK1 in EC tissues and matched noncancerous tissues. (D) Western blot was performed to determine the relative expression of CDK1 in EC cell lines (ECC-1, RL95-2, and AN3 CA) and normal T-HESC cells. Data were expressed as mean ± SD of three independent experiments. *p < 0.05, ** p < 0.01.

2818 miR-1271 and endometrial cancer adjacent normal tissues or T-HESC cells. These Over-expression of miR-1271 Suppressed results suggested that miR-1271 and CDK1 may EC Progression in vitro play an important role in EC. To analyze the effect of miR-1271 on prolifera- tion of EC cells, we transfected miR-1271 mimic CDK1 was a Direct Target of miR-1271 and negative control (NC) in RL95-2 cells. As To understand the regulating mechanisms of shown in Figure 3A, miR-1271 mimic was able miR-1271, we examined its potential targets by significantly increased the expression of miR- Target Scan. As shown in Figure 2A, the data 1271. Next, we performed MTT and colony for- predicted CDK1 as a putative target for miR- mation assays. The results indicated that a signif- 1271. To determine whether CDK1 was a target icant reduction in cell viability was observed in of miR-1271, we cloned the predicted CDK1 the RL95-2 cells transfected with miR-1271 com- 3′UTR binding site, as well as its mutant form pared with those transfected with the miR-NC (p to firefly luciferase reporter gene, respectively. < 0.01, Figure 3B). Also, miR-1271 over-expres- Co-transfection with miR-1271 inhibited the lu- sion decreased the clonogenicity of RL95-2 cells ciferase activity of the reporter containing the compared with NC cells (p = 0.01, Figure 3B). mutant 3’UTR but not the wild type in EC cell Moreover, flow cytometry was performed to as- (Figure 2B). In addition, endogenous CDK1 ex- sess whether this effect was mediated through the pression in RL95-2 cells transfected with miR- induction of apoptosis. As shown in Figure 3C, 1271 mimic was examined. As shown in Figure our results showed that up-reguation of miR-1271 2C, the results showed that enhanced expression increased RL95-2 cell apoptosis. More impor- of miR-1271 by miR-1271 mimics in the EC tantly, we found that restoration of CDK1 partial- cells leads to decreased CDK1 levels. Taken ly attenuated the effects of miR-1271 on RL95-2 together, our findings suggested that miR-1271 cells (Figure 3B, 3C and 3D). These together, our directly targets CDK1 expression by binding to results suggested that miR-1271 served as a tu- the 3′UTR region of CDK1. mor suppressor via targeting CDK1 in EC.

Figure 2. miR-1271 directly targets the CDK1 3’UTR. (A) the predictive miR-1271 binding site of AKT1 3’UTR and the corresponding mutant binding site were shown. (B) miR-1271 significantly suppressed the luciferase activity that carried wild-type CDK1 but not the mutant CDK1. (C) levels of CDK1 in RL95-2 cells transfected with miR-1271 mimics or negative control. *p < 0.05, ** p < 0.01.

2819 L. Li, Y.-W. Qu, Y.-P. Li

Figure 3. miR-1271 suppresses proliferation and colony formation, and induces apoptosis via targeting CDK1 in EC. (A) overexpression of miR-1271 in EC cells was confirmed by qRT-PCR. (B) Cell viability was assessed by MTT assay. (C) representative quantification of crystal violet-stained cell colonies. (D) apoptosis rate of transfected EC cells measured by flow cytometry. Data were expressed as mean ± SD of three independent experiments. p* < 0.05, ** p < 0.01.

Knockdown of miR-1271 Promoted EC tic alterations. Great efforts have been made to Progression in vitro understand the principles of molecular changes To explore the effect of miR-1271 in progres- during the progression of this tumor16. CDK1 is sion of EC, we transfected miR-1271 inhibitor a 34 kD protein encoded by cell division cycle and negative control (NC) in RL95-2 cells. RT- gene 2 (cdc2) and is a member of the Ser/Thr PCR confirmed the down-regulated expression protein kinase family17. It has been reported that of miR-1271 when EC cells was transfected with CDK1 may play a negative regulator in various miR-1271 inhibitor (Figure 4A). As shown in tumors, such as ovarian epithelial carcinoma18, Figure 4B and 4C, knockdown of miR-1271 sig- lung adenocarcinoma19, and endometrial cancer20. nificantly promoted EC cell growth by MTT Some previous reports21,22 also showed that some and colony formation assays. In addition, the re- miRNAs were involved in the progression of sults of flow cytometry revealed that knockdown tumors by through the modulation of CDK1. In of miR-1271 significantly suppressed apoptosis the present study, we firstly found that miR-1271 (Figure 4D). These results showed that miR-1271 served as a tumor suppressor in EC. Then, we served as a tumor suppressor in EC. searched the bioinformatics approach and found that miR-1271 could bind to the CKD1-3’UTR. Thus, we wondered whether miR-1271 could reg- Discussion ulate the expression of CKD1. MiR-1271 has been found to participate in the carcinogenesis The development of EC is a multistep pro- of some types of cancers. For instance, Zhong et cess with accumulation of genetic and epigene- al23 observed that the overexpression of miR-1271

2820 miR-1271 and endometrial cancer

Figure 4. Down-regulation of miR-1271 promotes proliferation and colony formation, and suppresses apoptosis in EC cells. (A) down-regulation of miR-1271 in EC cells was confirmed by qRT-PCR. (B) cell viability was assessed by MTT assay. (C) representative quantification of crystal violet-stained cell colonies.(D) apoptosis rate of transfected EC cells measured by flow cytometry. Data were expressed as mean ± SD of three independent experiments. *p < 0.05, ** p < 0.01. significantly inhibited proliferation and invasion order to explore the mechanism underlying cell in prostate cancer cells by targeting DIXDC1. proliferation, our attention focused on the asso- Zhou et al24 found that overexpression of miR- ciation between miR-1271 and CDK1. Our data 1271 suppressed the NSCLC growth in vitro showed that downregulation of CDK1 by miR- and in vivo, while the inhibition of miR-1271 1271 resulted in the inhibition of prostate cancer significantly increased NSCLC growth. Liu et cell proliferation. al25 indicated that the overexpression of miR-1271 inhibited the proliferation of ovarian cancer cells by targeting Cyclin G1. Furthermore, in their Conclusions clinical investigation, they found that high miR- 1271 expression was correlated with a high rate We for the first time found that miR-1271 was of patient survival. These findings have provided downregulated in EC cells and tissues, and sup- hints that miR-1271 may be associated with the pressed EC cell proliferation and induced apop- survival of cancer cells. In the present work, tosis by targeting CDK1. These findings might we firstly showed that miR-1271 was signifi- provide some new insights into the potential cantly downregulated in human EC tissues and novel treatment targets for EC. cells by TR-PCR. Our gain- and loss-of-function experiments demonstrated that up-regulation of miR-1271 inhibited cell proliferation and induced apoptosis in EC cells. Down-regulation of miR- Conflict of Interest 1271 exerted the contrary effect in EC cells. In The Authors declare that they have no conflict of interests.

2821 L. Li, Y.-W. Qu, Y.-P. Li

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