CircRHOT1 mediated cell proliferation, apoptosis and invasion of pancreatic cancer cells by sponging miR‐125a‐3p

Abstract Pancreatic cancer patients are asymptomatic at early stages and leading to late diagnoses. Additionally, pancreatic cancer easily metastasizes and is resistant to radiotherapy and chemotherapy. Therefore, it is critical to understand the underlying molecular mechanisms involved in pancreatic cancer to develop more efficient diagnostic and treatment strategies. In this study, we demonstrated that circRHOT1 was overexpressed in pancreatic cancer tissues and cell lines, and it was found to directly bind to miR‐125a‐3p, acting as an endogenous sponge to inhibit its activity. Knockdown of circRHOT1 expression significantly inhibited proliferation as well as invasion, and it promoted apoptosis of pancreatic cancer cells via the regulation of E2F3 through the targeting of miR‐125a‐3p. Taken together, our results showed that circRHOT1 plays critical roles in regulating the biological functions of pancreatic cancer cells, suggesting that circRHOT1 may serve as a potential diagnostic marker and therapeutic target for patients with pancreatic cancer.

structural features, circRNAs can act as microRNA sponges, RNAbinding protein sequestering agents and nuclear transcriptional regulators, meaning they can regulate gene expression at both the transcriptional and post-transcriptional levels. 13 Several types of circRNAs have been shown to play key roles in pancreatic cancer.
One study demonstrated that hsa_circ_0005397 is up-regulated in pancreatic ductal adenocarcinoma (PDAC). 14 hsa_circ_0005397 is derived from the RHOT1gene and has been termed circRHOT1.
The circRHOT1 expression was related to the pancreatic cancer cell proliferation, invasion and migration. 15 However, the function and potential mechanism of circRHOT1 in pancreatic cancer remain largely unknown.
In this study, we found that circRHOT1 was up-regulated in pancreatic cancer tissues and cell lines and that it directly binds to miR-125a-3p, acting as an endogenous sponge to inhibit miR-125a-3p activity. circRHOT1 mediates a regulatory pathway critical for the regulation of proliferation, invasion, the cell cycle and apoptosis of pancreatic cancer cells, which suggests that circRHOT1 might be a reasonable diagnostic and therapeutic target.

| Population
Twenty-eight pairs of PDAC fresh-frozen tissues, and adjacent non-

| Cell culture
The human pancreatic cancer cell lines (PANC-1, SW1990, COLO357 and CF-PAC1) and the human pancreatic ductal cell line (HPDE) were purchased from GeneChem (Shanghai, China). Cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) which was supplemented with 10% foetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% penicillin and streptomycin (Solarbio, Beijing, China), and they were maintained in a 37℃ incubator containing 5% CO 2 . The medium was replaced every 24-48 hours according to the cell density. Cells were observed under an inverted microscope and were digested with 0.25% trypsin (Gibco, Thermo Fisher Scientific) to enable passaging of the cells when they reached 80% confluence.

| Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent. Then, the concentration of RNA was measured using a spectrophotometer (Titertek-Berthold Colibri). Complementary DNA was synthesized using a PrimeScript RT reagent kit (Takara Bio Inc, Dalian, China), and quantitative real-time PCR was performed using SYBR Premix Ex Taq (Takara Bio Inc, Dalian, China). The threshold cycle (Ct) values for circRHOT1 and E2F3 were normalized against the Ct value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was an internal control, while miR-125a-3p was normalized against U6 snRNA, which was an internal control.
The relative RNA expression values were calculated using the 2 −ΔΔCt method.

| Western blot analysis
Protein was extracted using a Total Protein Extraction kit (KeyGEN Biotech, Nanjing, China), and the protein concentration was quantified using a BCA Protein Assay kit (KeyGEN Biotech). Each sample containing an equivalent amount of protein (20 µg) was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk powder for 1 hour at room temperature, the PVDF membranes were incubated overnight at 4°C with a rabbit antibody against E2F3 (1:1000; Affinity Biosciences, New Jersey, USA) and a rabbit monoclonal antibody against GAPDH (1:1000) (Beyotime Biotechnology, Beijing, China).
Then, the membranes were washed three times with TBS-T buffer, which was followed by incubation with a goat anti-rabbit secondary antibody (1:1000; Beyotime Biotechnology) for 1 hour at room temperature. Immunoreactive proteins were detected using an ECL Reagent (Affinity) and an automatic chemiluminescence image analysis system (Tanon 5200, Shanghai, China).

| Colony formation assay
Cell culture dishes (35 mm) were used, and each dish was covered with 2 mL of complete medium and 800 cells. After culturing for 14 days at 37°C, the colonies were fixed with 4% formaldehyde for half an hour, stained with 0.1% crystal violet solution for half an hour, imaged and counted.

| CCK-8 assay
Cell Counting Kit-8 (CCK-8) assays were performed with an Enhanced Cell Counting Kit-8 (Beyotime Biotechnology). PANC-1 cells were seeded into 96-well plates, and after adherence overnight, we transfected the cells according to the experimental design. Then, 10 µL of CCK-8 solution was added to each well. After 4 hours of incubation at 37°C with 5% CO 2 , the absorbance was measured at 450 nm by a microplate reader. We collected data once a day at the same time for 4 days.

| 5-Ethynyl-20-deoxyuridine assay
A Cell-Light 5-ethynyl-20-deoxyuridine (EdU) DNA Cell Proliferation kit from Donghuan (Shanghai, China) was used. PANC-1 cells were seeded into 24-well plates and then were transfected. When the cell density was close to 80%, the cells were incubated with serum-free DMEM supplemented with 10 μmol/L EdU for an additional 2 hours at 37°C, and then they were fixed with 4% formaldehyde. After EdU and DNA staining for 30 minutes each, images were immediately captured. All images were obtained with an Olympus FSX100 microscope (Olympus, Tokyo, Japan). The ratio of EdU-stained cells to Hoechst-stained cells was used to evaluate cell proliferation.

| RNA interference and stable transfection
Two small interfering RNAs (siRNAs) targeting the back-splice junction of circRHOT1 (si-circRHOT1-1 and si-circRHOT1-2) were designed and synthesized by Genechem. Through the detection, si-circRHOT1-1 knockdown efficiency was better than that of si-circRHOT1-2; the sh-circRHOT1 corresponding si-circRHOT1-1 was packaged into a GV248 lentiviral vector by Genechem. A miR-125a-3p mimic, an inhibitor and siRNAs targeting E2F3 (si-E2F3) were designed and constructed by GenPharma. The cells transfected with the sh-circRHOT1 lentivirus were cultured with 3 µL/mL puromycin for 4 days to generate a stably transfected cell line. Lipofectamine 3000 (Invitrogen, Carlsbad, USA) was used for siRNA and plasmid transfection.

| Dual-luciferase assay
Luciferase vectors with the 3'untranslated regions(3ʹUTR) of cir-cRHOT1 or E2F3 and their mutant versions, containing the Renilla luciferase gene (hRluc) and firefly luciferase gene (hLuc), were obtained from Genechem. 293T cells were plated in 24-well plates and were cultured overnight. Then, luciferase vectors were cotransfected into cells with the miR-125a-3p mimic or a mimic NC and were incubated for 48 hours. Luciferase assays were then performed using a Dual-Luciferase Reporter Assay System kit (E2920; Promega, Madison, USA). Firefly luciferase activity was normalized to Renilla luciferase activity and was expressed as a percentage of the control.

| Statistical analysis
Comparison of data between groups is presented as the mean ± SD.
Student's t tests, Fisher's exact tests and Mann-Whitney tests were performed by using SPSS (v.13.0.0; SPSS Inc, Chicago, IL, USA) to determine statistical significance. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant.
The primer sequences are listed as follows:

| CircRHOT1 is increased in PDAC tissues
Reverse transcription and quantitative real-time PCR (RT-qPCR) analysis was performed on 28 pairs of human PDAC specimens and their adjacent non-cancerous tissue samples to detect the expression of circRHOT1. CircRHOT1 expression was significantly upregulated in PDAC compared to the related adjacent non-cancerous tissues ( Figure 1A). We then divided these cases into negative and positive groups according to the ratio of circRHOT1 expression levels. Comparison of different clinical manifestations indicated that the expression level of circRHOT1 was correlated with lymphatic metastasis, but there was no statistical significance with tumour stage and size (Table 1). We then detected the expression of miR-125a-3p, which has potential binding sites for circRHOT1 in 28 PDAC and adjacent tissues ( Figure 1B). The expression of miR-125a-3p in PDAC tissues was significantly lower than that of adjacent tissues.

| CircRHOT1 overexpression affects the biological function of pancreatic cancer cells
To investigate the expression of circRHOT1 in pancreatic cancer cells, RT-qPCR analysis was performed. We confirmed the expres-  Figure 2C and 2D) and colony-forming capacity relative to that of the control cells ( Figure 2E). Additionally, knockdown of circRHOT1 significantly suppressed the invasiveness of PANC-1 cells ( Figure 2F). Moreover, this inhibition promoted apoptosis ( Figure 2G) and reduced the number of PANC-1 cells arrested in S phase ( Figure 2H).

| MiR-125a-3p has a crucial role in regulating the biological function in PANC-1 cells
By using TargetScan, miR-125a-3p was shown to have a binding site for circRHOT1 ( Figure 3A). Then, the expression levels of miR-125a-3p in HPDE and PANC-1 cells were examined by using RT-qPCR. The results indicated that the expression level of miR-125a-3p in PANC-1 cells was significantly decreased relative to that of HPDE cells ( Figure 3B). To investigate the function of miR-125a-3p in PANC-1 cells, a miR-125a-3p mimic and an inhibitor were used to regulate the expression of miR-125a-3p. The RT-qPCR results showed the efficiency of the miR-125a-3p mimic and inhibitor ( Figure 3C). Overexpression of miR-125a-3p reduced cell proliferation ( Figure 3D,E), reduced the colony-forming capacity ( Figure 3F) and suppressed the invasive potential of PANC-1 cells relative to the control cells ( Figure 3G); however, the opposite was true for the miR-125a-3p inhibitor. In addition, flow cytometry demonstrated that up-regulated miR-125a-3p promoted apoptosis ( Figure 3H) and reduced the number of PANC-1 cells arrested in S phase ( Figure 3I).

| CircRHOT1 regulates the expression of E2F3 by targeting miR-125a-3p in PANC-1 cells
To confirm the relationship between circRHOT1 and miR-125a-3p, RT-qPCR was used to detect the expression levels of miR-125a-3p and circRHOT1 after cells were treated with sh-circRHOT1, a miR-125a-3p mimic or a miR-125a-3p inhibitor. The results indicated that circRHOT1 knockdown partly rescued the expression of miR-125a-3p ( Figure 4A) and that circRHOT1 expression was negatively correlated with miR-125a-3p expression in PANC-1 cells F I G U R E 1 CircRHOT1 is increased in PDAC tissues. A, ΔCt value of circRHOT1 in PDAC tissues and adjacent normal tissues, as measured by RT-qPCR. B, ΔCt value of miR-125a-3p in PDAC tissues and adjacent normal tissues was measured by RT-qPCR. * P < .05, ** P < .01 ( Figure 4B). A luciferase reporter assay revealed that miR-125a-3phad binding sites for circRHOT1 ( Figure 4C). The above results suggest that circRHOT1 directly targets miR-125a-3p and negatively regulates it.
The probable binding sites in the target genes of miR-125a-3p were also predicted using TargetScan. The results showed that the E2F3 3ʹ-UTR was strongly linked tomiR-125a-3p ( Figure 4D), and the luciferase reporter assay confirmed this result ( Figure 4E).  Then, the expression of E2F3 in HPDE and PANC-1 cells was examined by using RT-qPCR and Western blot analysis. The results indicated that the expression of E2F3 in PANC-1 cells was increased relative to that in HPDE cells ( Figure 4F,G). The expression of E2F3 was decreased after PANC-1 cells were treated with sh-circRHOT1 ( Figure 4H,I). Additionally, the expression of E2F3was negatively correlated with miR-125a-3p expression when cells were treated with a miR-125a-3p mimic and an inhibitor ( Figure 4J,K). Moreover, RT-qPCR data and Western blot analysis showed that the miR-125a-3p inhibitor could limit the decrease in E2F3 expression induced by sh-circRHOT1 ( Figure 4L,M).

F I G U R E 3
MiR-125a-3p is crucial for regulating the biological function of PANC-1 cells. A, Putative complementary sites within circRHOT1 and miR-125a-3p were predicted by TargetScan. B, Relative expression of miR-125a-3pin human pancreatic ductal cell line (HPDE) and PANC-1 cells was measured by RT-qPCR. C, Relative expression levels of miR-125a-3p in PANC-1 cells after transfection were measured by RT-qPCR. D, Cell Counting Kit-8 assays were used to detect the viability of PANC-1 cells after transfection. E, 5-Ethynyl-20deoxyuridine assays were used to detect cell proliferation after transfection. F, Colony formation assays were used to detect clonogenic ability in PANC-1 cells after transfection G, Transwell assays were used to detect cell invasion capacities in PANC-1 cells after transfection. H and I, Flow cytometric assays were used to detect apoptosis and to assess the cell cycle after transfection. * P < .05, ** P < .01

| E2F3 is involved in modulating the biological functions of PANC-1 cells
To confirm whether E2F3 could modulate the biological function of PANC-1 cells, si-E2F3 was used to knock down the expression of E2F3 in PANC-1 cells. The RT-qPCR and Western blot results showed that the expression of E2F3 was significantly decreased 48h after transfection ( Figure 5A,B). The results from CCK-8 and EdU assays showed that the viability of PANC-1 cells was significantly decreased following treatment with si-E2F3 ( Figure 5C,D). E2F3 knockdown evidently suppressed the colony-forming capacity of PANC-1 cells ( Figure 5E). Additionally, decreased E2F3 suppressed the invasive potential of PANC-1 cells ( Figure 5F). Finally, flow cytometry demonstrated that knockdown of E2F3 promoted apoptosis ( Figure 5G) and reduced the number of PANC-1 cells arrested in S phase ( Figure 5H). The above results suggest that E2F3 is involved in modulating biological functions of PANC-1 cells.

| D ISCUSS I ON
Our study demonstrates that circRHOT1 is overexpressed in PDAC tissues, while miR-125a-3p is down-regulated. circRHOT1 is up-regulated Previous studies have shown that circRHOT1 is increased in pancreatic cancer tissues and cells. 14 Further, circRHOT1 promotes proliferation and invasion of pancreatic cancer cells. 16 In HCC, cir-cRHOT1 is significantly up-regulated and initiate NR2F6 expression to promote tumour progression, the expression of circRHOT1 was related to patient prognosis. 15 CircRNAs have been regarded as regulators or biological markers in many types of diseases, including pancreatic cancer. The classic function of circRNAs is their acting as a miRNA sponge to regulate the expression of miRNA target genes.
A previous study indicated that circ_0007534 regulates cell proliferation, apoptosis and invasion by sponging miR-625 and miR-892b in PDAC. 17 Another study suggested that circ_0006215 could regulate the expression of SERPINA4 by targeting miR-278a-3p in pancreatic cancer cells. 18 Our study shows a novel regulatory relationship between circRHOT1 and miR-125a-3p in pancreatic cancer. It has been reported that the expression of miR-125 is decreased in numerous F I G U R E 4 CircRHOT1 regulates the expression of E2F3 by targeting miR-125a-3p in PANC-1 cells. A, RT-qPCR was used to measure the relative expression of miR-125a-3pin PANC-1 cells after transfection. B, RT-qPCR was used to measure the expression level of circRHOT1 in PANC-1 cells after transfection. C, Dual-luciferase reporter assays were used to test the putative complementary sites within circRHOT1 with miR-125a-3p. D, Predicted binding sites of miR-125a-3p in the E2F3 3ʹUTRby TargetScan. E, Dual-luciferase reporter assays were used to test the putative complementary sites within miR-125a-3p and the E2F3 3ʹUTR. F and G, Relative expression of E2F3mRNA and protein in human pancreatic ductal cell line (HPDE) and PANC-1 cells was measured by RT-qPCR. H and I, RT-qPCR and Western blotting were used to measure the expression level of E2F3in PANC-1 cells after circRHOT1 knockdown. J and K, Relative expression of E2F3mRNA and protein measured by RT-qPCR and Western blot after transfection. L and M, Relative E2F3mRNA and protein expression was measured by RT-qPCR and Western blot after transfection. * P < .05, ** P < .01 kinds of cancer. 19 A recent study showed that miR-125asuppresses colorectal cancer progression by targeting VEGFA. 20 miR-125a suppresses the progression of bladder cancer by targeting FUT4. 21 These results suggest that miR-125a might serve as a tumour suppressor in cancers. In our study, miR-125a-3p was decreased in PANC-1 cells, and overexpression of miR-125a-3p reduced cellular proliferation and invasion and promoted apoptosis in PANC-1 cells. The qPCR and luciferase reporter assay results indicated that miR-125a-3p is a downstream target of circRHOT1. In addition, knockdown of miR-125a-3p could stifle the biological functions induced by inhibiting circRHOT1. These results suggest that circRHOT1 modulates biological functions of PANC-1 cells via regulation of miR-125a-3p.
TargetScan was employed to identify the downstream targets of miR-125a-3p, and E2F3 was selected for further analysis. Our current study indicates that the miR-125a-3p/E2F3 axis plays a critical role in the proliferation, apoptosis and invasion of PANC-1 cells. E2F3 is a member of the E2F family and serves as a transcription factor that could play an important role in cell proliferation, apoptosis and so on. 22 Numerous studies have shown that E2F3 functions in the control of tumour progression and is increased in different kinds of cancers. [23][24][25][26] Consistent with previous studies, the present study found that E2F3 was increased in PANC-1 cells. Additionally, knockdown of E2F3 could reduce proliferation and invasion and promote apoptosis of PANC-1 cells. The qPCR and Western blot results indicated that miR-125a-3p could regulate the expression of E2F3, and the luciferase reporter assay results showed that miR-125a-3p mimics obviously decreased the luciferase activity of E2F3-WT. Taken together, these data suggest that E2F3 acts as a direct target of miR-125a-3p. Our data showed that circRHOT1 inhibition decreased E2F3 expression, which could be reversed by treatment with miR-125a-3p inhibitors.
These results suggest that circRHOT1 performs biological functions in PANC-1 cells via regulation of the miR-125a-3p/E2F3 axis. However, our study has several limitations. Owing to the difficulty in collecting pancreatic cancer samples, the number of samples is low. Further study with a large sample size is needed to confirm these findings.

| CON CLUS IONS
Our data reveal that circRHOT1 is increased in PDAC tissues and cell lines. Additionally, circRHOT1 was correlated with lymphatic metastasis in PDAC, and it was found to regulate the proliferation, invasion and apoptosis of pancreatic cells by down-regulating miR-125a-3p to increase the expression of E2F3. Therefore, cir-cRHOT1 might be a reasonable diagnostic and therapeutic target for pancreatic cancer.

F I G U R E 5 E2F3 is involved in modulating the biological function of PANC-1 cells. A and B, Relative E2F3 expression was measured by
RT-qPCR and Western blot after E2F3 knockdown. C and D, Cell Counting Kit-8 and 5-ethynyl-20-deoxyuridine assays were used to detect the viability of PANC-1 cells after transfection. E, Colony formation assays were used to detect colony-forming capacity in PANC-1 cells after transfection. F, Transwell assays were used to detect cell invasion capacities in PANC-1 cells after E2F3 knockdown. G and H, Flow cytometric assays were used to detect apoptosis and to assess the cell cycle after E2F3 knockdown. * P < .05, ** P < .01