Hsa-circ-0001860 promotes Smad7 to enhance MPA resistance in endometrial cancer via miR-520h


 Background Medroxyprogesterone acetate (MPA) is one of the most commonly prescribed progestin for the treatment of endometrial cancer (EC). Despite initial benefits, many patients ultimately develop progesterone resistance. Circular RNA (circRNA) is a kind of noncoding RNA, contributing greatly to the development of human tumor. However, the role of circular RNA in MPA resistance is unknown. Methods We explored the expression profile of circRNAs in Ishikawa cells treated with (ISK/MPA) or without MPA (ISK) by RNA sequencing, and identified a key circRNA hsa_circ_0001860. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify its expression in MPA-resistant cell lines and tissues. CCK8, Transwell and flow cytometry were used to evaluate the functional roles of hsa_circ_0001860 in MPA resistance. The interaction between hsa_circ_0001860 and miR-520h was confirmed by bioinformatics analysis and luciferase reporter assay. Results The expression of hsa_circ_0001860 was significantly downregulated in MPA-resistant cell lines and tissues, and negatively correlated with lymph node metastasis and histological grade of EC. Functional analysis showed that hsa_circ_0001860 knockdown by shRNA promoted the proliferation, migration and invasion and inhibited the apoptosis of Ishikawa cells treated with MPA. Mechanistically, hsa_circ_0001860 promoted Smad7 expression by sponging miR-520h. Conclusion Hsa_circ_0001860 plays an important role in the development of MPA resistance in EC through miR-520h / Smad7 axis, and it could be targeted to reverse the MPA resistance in endometrial cancer.


Abstract Background
Medroxyprogesterone acetate (MPA) is one of the most commonly prescribed progestin for the treatment of endometrial cancer (EC). Despite initial bene ts, many patients ultimately develop progesterone resistance. Circular RNA (circRNA) is a kind of noncoding RNA, contributing greatly to the development of human tumor. However, the role of circular RNA in MPA resistance is unknown.

Methods
We explored the expression pro le of circRNAs in Ishikawa cells treated with (ISK/MPA) or without MPA (ISK) by RNA sequencing, and identi ed a key circRNA hsa_circ_0001860. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify its expression in MPA-resistant cell lines and tissues. CCK8, Transwell and ow cytometry were used to evaluate the functional roles of hsa_circ_0001860 in MPA resistance. The interaction between hsa_circ_0001860 and miR-520h was con rmed by bioinformatics analysis and luciferase reporter assay.

Results
The expression of hsa_circ_0001860 was signi cantly downregulated in MPA-resistant cell lines and tissues, and negatively correlated with lymph node metastasis and histological grade of EC. Functional analysis showed that hsa_circ_0001860 knockdown by shRNA promoted the proliferation, migration and invasion and inhibited the apoptosis of Ishikawa cells treated with MPA. Mechanistically, hsa_circ_0001860 promoted Smad7 expression by sponging miR-520h.

Conclusion
Hsa_circ_0001860 plays an important role in the development of MPA resistance in EC through miR-520h / Smad7 axis, and it could be targeted to reverse the MPA resistance in endometrial cancer.

Background
Endometrial cancer is one of the most common gynecological tumors in the United States 1 . In 2018, endometrial cancer affected 382,069 women worldwide and resulted in 89,929 deaths, and the incidence and mortality have been rapidly increasing in recent years [2][3] . More than 90% of endometrial cancers occurs in women over 45 years old, and about 7.1% of them are younger than 45 years old 4 . In order to preserve the fertility of young patients, progesterone such as medroxyprogesterone acetate (MPA) and megestrol acetate (MA), is regarded as the rst-line drug for conservative treatment [5][6][7] . In addition, patients in advanced stages who can't tolerate surgery also receive conservative treatment. Although 70% of the patients respond to MPA initially, 30% − 40% of them would recur, and 63% of the patients do not respond when they receive MPA treatment again 8-9 . Using a stable MPA-resistant Ishikawa cell, it has been shown that SIRT1 / FoxO1 / SREBP-1 as a pathway targeting PR is involved in the development of progesterone resistance in endometrial cancer cells 10 , but the molecular mechanism still remains unclear.
Therefore, it is of great signi cance to elucidate the mechanism and therapeutic target of MPA resistance in EC for individualized treatment of patients.
With the rapid development of RNA sequencing technology, a large number of previously known as "junk molecules" of non-coding RNA have been found to play important roles in human diseases, such as long non-coding RNA (lncRNA) and circRNA 11 . Characterized by covalent closed loop structure, circRNA has neither 5' end cap nor 3' end poly (A) tail. Therefore, it is di cult to be degraded by RNase R and relatively stable [12][13] . In addition, circRNA also has the characteristics of cell type and tissue speci city, spatiotemporal speci city, and evolutionary conservatism 14 . Therefore, these characteristics make it a potential and valuable biomarker for the prognosis and diagnosis in various carcinomas, such as hepatocellular carcinoma 15 , oral squamous cell carcinoma 16 and gastric carcinoma 17 .
In recent years, more and more studies have shown that circRNA plays important roles in the development of breast cancer 18 and gynecological cancer such as cervical cancer 19 , ovarian cancer 20 and endometrial cancer 21 . Moreover, circRNA regulates biological functions in a variety of ways, such as serving as microRNA sponges, gene transcription regulators, and protein decoys, and directly translating into protein 22 . Among them, miRNA sponge is the most common mechanism. For example, in endometrial cancer, circ_PUM1 can increase Notch3 by sponging miR-13, thus promoting the development of endometrial cancer 23 . However, the function of circRNA as miRNA sponge in the resistance of EC to MPA has not been elucidated.
In this study, we validated the differentially expressed circRNA hsa_circ_0001860 in MPA-sensitive ISK and MPA-resistant KLE and ISK PRB−/− cells. Functional test and luciferase reporter assay con rmed that hsa_circ_0001860 downregulation enhanced EC resistance to MPA through miR-520 h / Smad7 axis. These ndings may provide evidence to regulate MPA resistance of endometrial cancer by targeting circRNA hsa_circ_0001860 signaling pathway.

Patients and samples
Tissue samples and clinical data were collected from 113 endometrial cancer patients who received surgical treatment in the Shanghai International Peace Maternity and Child Health Hospital from December 2013 to December 2019. All patients were diagnosed according to histopathology report from biopsy after surgery, and none of them received chemotherapy or radiotherapy before operation. The tumor stages and histological grades were established in line with the criteria of Federation International of Gynecology and Obstetrics (FIGO) 2018 staging system. According to PR expression, patients were divided into MPA sensitive and MPA resistant groups. All tissue samples were stored at -80 °C until use.
The study was approved by the medical research ethics committee of the International Peace Maternal and Child Health Hospital, and the written informed consent of all patients was obtained when collecting specimens.

Cell Culture
Human EC cell lines including ISK and KLE were obtained from the American Type Culture Collection (ATCC, Manassas, vA, USA). MPA-resistant cell line ISK PRB−/− was established as previously described 10 .

RNA Isolation And Quantitative Real-time PCR (qRT-PCR) Assays
Total RNA was isolated using Trizol reagent (Takara, Dalian, China) and the RNA concentration was determined by NanoDrop ND-2000 (NanoDrop, USA). To quantify the amounts of mRNA and circRNA, 500 ng of RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Dalian, China).
Reverse transcription of miRNA was performed using a miScript II RT Kit (Qiagen). cDNA was ampli ed using Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). Real-time PCR was conducted with Quant Studio 7 Flex system (Life Technologies, USA) in accordance with the manufacturer's instructions. Actin was used as the control for the detection of mRNA and circRNA expression levels, while U6 was used as the control for miRNA expression analysis. The primer sequences used for qRT-PCR were listed in Table S1. The ΔΔCt method was used for quanti cation.

Cell Transfection
The EC cells planted on a six-well plate with 70-80% con uence were transfected with siRNA and miRNA mimics or inhibitors synthesized by GenePharma (Shanghai, China) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's instructions. The sequences used are listed in Tables S2  and S3. Two siRNA sequences for the hsa_circ_0001860 were used in this study (si-circ_0001860-2 has the highest inhibition e ciency and si-circ_0001860 mentioned in the article refers to si-circ_0001860-2).

In Silico Target Prediction And Luciferase Reporter Assay
The potential targets of hsa_circ_0001860 were predicted based on online software including CircBank 24 , CircInteractome 25 , and StarBase 26 . Finally, miR-520 h was predicted as a target gene of hsa_circ_0001860.
ISK cells were seeded in 24-well plates and cotransfected with corresponding plasmids and miRNA mimics. At 48 h after transfection, luciferase reporter assays were conducted using a dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer's instructions. Relative luciferase activity was normalized to Renilla luciferase activity.

Western Blot Analysis
Treated cells were lysed in RIPA buffer containing protease inhibitor phenylmethanesulfonyl uoride (Beyotime, Nanjing, China). Protein samples were loaded into the 10% sodium dodecyl sulfatepolyacrylamide (SDS-PAGE) gel and subjected to electrophoresis at 120 V, and then transferred to polyvinylidene uoride (PVDF) membrane (Millipore, Billerica, MA). The membranes were blocked with 5% BSA in TBST buffer and incubated with speci c primary antibodies at 4 °C overnight. The next day, membranes were washed for 15 min 3 times in TBST and incubated with secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by an enhanced chemiluminescence (ECL) system and imaged with Amersham Imager 600. GAPDH was used as loading control. Detailed information of antibodies used in this study was provided in Table S4.

Cell Proliferation And Cytotoxicity Assay
The cell proliferation and cytotoxicity was measured using Cell Counting Kit-8(CCK8) following the manufacturer's directions (Yeasen, Shanghai, China). The absorbance values were measured at 450 nm using a SpectraMax 190 microplate reader (Bio-Rad Model 680).

Migration And Invasion Assays
In the transwell migration and invasion assay, the upper transwell chambers (8-µm pore) were coated with 50 µl of Matrigel at a dilution of 1:6 (BD Biosciences, San Jose CA, USA). A total of 1 × 10 5 cells were seeded into the upper chamber of a 24-well chemotaxis chamber with polycarbonate lters (8-µm pore) (Corning Incorporated, Glendale, AZ, USA). DMEM/F12 supplemented with 10% FBS was added to the lower chamber. Then, cells were treated with MPA (10 µM) for 24 h or 48 h. After that the treatment, cells on the upper side of the chamber were removed, and cells on the lower side were xed with 4% paraformaldehyde, stained with crystal violet, and photographed under a microscope at 100 × magni cation. The number of crystal violet-stained cells was counted in ve elds from each well.

Apoptosis Assay
Cell apoptosis was detected using a Annexin V-PE/7-AAD Detection Kit (Yeasen, Shanghai, China) according to the manufacturer's instructions. Brie y, after incubation with MPA (10 µM) or dimethyl sulfoxide (control) for 48 h, cells were trypsinized, washed and resuspended in binding buffer. Next, 5 µl of Annexin V-PE and 10 µl of 7-AAD were added to the cell suspension and incubated in the dark at 4 °C for 15 min. A FACScan ow cytometer and FlowJo software (Tree Star Inc., Ashland, OR) were used to analyze the cells.

Statistical analysis
All experiments were performed in triplicate. Data were analyzed with SPSS software (version 19.0) (SPSS, Inc., Chicago, IL, USA) and presented as the mean ± SD. The statistical signi cance of the results was calculated using an unpaired Student's t-test. Clinicopathological features were analyzed by a χ2 test. A P value < 0.05 was considered statistically signi cant.

Results
Hsa_circ_0001860 is downregulated in MPA-resistant EC cells and tissues and negatively correlated with lymph node metastasis and histological grade In our previous study, we explored the expression pro le of circRNAs in Ishikawa cells treated with (ISK/MPA) or without MPA (ISK) by RNA sequencing, and identi ed 87 differentially expressed circRNAs. We further veri ed 20 dysregulated circRNAs by qRT-PCR and found that hsa_circ_0046843 and hsa_circ_0001860 were the most signi cant dysregulated circRNAs. Bioinformatics analysis such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and circRNA-miRNA interaction network analysis revealed that hsa_circ_0001860 was the most signi cantly upregulated circRNA and the key circRNA related to progesterone treatment in EC (data not shown).
To further study the role of hsa_circ_0001860 in progesterone resistance, we used qRT-PCR to verify its expression in MPA-resistant cell lines and tissues. Our data showed that Hsa_circ_0001860 was highly expressed in MPA-sensitive EC cell lines (ISK) compared with MPA-resistant EC cell lines (ISK PRB−/− and KLE). Its expression was dramatically increased in ISK by MPA treatment, however, it was not changed by MPA in ISK PRB−/− and KLE ( Fig. 1A and 1B). Moreover, the expression of Hsa_circ_0001860 was signi cantly decreased in MPA-resistant EC tissue compared with MPA-sensitive EC tissue (Fig. 1C). To explore the correlation between hsa_circ_0001860 expression and clinicopathological parameters, the median hsa_circ_0001860 expression value was used as the cutoff threshold to categorize all patients with EC. The results showed that the level of hsa_circ_0001860 was negatively correlated with stage, histological grade and lymph node metastasis (Table 1). Downregulation of hsa_circ_0001860 promotes proliferation and inhibits apoptosis of endometrial cancer cells Given that hsa_circ_0001860 was downregulated in MPA-resistant cell and tissue, we next examined the effect of hsa_circ_0001860 knockdown on EC cell lines, which were transfected with hairpin RNA (sh-circ_0001860) or the vector control (sh-NC). After transfection, the expression of hsa_circ_0001860 was dramatically decreased in sh-circ_0001860-transfected cells compared with sh-NC-transfected cells, indicating the successful knockdown by hsa-circ_0001860 ( Fig. 2A). It was further revealed that the proliferation was increased and the apoptosis was inhibited when circ_0001860 was knocked down. In line with this, the overexpression of hsa_circ_0001860 in ISK PRB−/− and KLE cells decreased the proliferation and promoted apoptosis ( Fig. 2B and 2D). However, little change in MPA-sensitivity was observed in these functional assays in ISK, ISK PRB−/− and KLE ( Figure S1).
Cell migration and invasion experiments showed that MPA could signi cantly inhibit the migration and invasion of MPA-sensitive EC cell lines (ISK) compared with DMSO, whereas no effect was observed on MPA-resistant EC cell lines (ISK PRB−/− and KLE). In order to further explore the effect of hsa_circ_0001860 on MPA-sensitivity in the migration and invasion of EC cells, we downregulated hsa_circ_0001860 in ISK cells, and upregulated hsa_circ_0001860 in ISK PRB−/− and KLE cells. We found that the downregulation of hsa_circ_0001860 abolished MPA-induced reduction of the migration and invasion of ISK cells (Fig. 3A and 3B). On the other hand, overexpression of hsa_circ_0001860 in MPA-resistant ISK PRB−/− and KLE cells promoted MPA-induced migration and invasion ( Fig. 3C and 3D). These results showed that EC cell sensitivity to MPA was mediated by hsa_circ_0001860.
Hsa_circ_0001860 regulates tumor progression and MPA sensitivity of EC Cells via binding to miR-520 h We next tried to predict the potential targets of hsa_circ_0001860 using the CircBank, CircInteractome and StarBase (Fig. 4A) and identi ed miR-520 h as a potential target gene of hsa_circ_0001860, which has a binding site for miR-520 h (Fig. 4B). Luciferase reporter assay demonstrated that miR-520 h expression signi cantly reduced the luciferase activity of the reporter in ISK cells co-transfected with WT but not MUT, suggesting that hsa_circ_0001860 may function as a sponge for miR-520 h (Fig. 4C). Then we investigated the biological functions of miR-520 h by knocking down miR-520 h with miR-520 h inhibitor in ISK cells transfected with sh_circ_0001860. It was found that miR-520 h inhibitor can reverse the effects of sh_circ_0001860 on promoting ISK cell proliferation and inhibiting its apoptosis ( Fig. 4D and   4E). Furthermore, knockdown of miR-520 h in sh_circ_0001860-transfected ISK cells rendered them sensitivity to MPA as evidenced by the inhibitory effect of MPA on the migration and invasion of miR-520 inhibitor-treated cells ( Fig. 4F and 4G).
Hsa_circ_0001860 regulates Smad7 expression and activates the Smad7/EMT signaling pathway It has been reported that miR-520 h enhances EOC cell dissemination and induces EMT in vivo by suppressing Smad7 expression 27 . We hypothesized that hsa_circ_0001860 could regulate tumorigenesis, migration and invasion of EC cells mediated by MPA by promoting Smad7 expression via acting as a sponge for miR-520 h. We examined the effect of hsa_circ_0001860 on the levels of downstream protein Smad7 of miR-520 h using Western blotting and found that knockdown of hsa_circ_0001860 decreased the levels of Smad7 and in uenced EMT signaling pathway-related proteins such as phosphorylated Smad2/3, E-cadherin and N-cadherin. Meanwhile, concurrent knockdown of miR-520 h and hsa_circ_0001860 reversed hsa_circ_0001860 knockdown-induced decrease of Smad7 expression ( Fig. 5A and 5B). These results suggest that circRNA hsa_circ_0001860 functions as a ceRNA to regulate Smad7 expression, activate the Smad7/EMT signaling cascade, and promote MPA sensitivity by targeting miR-520 h.

Discussion
EC is one of the most common gynecologic malignancies. Progestin therapy drugs including MPA and MA are often used to preserve fertility for young patients. However, almost a third of these patients eventually developed MPA resistance 8 . There are several mechanisms that underlie the acquired resistance to MPA, such as PR dysregulation 28 , Immune system and in ammatory response 29 and the activation of lipid metabolism 30 . Recently, some studies show that ncRNAs such as miRNAs and long ncRNAs (lncRNAs) also play vital roles in MPA resistance. For example, HOTAIR and LSD1 collaboratively repress PRB expression and thus reduce progesterone sensitivity in endometrial carcinoma cells 31 . CHOP and Lnc-CETP-3 might be involved in progesterone-PRB pathway to activate ER stress and provide therapeutic targets for EC patients with negative PRB expression 32 .
CircRNA has been considered to be essential in the carcinogenesis and tumor progression of EC 23 .
Besides, some studies also suggest that it could be a biomarker candidate for diagnosis and treatment of EC 33,34 . However, the function of circRNAs in MPA resistance remains unknown. In this study, we demonstrated that circRNA expression is associated with MPA resistance in EC. We identi ed a novel circRNA hsa_circ_0001860 that was downregulated in tissue samples from CircRNA can participate in biological functions in a variety of ways, and the most common way is acting as miRNA 'sponges' and regulate the expression and activity of the target genes 22 . In our study, we revealed that hsa_circ_0001860 acted as a miRNA sponge for miR-520 h. It is known that miRNAs participate in a majority of biological processes via regulating target gene expression 35 . MiR-520 h has also been studied in various cancers. A recent study shows that miR-520 h promotes the drug resistance of human breast cancer cells through protecting cells from paclitaxel-induced apoptosis by targeting Death-associated protein kinase 2 (DAPK2) 36 . In addition, miR-520 h also promotes EOC progression by downregulating Smad7 and activating the TGF-β signaling pathway 27 . Smad7 inhibits the TGF -β / Smad signal pathway by preventing the formation of Smad2 / 4 complex and nuclear translocation after phosphorylation of Smad2 and Smad3, thus inhibiting EMT 37 .
In our present study, we demonstrated that downregulating miR-520 h reversed MPA resistance and promoted MPA-induced migration and invasion in ISK-sh-circ_0001860 cells. Moreover, downregulation of hsa_circ_0001860 suppressed Smad7 protein expression, which could be reversed by the concurrent downregulation of miR-520 h. Therefore, our study presented a model of EMT process in EC cells treated with MPA, in which hsa_circ_0001860 may play a crucial role in EC metastasis and MPA resistance (Fig. 6).

Conclusion
In conclusion, we show that hsa_circ_0001860 plays an important role in the resistance of EC to MPA through miR-520 h / Smad7 axis, and it could be developed into a novel marker and therapeutic target for MPA-resistant endometrial cancer.

Declarations
Availability of data and materials Not applicable.

Ethics approval and consent to participate
The study was approved by the medical research ethics committee of the International Peace Maternal and Child Health Hospital, and the written informed consent of all patients was obtained when collecting specimens.

Consent for publication
All the authors agree to the publication clause.

Competing interests
The authors declare that there are no con icts of interest.