LncRNA ABHD11‐AS1 promotes the development of endometrial carcinoma by targeting cyclin D1

Abstract To investigate the expression, role and mechanism of action of long non‐coding RNA (lncRNA) ABHD11‐AS1 in endometrial carcinoma. The expression of lncRNA ABHD11‐AS1 was quantified by qRT‐PCR in human endometrial carcinoma (n = 89) and normal endometrial tissues (n = 27). LncRNA ABHD11‐AS1 was stably overexpressed or knocked‐down in endometrial carcinoma cell lines to examine the cellular phenotype and expression of related molecules. Compared to normal endometrial tissue, lncRNA ABHD11‐AS1 was significantly overexpressed in endometrial carcinoma. Overexpression of lncRNA ABHD11‐AS1 promoted the proliferation, G1‐S progression, invasion and migration of endometrial cancer cells; inhibited apoptosis; up‐regulated cyclin D1, CDK1, CDK2, CDK4, Bcl‐xl and VEGFA; and down‐regulated p16, while ABHD11‐AS1 down‐regulation has the opposite effect. RNA pull down demonstrated that lncRNA ABHD11‐AS1 binds directly to cyclin D1. Knockdown of cyclin D1 can reverse the effect of ABHD11‐AS1. Overexpression of lncRNA ABHD11‐AS1 increased the tumorigenicity and up‐regulated cyclin D1 in an in vivo model of endometrial cancer in nude mice. LncRNA ABHD11‐AS1 functions as an oncogene to promote cell proliferation and invasion in endometrial carcinoma by positively targeting cyclin D1.


| INTRODUCTION
The incidence of endometrial cancer is increasing year by year.
Endometrial cancer is the second leading cause of deaths due to gynaecological cancer worldwide. 1 Early-stage endometrial cancer has a high survival rate, while approximately 30% of patients are diagnosed with advanced stage disease. 2 Long non-coding RNAs (lncRNAs) are a class of RNA molecules over 200 nucleotides long without protein-coding ability that are expressed in a wide range organisms and tissues. 3,4 LncRNAs represent a new frontier in molecular biology and play significant roles in epigenetic, transcriptional and post-transcriptional regulation of gene expression, are involved in normal cell physiology and participate in the development of various diseases, including cancer. 5 The lncRNA ABHD11-AS1 has been found to be overexpressed in gastric cancer, 6 ovarian cancer 7 and bladder cancer tissues. 8 However, the role of ABHD11-AS1 in endometrial cancer has not been reported. Therefore, we investigated the expression, function and mechanism of action of lncRNA ABHD11-AS1 in endometrial carcinoma.

| Tissue specimens
Twenty-seven normal endometrial specimens that were collected from the normal endometrial tissue of patients with uterine fibroids undergoing hysterectomy, and 89 endometrial carcinomas were collected from patients undergoing surgical resection at the First Affiliated Hospital of China Medical University (Shenyang, Liaoning, China). No patients received pre-operative chemotherapy or radiotherapy. All specimens were confirmed by two pathologists. This research programme (No. 2016-32-2) was approved by the Chinese Medical University Ethics Committee. All tissue samples were processed in accordance with ethical and legal standards.

| Cell proliferation assays
Cells were trypsinized, seeded into 96-well plates at a density of 3000 cells/well, allowed to adhere, transfected and cultured for 0, 24, 48 or 72 hours. After adding 20 lL of MTT (5 mg/mL), the cells were incubated at 37°C for 2-4 hours, the medium was discarded, and 150 lL of dimethyl sulphoxide (DMSO) was added under dark conditions. Finally, the OD values were measured at 490 nm using a spectrophotometer (BioTek Instruments, Winooski, VT, USA). Each experiment was performed using triplicate wells (at least).

| Cell cycle assays
Control and transfected cells were cultured in six-well plates for 48 hours, trypsinized, centrifuged at 231 g for 5 minutes, washed twice with PBS; 70% of ice-cold ethanol was added, incubated at À20°C for 2 hours or overnight, washed twice with PBS; cell cycle detection kit (BD, New Jersey, USA) was added, incubated for 30 minutes at 4°C; and cell cycle analysis was performed by flow cytometry.

| Apoptosis assays
Quantification of apoptosis was performed using flow cytometry after staining with annexin V-labelled with 7AAD and PE (BD Biosciences) according to the manufacturer's instructions. Cells were harvested 48 hours after transfection, washed twice with cold PBS, resuspended, and then a mixture of 100 lL of 1 9 Binding Buffer and 5 lL of equal amounts of Annexin V-PE and 7AAD was added.
After incubation in the dark for 15 minutes, 400 lL of 19 Binding Buffer was added, and the cells were analysed by flow cytometry within 1 hour.

| Wound-healing assay
Cells were seeded in six-well plates at a density of 10 6 per well, allowed to adhere, and scratches were created in the monolayers

| Real-time PCR
Tissue specimens or cells were placed into microcentrifuge tubes with 1 mL of TRIzol (Takara, Shiga, Japan), the tissues were cut finely, and then the samples were shaken for 30 seconds. Chloroform was added, mixed thoroughly, incubated at room temperature for 10-15 minutes and centrifuged at 14803 g for 20 minutes. The upper layer (500 lL) was transferred to a new microcentrifuge tube; an equal volume of isopropyl alcohol was added, incubated at À20°C for 30 minutes and centrifuged at 14803 g for 20 minutes; the supernatant was discarded; 1 mL of 75% ethanol was added and centrifuged at 5782 g for 20 minutes; the supernatant was discarded; and the pellet was air-dried and dissolved in 10 lL of DEPC water. One microlitre of RNA was added to 79 lL of DEPC water, and the OD260/OD280 ratio was assessed to determine RNA concentration and quality.
Total RNA was reverse transcribed into cDNA using avian myeloblastosis virus transcriptase and random primers (Takara, Shiga, Japan) according to the manufacturer's instructions. The target gene was amplified by real-time quantitative PCR with SYBR Premix Ex Taq TM II kit (Takara). The relative expression of the target genes was determined by comparing the threshold cycle (Ct) values of the target genes to that of 18S rRNA (18S) using the 2 ÀDDCt method (Gen-ePharma).

| RNA pull-down assays
Streptavidin beads (BEAVER, Suzhou, China) were used to capture biotin-labelled ABHD11-AS1; IgG was used as a control. The biotinylated nucleic acid compound was incubated with protein lysates prepared from Ishikawa cells transfected with ABHD11-AS1 for 40 minutes in a 42°C water bath to specifically capture ABHD11-AS1 and the corresponding bound proteins. After elution of the beads, the protein samples were detected by Western blotting; the transfected protein in the cell samples was used a positive control; and IgG was used as a negative control.

| Statistical analysis
Data were analysed using SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). Correlations were analysed using Spearman's correlation test. Two-tailed t tests were used to compare mean values.
At least three replicates were performed for each group; at least three independent experiments were performed for each assay. All data are expressed as mean AE standard deviation; P < .05 was considered significant.

| LncRNA ABHD11-AS1 is overexpressed in endometrial carcinoma
Real-time PCR revealed the expression of ABHD11-AS1 was significantly higher in endometrial carcinoma than in normal endometrial tissues ( Figure 1A, P < .05).

| LncRNA ABHD11-AS1 promotes proliferation, cycle, inhibits apoptosis and enhances the invasive and metastatic potential of endometrial cancer cells
The expression of ABHD11-AS1 was lower in Ishikawa cells than HEC-1B cells (Figure 2A  The wound-healing assay and the Transwell assay revealed that ABHD11-AS1 transfection increased the migratory ability and invasive ability ( Figure 4A,C, P < .05), whereas si-ABHD11-AS1 transfection reduced the migratory ability and invasive ability ( Figure 4B,D, P < .05).

| LncRNA ABHD11-AS1 promotes endometrial tumorigenicity in vivo
Tumour xenograft volume in nude mice treated with ABHD11-AS1 was greater than that in mock nude mice ( Figure 5A,B,C, P < .05).
The growth rate was also faster than that in the mock group (Figure 5D, P < .05).

| LncRNA ABHD11-AS1 is coimmunoprecipitated with cyclin D1
RNA pull-down assays were performed to detect the protein that interacted with ABHD11-AS1. Protein from RNA pull-down assays with biotin-labelled ABHD11-AS1 against ABHD11-AS1 was used for Western blot, which demonstrated an enrichment of cyclin D1 compared with IgG ( Figure 6B). Western blotting of the xenograft tumour tissues from the nude mouse model showed that overexpression of ABHD11-AS1 increased the expression of cyclin D1, CDK1, CDK2, CDK4, Bcl-xl and VEGFA and decreased the expression of p16 ( Figure 6C).

| LncRNA ABHD11-AS1 promotes the development of endometrial cancer by targeting cyclin D1
A siRNA was used to silence cyclin D1 in ABHD11-AS1-overex- These observations are consistent with the reported roles of ABHD11-AS1 in gastric cancer, ovarian cancer and bladder. [6][7][8] LncRNAs participate in various physiological and pathological processes, including epigenetic regulation and the development of cancer. In addition, lncRNAs have the unique ability to interact directly with both nucleic acids and proteins and have discontinuous effects on many biological processes. 4 Several studies have shown lncRNAs play important roles in the regulation of protein expression. [10][11][12]14 In this study, Western blotting revealed overexpressing  In conclusion, this study reveals ABHD11-AS1 is up-regulated and promotes tumorigenesis in endometrial carcinoma by positively targeting cyclin D1. This is the first demonstration of an oncogenic role for ABHD11-AS1 in endometrial carcinoma; this finding could potentially pave the way for the development of novel strategies for diagnosis and treatment.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The research protocol was approved by the China Medical University Ethics Committee (No: 2016-32-2).

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
The data sets used and/or analysed during the current study available from the corresponding author on reasonable request.

CONFLI CT OF INTERESTS
The authors have no conflict of interests to declare.