Long non‑coding RNA ASAP1‑IT1 suppresses ovarian cancer progression by regulating Hippo/YAP signaling
- Authors:
- Published online on: February 8, 2021 https://doi.org/10.3892/ijmm.2021.4877
- Article Number: 44
Abstract
Introduction
Ovarian cancer is the 7th most common form of cancer encountered in women worldwide, with an estimated 295,000 new cases and 185,000-related deaths in 2018 (1). Epithelial ovarian cancer is the most prevalent type of ovarian cancer, which can be further classified into endometrioid, clear-cell, mucinous, high-grade and low-grade serous carcinomas (2). Despite advancements being made in the understanding of ovarian cancer pathology and the application of novel early diagnostic and treatment strategies for patients with ovarian cancer, the majority of the patients are diagnosed at an advanced stage of the disease and the 5-year overall survival rate has been estimated to <50% (3). Therefore, the identification of novel targets for the diagnosis and treatment of patients with ovarian cancer is of utmost importance.
Long non-coding RNAs (lncRNAs) are single-stranded RNA molecules that are ≥200 nucleotides in length (4). According to the competing endogenous RNA hypothesis, certain lncRNAs contain miRNA response elements and can compete for binding to miRNAs with mRNAs, resulting in the regulation of gene expression (5). In doing so, lncRNAs are implicated in almost all physiological processes (6). Recent studies have indicated that the aberrant expression of lncRNAs is involved in cancer initiation and development, and notably in cancer cell proliferation and apoptosis (7). For example, lncRNA DNM3OS has been shown to be involved in the epithelial-to-mesenchymal transition process and to promote ovarian cancer cell migration and invasion (8). lncRNA ABHD11-AS1 has been shown to facilitate ovarian cancer cell proliferation, migration and invasion, and inhibit cell apoptosis via the upregulation of RhoC expression (9). lncRNA ASAP1-IT1 is an intronic transcript of the ASAP1 gene (10). A previous study indicated that ASAP1-IT1 expression was decreased in high-grade ovarian tumors compared with the corresponding expression noted in low-grade tumors (11). A high expression of ASAP1-IT1 has been shown to be associated with the optimal prognosis of patients with ovarian cancer (11). However, the mechanisms through which ASAP1-IT1 contributes to ovarian cancer progression remain unknown.
Cell number and cell size are tightly controlled by the Hippo pathway (12). The dysregulation of the Hippo pathway leads to the overgrowth of cells and resistance to cell apoptosis (13). Accumulating evidence has suggested that the overexpression of Yes associated protein 1 (YAP1), which is the downstream effector of the Hippo pathway, contributes to cancer development and initiation in different organs, including the colon, bladder, breast and ovaries (14-17). The higher expression of YAP1 has been observed in ovarian cancer tissues and cells, and is considered critical for cancer cell proliferation and survival (18). Among several tumor suppressive kinases of the Hippo pathway, the loss of large tumor suppressor 2 (LATS2) expression has been considered a major cause of YAP1 overexpression in cancer cells (19,20). Decreased expression of LATS2 was also reported in ovarian cancer (21). However, the regulation of LATS2/YAP1 signaling by lncRNAs in ovarian cancer remains elusive.
In the present study, the overexpression of ASAP1-IT1 was found to inhibit cell proliferation and induce apoptosis by sponging miR-2278. Concomitantly, it was able to increase LATS2 expression in ovarian cancer. The findings presented herein indicate that the ASAP1-IT1/miR-2278/LATS2 axis may play a key role in ovarian cancer.
Materials and methods
Clinical samples
Consecutive patients with ovarian cancer (n=58) who were treated at the China-Japan Union Hospital of Jilin University between January, 2015 and January, 2017, were used in the present study. The healthy tissues (n=58) were those 2 cm away from the tumors. The clinical stage and metastatic status of the cancer was determined by surgical evaluation, whereas histopathological analysis was conducted by 2 gynecological pathologists to assess cancer type and grade, independently. The extracted tissue samples were stored at -80°C. All the participants in the present study provided written informed consent prior to the surgery and the study conduct. Ethical approval was also provided for their participation by the Ethical Committee of The China-Japan Union Hospital of Jilin University.
Cell lines and cell culture
The human ovarian surface epithelial cell line HOSEpiC was purchased from ScienCell. The human ovarian serous cystadenocarcinoma cell line, SKOV3, and the human ovarian serous adenocarcinoma cell line, OVCAR3, were obtained from the American Type Culture Collection (ATCC). These cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) and supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were maintained in an incubator at 37°C with 5% CO2.
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was isolated from the HOSEpiC, OVCAR3, SKOV3 cells and tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. RNA was reverse transcribed into first-stranded cDNA using the RevertAid RT Reverse Transcription kit (cat. no. K1691, Thermo Fisher Scientific, Inc.). RT-qPCR was carried out with TB Green® Premix Ex Taq™. The reaction conditions included the following steps: Step 1: 95°C for 30 sec; step 2: 35 cycles of 95°C for 10 sec and 60°C for 30 sec. GAPDH was used as the internal control for lncRNA and mRNA, while U6 was used as the control for miRNA expression. The relative expression of each gene was calculated with the 2−ΔΔCq method (22). The primer sequences are listed in Table I.
Overexpression of ASAP1-IT1
The full length of ASAP1-IT1 was amplified from HOSEpiC cDNA, digested by restriction endonucleases EcoRI (NEB) and XhoI (NEB) and ligated into the pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific). The primer sequences were as follows: ASAP1-IT1 forward, 5′-GGG TAC CCA AAT GGG AAA AAA A-3′ and reverse, 5′-GGA ATT CCA AAG ACT ATC ACA-3′. For overexpression experiments, 2 µg pcDNA3.1-ASAP1-IT1 were transfected into the OVCAR3 and SKOV3 cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The cells were allowed to grow for 48 h and RT-qPCR was performed to confirm the overexpression of ASAP1-IT1 as described above.
Overexpression and downregulation of miR-2278
miR-NC mimic, miR-2278 mimic, miR-NC inhibitor and miR-2278 inhibitor were purchased from Suzhou GenePharma Co., Ltd. For transfection, 50 nM miR-NC mimic (5′-GUG GAU UUU CCU CUA UGA UUU-3′), miR-2278 mimic (5′-GAG AGC AGU GUG UGU UGC CUG G-3′), miR-NC inhibitor (5′-UUC UCC GAA CGU GUC ACG UUU-3′) or miR-2278 inhibitor (5′-CCA GGC AAC ACA CAC UGC UCU C-3′) were transfected into 1×106 OVCAR3 or SKOV3 cells in 6-well plates using Lipofectamine 300 (Invitrogen; Thermo Fisher Scientific, Inc.). Following 48 h of cell growth, the cells were harvested and the transfection efficiency was determined by RT-qPCR as described above.
Small interference RNA mediated silencing of LATS2
Control siRNA (5′-UUC UCC GAA CGU GUC ACG UTT-3′) and LATS2 siRNA (5′-UAC CAUA AAU ACA AUC UUC TT-3′) were synthesized and purchased from Suzhou GenePharma Co., Ltd. A total of 50 nM control siRNA or LATS2 siRNA were transfected into 1×106 OVCAR3 or SKOV3 cells in 6-well plates using Lipofectamine 300 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, cells were harvested and the transfection efficiency was determined by western blot analysis as described below.
Cell proliferation assay
The proliferative ability of the OVCAR3 and SKOV3 cells was detected using the CCK-8 kit (Dojindo Molecular Technologies, Inc.). Briefly, 10 µl CCK-8 solution were added into the culture medium and sustained for 1 h at 37°C. The absorbance at 450 nM of the medium was measured using a microplate reader (iMark™, Bio-Rad Laboratories, Inc.).
Cell apoptosis assay
The percentage of apoptotic cells was detected by flow cytometry. OVCAR3 and SKOV3 cells were incubated for 48 h at 37°C following transfection, harvested and stained with propidium iodide (PI) and Annexin-V provided by the Annexin-V Apoptosis Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The stained cells were examined by flow cytometry on a BD FACSVerse flow cytometer (BD Biosciences). PI+/Annexin-V+ and PI-/Annexin-V+ cells were considered as apoptotic cells.
Bioinformatics analysis
The sequence of ASAP1-IT1 was entered into the online miRDB software (http://mirdb.org/) (23) to predict potential binding miRNAs. The potential target genes of miR-2278 were predicted with the TargetScan software (http://www.targetscan.org/vert_72/) (24).
Western blot analysis
Tissues and cells were subjected to protein extraction using RIPA lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The lysates were quantified with a BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 20 µg proteins were loaded into each lane of the 8% SDS-PAGE gel and electrophoresis was performed. The proteins were transferred to a PVDF membrane. Subsequently, the membrane was blocked in 5% non-fat milk for 1 h at room temperature and incubated with primary and secondary antibodies at room temperature for 1 h at 37°C. The blots were developed using the ECL Western blot substrate (Pierce; Thermo Fisher Scientific, Inc.). The results were quantified using ImageJ software (version 1.8, National Institutes of Health). The antibodies used were as follows: YAP1 (cat. no. 14074, 1:1,000), p-YAP1 (Ser127l; cat. no. 13008, 1:1,000), LATS2 (cat. no. 5888, 1:1,000) and GAPDH (cat. no. 97166, 1:10,000) antibodies were purchased from Cell Signaling Technology, Inc; p-LATS2 (T1079 + T1041; cat. no. ab111344, 1:1,000) antibody was bought from Abcam. HRP-conjugated secondary antibodies against mouse (cat. no. L3032-2, 1:5,000) and rabbit (cat. no. L3012-2, 1:5,000) were products of Signalway Antibody LLC.
Dual luciferase reporter assay
The full length of ASAP1-IT1 was sub-cloned from pcDNA3.1 into the pmirGLO plasmid (Promega Corporation). The 3′UTR of LATS2 was amplified from HOSEpiC cDNA, digested with restriction endonucleases NheI (NEB) and SalI (NEB) and then ligated into the pmirGLO plasmid. Point mutations were introduced into the putative binding site for miR-2278 on ASAP1-IT1 and LATS2 3′UTR with a QuickChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) following the manufacturer's protocol. OVCAR3 and SKOV3 cells were seeded at a density of 5×105 cells in 24-well plates and transfected with 1 µg pmirGLO-ASAP1-IT1-WT, pmirGLO-ASAP1-IT1-Mut, pmirGLO-LATS2 3′UTR-WT or pmirGLO-LATS2 3′UTR-Mut. The cells were incubated for 48 h following transfection and subsequently collected for the determination of the relative luciferase activity, which was detected with a Dual Luciferase Reporter System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.
Statistical analysis
Graphpad Prism 6 was used to analyze the data and the data are presented as the means ± SD. Differences between 2 groups were compared with the Student's t-test and those between multiple groups were compared with one-way ANOVA followed by a Tukey's post hoc test. The log rank test and Kaplan-Meier curve were used for survival analysis and the graphs were prepared using Graphpad Prism 6. The association between ASAP1-IT1, miR-2278 and LATS2 expression with the clinicopathological characteristics of 58 ovarian tumors was examined using the Chi-squared test. The correlation between the differences in the mRNA expression levels of ASAP1-IT1, miR-2278 and LATS2 were analyzed by the Pearson's correlation analysis. A P-value <0.05 (P<0.05) was considered to indicate a statistically significant difference.
Results
High ASAP1-IT1 levels predict the optimal prognosis of patients with ovarian cancer
To examine the role of ASAP1-IT1 in ovarian cancer, 58 pairs of ovarian tumors were collected and matched with healthy tissues from the patients. The expression levels of ASAP1-IT1 were downregulated in ovarian tumor tissues compared with the non-cancer specimens (Fig. 1A). No significant associations were observed between ASAP1-IT1 expression and the clinical parameters of pathological type, patient age and lymph node metastasis (Table II). However, disease stage and differentiation status were associated with ASAP1-IT1 expression (Table II). Subsequently, the patients were divided into the ASAP1-IT1 high and low expression groups according to the median expression of ASAP1-IT1. The results of Kaplan-Meier analysis indicated that a high expression of ASAP1-IT1 was associated with the optimal prognosis of patients with ovarian cancer, whereas the 3-year survival rate was estimated at 85 and 45% in the high and low expression groups, respectively (Fig. 1B). These results highlighted the potential involvement of ASAP1-1T1 in the progression of ovarian cancer.
 | Table IIAssociation of ASAP1-IT1, miR-2278 and LATS2 expression with the clinicopathological characteristics of 58 patients with ovarian tumors. |
Overexpression of ASAP1-IT1 inhibits ovarian cancer cell proliferation and induces apoptosis
To investigate the biological function of ASAP1-IT1 in ovarian cancer cells, RT-qPCR was conducted to detect ASAP1-IT1 expression in normal ovarian cells and ovarian cancer cells. It was found that ASAP1-IT1 expression was decreased in OVCAR3 and SKOV3 cells, which are 2 ovarian cancer cell lines, compared with the corresponding expression noted in the HOSEpiC cell line (Fig. 2A). The vector containing the full length of ASAP1-IT1 was transfected into the OVCAR3 and SKOV3 cells in order to increase ASAP1-IT1 expression (Fig. 2B and C). The proliferative ability of the OVCAR3 and SKOV3 cells was detected by CCK-8 assay and the data indicated that it was significantly inhibited following ASAP1-IT1 overexpression (Fig. 2D and E). Moreover, flow cytometric analysis indicated an increase in the number of apoptotic cells observed in the OVCAR3 and SKOV3 pcDNA3.1-ASAP1-IT1-transfected cell lines (Fig. 2F and G). These data suggested that ASAP1-IT1 was involved in ovarian cancer cell proliferation and survival.
ASAP1-IT1 inactivates YAP1 signaling in ovarian cancer cells
Hippo/YAP1 signaling is critical for ovarian cancer cell proliferation and survival (17). In the present study, western blot analysis was used to detect the expression levels of LATS2, which is the major kinase component of the Hippo pathway (19). Moreover, the expression of YAP1 was detected in ovarian cancer cells following ASAP1-IT1 overexpression. ASAP1-IT1 overexpression increased LATS2 and p-LATS2 expression, and decreased YAP1 protein expression in the OVCAR3 and SKOV3 cells (Fig. 3A and B). An increase in the LATS2 mRNA levels was also observed (Fig. 3C and D). In addition, the mRNA expression levels of SGK1, FGF2 and ETV5, which are 3 conserved YAP1 downstream genes involved in ovarian cancer cell apoptosis (25-28), were decreased in the OVCAR3 and SKOV3 cells (Fig. 3E and F). These data indicated that ASAP1-IT1 upregulated LATS2 expression and inactivated YAP1 signaling in ovarian cancer cells.
ASAP1-IT1 sponges miR-2278 in ovarian cancer cells
Bioinformatic analysis was used to predict potential miRNAs that may interact with ASAP1-IT1. Among the candidate miRNAs, the ASAP1-IT1 sequence harbored the miRNA responsive element for miR-2278 (Fig. 4A). The expression of the latter has been shown to be upregulated in the serum from patients with ovarian cancer (29). It is interesting to note that in the present study, high expression levels of miR-2278 were associated with an advanced FIGO tumor stage (Table II). The overexpression of ASAP1-IT1 decreased miR-2278 expression in OVCAR3 and SKOV3 cells (Fig. 4B and C). Subsequently, miR-2278 inhibitor was used to decrease miR-2278 expression in OVCAR3 and SKOV3 cells (Fig. 4D and E). The downregulation of miR-2278 increased ASAP1-IT1 expression in these cells (Fig. 4F and G). In addition, transfection with miR-2278 mimic increased miR-2278 expression in the OVCAR3 and SKOV3 cells (Fig. 4H and I). In the OVCAR3 cells, the overexpression of miR-2278 decreased the relative luciferase activity of the ASAP1-IT1-WT cells, whereas this effect was not noted in the ASAP1-IT1-Mut cells that harbored mutations in the putative binding site (Fig. 4J). Similar results were observed in SKOV3 cells (Fig. 4K), suggesting a direct interaction between ASAP1-IT1 and miR-2278. Furthermore, a significant negative correlation between ASAP1-IT1 and miR-2278 expression was noted in the clinical samples (Fig. 4L). In contrast to the ASAP1-IT1 cells, a high expression of miR-2278 was associated with a poor prognosis of patients with ovarian cancer (Fig. 4M).
ASAP1-IT1 upregulates LATS2 expression via sponging miR-2278
Bioinformatics analysis indicated a putative binding site of miR-2278 in the 3′UTR of LATS2 mRNA (Fig. 5A), suggesting that ASAP1-IT1 may regulate LATS2 via miR-2278. The downregulation of miR-2278 increased LATS2 mRNA expression in OVCAR3 and SKOV3 cells (Fig. 5B and C). In addition, western blot analysis confirmed an elevation of LATS2 protein expression in OVCAR3 and SKOV3 cells transfected with the miR-2278 inhibitor (Fig. 5D and E). In contrast to these observations, the overexpression of miR-2278 decreased LATS2 mRNA (Fig. 5F and G) and protein expression levels (Fig. 5H and I). Dual luciferase reporter assay indicated that miR-2278 reduced the relative luciferase activity of the plasmid containing the LATS2 3′UTR sequence, which was reversed when the ASAP1-IT1 overexpression plasmid was transfected into OVCAR3 and SKOV3 cells (Fig. 5J and K). Furthermore, a positive correlation was noted between LATS2 mRNA and ASAP1-IT1 expression in the clinical samples (Fig. 5L). In contrast to these observations, the miR-2278 expression negatively correlated with the LATS2 mRNA levels in these samples (Fig. 5M). LATS2 expression was also negatively associated with the overall survival status of the patients (Fig. 5N). However, LATS2 mRNA expression was not associated with the parameters pathological type, age, FIGO stage, differentiation status and lymph node metastasis (Table II). The data indicated that miR-2278 directly suppressed LATS2 expression and suggested that ASAP1-IT1 may inactivate YAP1 signaling by binding to miR-2278.
ASAP1-IT1 inactivates YAP1 signaling via the upregulation of LATS2 expression
To explore whether ASAP1-IT1 can downregulate YAP1 via LATS2, LATS2 siRNA was used to silence LATS2 expression in OVCAR3 and SKOV3 cells (Fig. 6A and B). The silencing of LATS2 attenuated the downregulation of YAP1 protein expression induced by ASAP1-IT1 overexpression in OVCAR3 and SKOV3 cells (Fig. 6C and D). The Hippo pathway suppressed YAP1 activity via the phosphorylation of YAP1 to induce its translocation from the nucleus to the cytoplasm. Taken together, these data indicated that ASAP1-IT1 inactivated YAP1 signaling via the upregulation of LATS2 expression.
ASAP1-IT1 suppresses ovarian cancer progression via the regulation of LATS2 expression
The results of cell proliferation assay indicated that the silencing of LATS2 attenuated the growth inhibitory effects of ASAP1-IT1 overexpression on the OVCAR3 and SKOV3 cells (Fig. 7A and B). Furthermore, LATS2 silencing inhibited the apoptosis of OVCAR3 and SKOV3 cells induced by ASAP1-IT1 overexpression (Fig. 7C and D), suggesting that LATS2 is essential for the tumor suppressive role of ASAP1-IT1 in ovarian cancer.
Discussion
Accumulating evidence has indicated that lncRNAs are mainly involved in the initiation and development of ovarian cancer by controlling signaling networks and promoting sustained cancer cell proliferation and resistance to cell apoptosis (18,30,31). The inactivation of uncontrolled cell growth signaling and the induction of cancer cell death are critical for improving the prognosis of patients with ovarian cancer (32). Therefore, investigations into the biological roles and molecular mechanisms of lncRNAs is crucial for providing insight into the treatment of ovarian cancer.
Due to the oncogenic potential of ASAP1 (33), ASAP1-IT1, the intronic transcript of ASAP1 (11), has gained considerable attention in cancer research over the past years. The aberrant upregulation of ASAP1-IT1 expression has been observed in several cancer types, including bladder cancer (10), non-small cell lung cancer (34) and cholangiocarcinoma (35). In non-small cell lung cancer cells, ASAP1-IT1 facilitates cell proliferation, migration and invasion via the activation of the PI3K/AKT pathway (34). ASAP1-IT1 positively regulates Hedgehog signaling in cholangiocarcinoma cells, leading to an enhanced cell proliferation and migration (35). In contrast to these observations, ASAP1-IT1 expression is downregulated in ovarian cancer and is associated with the optimal prognosis of patients with this disease (11). Moreover, the results demonstrated that the upregulation of ASAP1-IT1 expression in ovarian tumors was associated with optimal prognosis. In addition, high expression levels of ASAP1-IT were associated with early-stage tumors and an optimal differentiation status. The overexpression of ASAP1-IT1 significantly inhibited ovarian cancer cell proliferation and induced cell apoptosis. The data suggested the involvement of ASAP1-IT1 in ovarian cancer progression.
The molecular mechanisms of ASAP1-IT1 in ovarian cancer cells have not yet been previously examined, at least to the best of our knowledge. The transcription co-activator, YAP1, plays a pivotal role in ovarian cancer initiation, proliferation and survival (36). The loss of LATS2 is essential for the malignant transformation of ovarian cells via the regulation of YAP1 expression (37). The present study indicated that ASAP1-IT1 upregulated LATS2 mRNA and protein expression, while it suppressed YAP1 protein expression. This suggested that ASAP1-IT1 may promote LATS2 mRNA stability, activate the Hippo pathway, destabilize YAP1 protein and inactivate YAP1 signaling. By using bioinformatic analysis, several miRNAs were predicted to interact with ASAP1-IT1. Among the list of miRNAs, miR-2278 was a highly ranked potential target of ASAP1-IT1. According to the microarray data derived from the sera of 168 high-grade serous ovarian carcinoma patients and 65 healthy controls, miR-2278 has been shown to be one of the top upregulated miRNAs identified (29). Currently, the association between miR-2278 and LATS2 has not yet been reported in any cell type, at least to the best of our knowledge. Bioinformatics analysis indicated that miR-2278 may target LATS2. By using RT-qPCR, western blot analysis and dual luciferase reporter assays, the data of the present study confirmed that LATS2 was a target of miR-2278 and that ASAP1-IT1 upregulated LATS2 expression. The Hippo pathway phosphorylates YAP1, leading to translocation of YAP1 from the nucleus to the cytoplasm, which is required for subsequent degradation (19). In the present study, ASAP1-IT1 overexpression induced the translocation of YAP1, which was reversed following LATS2 the silencing in OVCAR3 cells. Furthermore, silencing of LATS2 attenuated the biological function of ASAP1-IT1 in ovarian cancer cells. Previous studies have indicated that YAP1 is directly regulated by miRNA/lncRNA in ovarian cancer (17,18). The findings of the present study suggested that ASAP1-IT1 inhibited ovarian cancer progression by sponging miR-2278 and inducing the upregulation of LATS2 expression, the activation of the Hippo pathway and the inactivation of YAP1.
Taken together, the present study revealed that the ASAP1-IT1/miR-2278/LATS2 axis was involved in the regulation of ovarian cancer progression. Due to the complexity of signaling transduction in cancer cells, the current findings will be further extended in future studies in order to examine the molecular mechanisms of ASAP1-IT1 in ovarian cancer.
Funding
No funding was received.
Availability of data and materials
They are available at special request.
Authors' contributions
KW and YZ contributed to the experiment design and the performance of the experiments. KW and YBH collected the specimens and clinical information of the patients. CY supervised the study, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All procedures performed in the present study involving human participants were supervised and approved by the Ethical Committee of The China-Japan Union Hospital of Jilin University. Written informed consent for the publication of all data was obtained from all patients prior to participate the current study.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Not applicable.
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