Circ_PUM1 promotes the development of endometrial cancer by targeting the miR‐136/NOTCH3 pathway

Abstract Endometrial cancer is one of the most common gynaecological malignancies and the sixth most common cause of cancer‐related death among women. Here, we define the role and molecular mechanism of circ_0000043 (hereafter referred to as circ_PUM1) in the development and progression of endometrial carcinoma. QRT‐PCR was used to detect the expression of circ_PUM1 in normal endometrial tissue and endometrial carcinoma tissues. Changes in cell function and tumorigenicity in nude mice were examined after circ_PUM1 overexpression or knockdown. Bioinformatic analysis and dual‐luciferase reporter assay were used to predict and analyse the miRNAs that circ_PUM1 binds. Gene expression changes were analysed using Western blot. Circ_PUM1 was expressed at significantly higher levels in endometrial cancer tissues than in normal tissues. Up‐regulation of circ_PUM1 promoted the proliferation, migration and invasion of endometrial carcinoma cells. Opposite results were observed with circ_PUM1 knockdown, and the tumorigenic ability of endometrial cancer cells after circ_PUM1 knockdown was reduced compared to control cells. Circ_PUM1 is capable of binding to miR‐136, and up‐regulating its target gene NOTCH3, which can be reversed by overexpression of miR‐136. Circ_PUM1 can compete with miR‐136, leading to up‐regulation of NOTCH3, and thereby promote the development of endometrial cancer.

circular RNAs have been identified in mammalian cells, and the field has recently become a research hotspot. Circular RNAs are comprised of exons or introns generated by reverse shearing and other mechanisms, do not have 5' caps or poly(A) tail structures and are highly stable. 2 These properties make circular RNAs a highly efficient and competitive endogenous RNA. Circular RNAs may function as sponges by adsorbing corresponding miRNAs, 3 releasing expression of downstream target proteins and participating in various biological processes. It is reported that circRNAs play a pivotal role in the occurrence and development of a variety of cancers, including liver cancer, colon cancer, gastric cancer and others. [4][5][6] We focus on a circRNA PUM1, which is reported to promote the malignant behaviour of lung adenocarcinoma and ovarian cancer, 7,8 but related studies in endometrial carcinoma have not yet been reported.
This research aimed to explore the role of circRNA PUM1 in the development and progression of endometrial cancer.

| Specimens
A total of 69 cases of endometrial carcinoma tissue specimens were collected from patients who underwent surgery from

| Cell culture and transfection
Endometrial cancer cell line HEC-1B and Ishikawa human endometrial carcinoma cells were separately cultured in high glucose DMEM medium and RPMI-1640 medium containing 1% streptomycin and penicillin, and 10% foetal bovine serum (FBS). The cells were placed in a 5% CO 2 , 37°C incubator for maintenance culture, and the media were changed once every 24-48 hours depending on cell confluence.
When adherent cells approached confluence, they were digested with trypsin and subcultured. All operations of cell culture were performed under aseptic conditions in a clean hood, and all subsequent experiments were performed using cells in logarithmic growth phase. Cell transfection was performed using LipofectamineTM 3000 according to the manufacturer's instructions. The circ_PUM1 plasmid sequence is shown in Table S3.

| Cell proliferation assay
Cell proliferation was analysed by MTT assay. The log phase cells were trypsinized and counted. 3 × 10 3 cells were distributed in each well of 96-well plates, along with 100 µL of culture media. The experiment was divided into two groups, the transfection negative control group and the transfection experimental group, and measurements were taken at 4 time-points: 0, 24, 48 and 72 hours. At each time-point, 20 µL MTT solution was added and cells were cultured at 37°C for 4 hours, the media were discarded, 150 µL DMSO was added, and the OD value was measured at 490 nm. The proliferation of cells in the negative control group and the experimental group was compared. Each experiment was repeated in triplicate.

| Wound healing assay
A total of 10 6 cells were added to each well of 6-well plates with 2 mL of culture media, and allocated into a negative control group F I G U R E 1 Circ_PUM1 is overexpressed in endometrial carcinoma. Circ_PUM1 was significantly higher in endometrial cancer than in normal endometrial tissue (A). The expression of Circ_PUM1 was highest in HEC-1B, second in Ishikawa, third in EEC and lowest in hESC in endometrial cell lines (B). *P < .05 Experiments were performed in triplicate.

| Invasion assay
The

| Apoptosis assay
A total of 10 4 cells were added to each well of a 6-well plate with 2 mL of culture medium. The cells were divided into a negative control group and a experimental group. After 48 hours, the cells were

| qRT-PCR
Total RNA was isolated from endometrial cancer cell lines using Trizol reagent; cDNA was reverse transcribed using myeloblastosis virus transcriptase and random primers (Takara) according to the manufacturer's instructions. Genes of interest were amplified by real-time quantitative PCR using a SYBR Premix Ex Taq™ II Kit (Takara). The expression level of each target gene was normalized to that of 18s mRNA.

| Western blotting
The total protein in a sample was collected with RNA lysate and subsequently quantified and denatured; proteins were separated by SDS-PAGE and transferred to Hybond membrane (Amersham), followed by blocking for 1 hour with 3% bovine serum albumen. Then incubated with primary antibodies against NOTCH3 (1:1000; Proteintech, Proteintech Group) for 2 hours, washed with TBST buffer 3 times, incubated with HRP-conjugated anti-rabbit secondary antibodies for 2 hours, washed three times and visualized using ECL luminescence solution.

| Dual-luciferase reporter assay
Luciferase reporter assay was conducted to evaluate that miR-136 has a binding site within the 3' untranslated region (UTR) of circ_ PUM1. HEK293T cells were seeded into 24-well plates and cotransfected with 50 nmol/L miR-136, or negative control and 600 ng wild type circ_PUM1 3' UTR or mutant control dual-luciferase vector.
After 48 hours, the samples were measured for luciferase activity using a Dual-Luciferase Reporter Assay System (Promega).

| Statistical analysis
Data are presented as the mean ± SEM deviation from at least three separate experiments and were analysed using SPSS 20.0 software (SPSS Inc). Statistical comparison of means between groups was performed using Student's t test. All P-values are two-sided; P < .05 was considered statistically significant.

| Circ_PUM1 is highly expressed in endometrial carcinoma tissues and endometrial cancer cell lines
We detected the expression of circ_PUM1 by qRT-PCR in 16 normal endometrial tissues and 69 endometrial carcinoma tissues.
Circ_PUM1 expression was significantly higher in endometrial carcinoma than in normal endometrial tissue ( Figure 1A, *P < .05), details could be found in Tables S1 and S2. We detected the expres-

| Circ_PUM1 promotes the migration and invasion of endometrial cancer cells
Through scratch assays and transwell migration experiments, we verified that cell migration and invasion were increased after upregulation of circ_PUM1 expression ( Figure 3A,B,E,F, *P < .05).

| Disruption of circ_PUM1 structure decreases tumorigenic capacity of HEC-1B cells in nude mice
Compared with HEC-1B cells transfected with sh-NC, HEC-1B cells transfected with sh-circ_PUM1 showed significantly smaller tumour volumes and a relatively slow growth rate during the same time interval ( Figure 4A-C, *P < .05).

| Circ_PUM1 is capable of functioning as a molecular "sponge" via binding to miR-136
Bioinformatics software predicted miRNAs ( Figure 5A) that may bind to circ_PUM1. Dual-luciferase reporter gene results suggest that circ_PUM1 is capable of acting as a 'sponge,' binding to miR-136 ( Figure 5B, *P < .05).

| D ISCUSS I ON
Due to the lack of a free 3' or 5' end, circRNA is resistant to the normal mechanisms of linear RNA decay and therefore has a long half-life. 10 Due to the development and extensive use of RNA sequencing technology, especially those that do not rely on poly(A) purification, 11 the expression profiles and biological functions of cir-cRNAs have become a hot topic. Studies have shown that dysregulation of circRNAs is seen in a variety of cancers, [12][13][14] but studies in gynaecological malignancies are rare.
We screened for abnormally expressed circRNAs in normal ovarian and ovarian carcinoma tissues by microchips. Among the identified circRNAs, circ_PUM1 was significantly up-regulated in ovarian carcinoma tissues. 8 We wondered whether the circRNA was also HEC-1B cells with circ_PUM1 knockdown were injected subcutaneously into nude mice, and the tumour volume was significantly smaller than that of the control group at the same time-point.
Together, these data suggest that circ_PUM1 plays a role in the de- activity. 18 We wondered if circ_PUM1 also carries this function and used a bioinformatics approach to predict its miRNA binding sites. We analysed the predicted miRNAs and their corresponding downstream proteins in detail, combined with dual-luciferase reporter assay for verification. Our results show that miR-136 can bind to circ_PUM1. The role of miRNAs in gene dysregulation in cancer is critical. [19][20][21] MiR-136 has also been studied in various cancers. MiR-136 has been identified as a tumour suppressor gene in various adenocarcinomas such as breast cancer, colon cancer and lung cancer. [22][23][24] In addition, miR-136 can inhibit the activity of ovarian cancer stem cells and enhance the sensitivity of ovarian cancer to paclitaxel by targeting Notch signalling affects a variety of cellular processes, including maintenance of undifferentiated states, participating in cell fate decisions, inducing terminal differentiation and other functions involved in the progression of cancer. 25 Notch was originally found to be an oncogene in T cell leukaemia 26 and mediates TGF-α-induced changes in epithelial differentiation during pancreatic tumorigenesis. 27 NOTCH3 is more strongly expressed in endometrial cancer cells than in normal endometrial gland cells and is associated with poor prognosis. 28 Based on these data, we examined changes in NOTCH3 expression in Ishikawa and HEC-1B after overexpressing circ_PUM1 or knocking out circ_PUM1 by Western blot. We found that up-regulation of circ_ PUM1 promoted NOTCH3 protein expression compared with the control group, while circ_PUM1 knockdown yielded the opposite result.
Up-regulation of miR-136 in Ishikawa cells overexpressing circ_PUM1 reversed the up-regulation of NOTCH3 protein by circ_PUM1.
In conclusion, our data suggest that circ_PUM1 plays a critical role in the development and progression of endometrial cancer, mainly by adsorbing miR-136 via a 'sponge' effect and thereby promoting expression of the target gene NOTCH3. To the best of our knowledge, circ_PUM1 is the first circRNA to be studied in endometrial cancer and shown to be differentially expressed and functionally important. This research will provide new directions for improving and expanding molecular targets for the treatment of endometrial cancer.

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.