ADAMTS9-AS1 Constrains Breast Cancer Cell Invasion and Proliferation via Sequestering miR-301b-3p

Objective: For determination of how ADAMTS9-AS1/miR-301b-3p/TGFBR2/JAK STAT signaling axis modulates progression of breast cancer cells. Methods: Target lncRNA was determined by differential analysis of breast cancer expression data and survival analysis. Differentially expressed miRNAs and target mRNAs that had binding sites with target lncRNA were predicted. GSEA software was used to carry out pathway enrichment analysis for mRNAs. Binding of the researched genes were tested with RNA binding protein immunoprecipitation (RIP). How miR-301b-3p bound TGFBR2 mRNA was tested by dual-luciferase method. Transwell, colony formation, EdU approaches were employed for verification of invasion and proliferation of breast cancer cells in each treatment group. Results: Markedly inactivated ADAMTS9-AS1 in breast cancer pertained to patient’s prognosis. MiR-301b-3p was capable of binding TGFBR2/ADAMTS9-AS1. However, overexpression of ADAMTS9-AS1 stimulated miR-301b-3p binding ADAMTS9-AS1 and repressed miR-301b-3p binding TGFBR2 mRNA. ADAMTS9-AS1 interference enhanced cancer proliferation and invasion, facilitated levels of KI67, PCNA, MMP-9 and MMP-2, and activated the JAK STAT signaling pathway. While silencing miR-301b-3p reversed the effect of ADAMTS9-AS1 interference. In addition, TGFBR2 interference or restraining JAK STAT signaling counteracted the effect of ADAMTS9-AS1. Conclusion: ADAMTS9-AS1 could sequester miR-301b-3p to inhibit progression of breast cancer via TGFBR2/JAK STAT pathway. This study supplies a rationale for incremental apprehension of ADAMTS9-AS1 in breast cancer progression.


INTRODUCTION
As the most prevalent female malignant tumor globally, morbidity of breast cancer has been steadily increasing since 1987, and its incidence accounts for approximately 24.5% of all women's malignancies (Sung et al., 2021). Not a single-gene disorder, breast cancer is a diversified disease classified by histology, immunopathology, mRNA expression profile and miRNA expression features rather than a monogenic disease (Laulin and Brudieux, 1990). As molecular biological research deepens, abnormal signaling pathways of tumor inhibitor genes and oncogenes manifest connection with breast cancer development (Boo et al., 2016;Gilam et al., 2016;Nassar et al., 2017;Sun et al., 2017). So far, the specific molecular mechanism for breast cancer progression has not been completely clarified.
Long non-coding RNAs (lncRNAs) exert a role in expression modulation at genetic levels and also exert functions on tumorrelevant cancer cell behaviors (Mercer et al., 2009;Fatica and Bozzoni, 2014). More and more evidence demonstrated association lncRNA-miRNA-mRNA axis maintains in tumor development. As a critical post-transcriptional regulator, the activity of miRNA can be sequestered by lncRNA (Wang et al., 2018;Peng et al., 2019;Shen et al., 2019). Such lncRNA is called competitive endogenous RNA (ceRNA). ADAMTS9-AS1 is an antisense lncRNA generated by reverse transcription of protein-coding gene ADAMTS9 and closely linked with various tumors. ADAMTS9 is a tumor inhibitor in assorted cancer cells: esophagus carcinoma (Lo et al., 2007), gastric cancer (Du et al., 2013), breast cancer (Ocak et al., 2013) and nasopharyngeal carcinoma (Lung et al., 2008;Sheu et al., 2009). Nevertheless, there is a shortage of deep research about the function of lncRNA ADAMTS9-AS1 in tumors. ADAMTS9-AS1 was found in esophageal squamous cell carcinoma at first (Li et al., 2017). It is greatly important for ectoderm and epithelial cells, as well as a novel biomarker for esophageal squamous cell carcinoma (Li et al., 2017). A study comprehensively analyzed lncRNA and found that ADAMTS9-AS1 is central in differential coexpression network in ovarian cancer (Wang et al., 2016). Few references have deeply researched ADAMTS9-AS1 in breast cancer yet. Here, such work was performed, which might conduce to biological understanding of this lethal disease.
Here, on the basis of the downloaded bioinformatics data, LncRNA ADAMTS9-AS1 was determined using differential analysis as well as survival analysis. Its role in breast cancer was explored by cell functional experiments. In an effort to scrutinize its downstream mechanism, bioinformatics databases were applied to predict differential miRNA and its target gene that had binding sites with ADAMTS9-AS1. The evidence proved that ADAMTS9-AS1 restrained invasion and proliferation of cancer cells through sequestering miR-301b-3p to target TGFBR2 and regulate JAK STAT signaling pathway. The results provide a foundation for further revealing of the mechanism of ADAMTS9-AS1 in breast cancer.

Bioinformatics Analysis
The Cancer Genome Atlas (TCGA) 1 provided expression data of lncRNAs, mRNAs and miRNAs related to breast cancer. Clinical information was offered in Supplementary Table 1. Differential expression analysis was performed with edgeR (| logFC| > 2, padj < 0.05). Differentially expressed lncRNAs 1 https://portal.gdc.cancer.gov/ (DElncRNAs) and miRNAs (DEmiRNAs) that had binding sites were screened through miRcode. 2 Downstream regulatory target genes of DEmiRNAs were predicted by miRDB, 3 TargetScan, 4 along with miRTarBase. 5 The predicted mRNAs were intersected with DEmRNAs to obtain target DEmRNAs of DEmiRNAs. Pathway enrichment analysis of unigenes was conducted by using Gene Set Enrichment Analysis (GSEA).

Cell Culture and Transfection
Mammary epithelial cells MCF10A (CBP60419) and breast cancer cells MDA-MB-231 (CBP60382), BT-549 (CBP60357), MDA-MB-468 (CBP60387) and MCF-7 (CBP60380) were cultured in complete mediums. All mediums and cell lines were purchased from Nanjing Cobioer Biotech. Cells were inoculated in 6-well plates at 3 × 10 5 each well until cell growth density reached 70-90%. Then, cells were subjected to transfection utilizing Lipofectamine 2000 (11668-019, Invitrogen, California, United States). Next, 250 µl Opti-MEM (11668-019, Invitrogen, California, United States) medium was used to release 4 µg target plasmids and 10 µl Lipofectamine 2000, respectively, and mixed well by flicking. After being maintained at room temperature for 5 min, two liquids were mixed evenly, and added into cell culture wells after 20 min. Culture plates were shaken gently and mixed evenly. Then plates were generally cultured in an incubator. After 24 h, medium was changed and cells were collected 36-48 h later.

Quantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was detached from cells (1 × 10 6 per well) with Trizol (No. 16096020, Thermo Fisher Scientific, New York, United States). Next, 5 µg samples were chosen for reverse transcription to synthesis into cDNA following the instruction of cDNA kit (K1622; Fermentas Inc., Ontario, CA, United States). MiR-301b-3p expression was detected by qPCR on TaqMan MicroRNA Assay with cDNA as the template. The reaction of qRT-PCR was as follows: 95 • C for 2 min, then 45 cycles of 95 • C for 15 s, 60 • C for 45 s, 72 • C for 45 s. U6 was taken as the internal reference to normalize results.
qRT-PCR was performed on ADAMTS9-AS1 and TGFBR2 according to the instruction of TaqMan Gene Expression Assays protocol (Applied Biosystems, Foster City, CA, United States).
GAPDH was taken as the internal reference. PCR procedures were designed as follows: 95 • C for 10 min, 35 cycle of 95 • C for 15 s, 60 • C for 30 s, 72 • C for 45 s. All qRT-PCR was set with 3 parallel wells. Primers were designed as shown in Table 1 and synthesized by TAKARA Company (Beijing, China). Relative transcription level of target gene was formulated by relative quantitative method (2 − CT ). Ct = Ct (experimental group)− Ct (control group), Ct = Ct (target gene)−t (internal reference), relative transcription level of target mRNA = 2 − Ct .

Western Blot
Total proteins were acquired from cells (1 × 10 6 per well), and protein concentration was determined by bicinchoninic acid (BCA) kit (Thermo, United States). Next, 30 µg total proteins were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at constant 80 V for 35 min and then 120 V for 45 min. The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Amersham, United States). The membrane was blocked with 5% skim milk at normal temperature for 1 h. It was followed by an overnight incubation at 4 • C overnight with rabbit anti-TGFBR2, Ki67, PCNA, MMP-2, MMP-9, JAK1, p-JAK1, JAK2, p-JAK2, STAT1, p-STAT1, STAT3, p-STAT3, β-actin. All antibodies were offered by Abcam (Cambridge, United Kingdom). The membrane was washed with Tris-buffered saline with 0.1% Tween R 20 detergent (TBST) three times (10 min each time). Afterward, secondary antibody goat anti-rabbit IgG H&L labeled by horseradish peroxidase (HRP) was added for 1 h of incubation at room temperature. Phosphate buffered saline with Tween 20 (PBST) solution was used for 3 washes of the membrane (each time 10 min). Chemiluminescence apparatus (GE, United States) was applied for scanning and development. Protein bands were gray-scale scanned with Image Pro Plus 6.0 (Media Cybernetics, United States) software to analyze relative protein expression.

In vitro Colony Formation Assay
The transfected breast cancer cells (1 × 10 4 per well) were inoculated into 12-well plates and cultured at 37 • C with 5% CO 2 for 2 weeks. Then cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of colonies, whose cells are more than 50, was manually counted.

EdU Assay
Cells (3 × 10 5 per well) to be tested were seeded in 24well plates with 3 parallel wells. EdU (C10310-1, RiboBio, Guangzhou, China) was added into the culture solution to achieve a concentration of 10 µmol/l for 2 h of incubation in the incubator. After removing medium, cells undergone 15 min of fixation with PBS + 4% paraformaldehyde at common temperature for and twice washes with PBS + 3% bovine serum albumin (BSA). PBS containing 0.5% Triton-100 was added for 20 min of incubation at room temperature and then PBS with 3% BSA was used to wash cells twice. Each well was added with 100 µl Apollo R 567 (C10310-1, RiboBio, Guangzhou, China) for 30 min of incubation in the dark at room temperature. Thereafter, cells were washed with PBS containing 3% BSA. DIPA (C0065, Solarbio, Beijing, China) was added to stain nucleus for 5 min. Next, cells were washed with PBS in triplicate and sealed. Cells were observed under a fluorescent microscope (Type: FM-600, Shanghai PuDa Company) and positive cell number in each field was recorded. Under the microscope, total cells presented as blue and positive cells presented as red. Percentage of EdU stained positive cells was calculated in 3 random fields of each well. EdU positive rate = (EdU positive nucleus number/total nucleus) × 100%.

Transwell Assay
Extracellular matrix (ECM) gel was maintained at 4 • C overnight and then attenuated with serum-free medium at 1:9 ratio to 1 mg/ml. Each polycarbonate membrane of the 24-well Transwell chamber was added with 40 µl ECM gel and maintained generally in the incubator for 5 h. Polymerized gel was prepared by ECM gel and redundant solution was removed. Then, 70 µl/chamber pure Dulbecco's Modified Eagle Medium (DMEM) was added. Cells (1 × 10 4 per well) were incubated in an incubator at 37 • C for 0.5 h to rehydrate Matrigel and redundant culture solution was removed. Cells in each group were starved for 24 h without serum, digested, centrifuged and resuspended with DMEM without FBS to a final concentration of 2.5 × 10 5 cells/ml. The upper chamber where the basement membrane was hydrated was added with added 0.2 ml suspension, while the lower chamber was added with 700 µl pre-cooled DMEM containing 10% FBS. Then, plates were generally cultured in a moist incubator for 24 h. Thereafter, the chamber was discarded, and cells in the chamber and on the membrane were wiped by a wet cotton swab. The remaining cells were fixed with methyl alcohol for 30 min, stained with 0.1% crystal violet for 20 min and dried in inverted position. Cells were observed and photographed under an inverted microscope. Average number of cells that passed through the membrane was counted in 5 random fields.

RNA Binding Protein Immunoprecipitation
Co-transfection was, respectively, undertaken on MCD-7 cells (3 × 10 5 per well) with mimic NC/miR-301b-3p mimic along with oe-NC/oe-ADAMTS9-AS1. The binding between miR-301b-3p with ADAMTS9-AS1 and TGFBR2 was examined using RIP kit. MCF-7 cells were washed with pre-cooled PBS and then the supernatant was removed. Cells were lysed with equivalent volume of lysis solution (P0013B, Beyotime) in ice-bath for 5 min and centrifuged at 4 • C and 14,000 rpm for 10 min. The cell extract was incubated with antibodies for co-precipitation. Specific steps were: 50 µl magnetic beads were taken from each co-precipitation reaction system, washed and resuspended in 100 µl RIP Wash Buffer. According to experimental groups, 5 µg antibodies were added for binding. Magnetic beads-antibody complex was resuspended in 900 µl RIP Wash Buffer after being washed. Thereafter, 100 µl cell extract was added for an overnight incubation at 4 • C. Samples were placed on the magnetic drill to collect magnetic beads-protein complex. The antibodies used in RIP were: Ago2 (ab32381, 1:50, Abcam, United Kingdom) which was evenly shaken at room temperature for 30 min, and IgG (1:100, ab172730, Abcam, United Kingdom) as the NC. Samples were digested by protease K. Then, RNA was extracted from the samples for succeeding analyses.

Dual-Luciferase Reporter Gene Assay
The whole length of 3 UTR of TGFBR2 gene was amplified. PCR products were cloned into multiple cloning sites on the downstream of pmirGLO luciferase gene (Promega, WI, United States) by using restriction enzyme sites, to construct TGFBR2-wild type (WT) (CCAGCUAUGA CCACAUUGCACUU) group. Binding sites of miR-301b-3p and its target gene were predicted by target gene databases. TGFBR2-mutant (MUT) vector (CCAGCUAUGACCACAUAC GUGAU) was constructed by PCR site-directed mutation method. Renilla luciferase expression vector pRL-TK (TaKaRa, Dalian, China) was taken as the internal reference. HEK-293T cells (3 × 10 5 per well) were co-transfected with mimic NC/miR-301b-3p mimic and luciferase reporter vectors, respectively. Dual Luciferase activity was tested by using Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States).

Statistical Analysis
Results were treated with SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, United States). Three times of biological repetitions and technical repetitions were required for each assay. Measurement data were shown as mean ± standard deviation. Comparison of two or more groups were, respectively, Student's t-test and analysis of variance (ANOVA). Repeated-measures ANOVA was adopted to compare results at each time point. Patient's overall survival curve was calculated by Kaplan-Meier. Patient's survival differences were analyzed by log-rank. Analysis method of enumeration data was chi-square test. P < 0.05 stands for statistical significance.

Inactivated ADAMTS9-AS1 in Breast Cancer
There were 1,017 DElncRNAs, 74 DEmiRNAs and 2,161 DEmRNAs, as offered by Figure 1A and detailed in Supplementary Tables 2-4. miRcode database was used to compare the results, finding that 60 lncRNAs and 18 miRNAs had binding sites (Supplementary Table 5). Low and high ADAMTS9-AS1 groups were classified by median ADAMTS9-AS1 level. Survival analysis of the 60 lncRNAs revealed notably lower survival of low ADAMTS9-AS1 expression group. Furthermore, ADAMTS9-AS1 levels noticeably pertained to clinical M stage, and patients in high ADAMTS9-AS1 group were mostly in M0 stage ( Figure 1B). Hence the downregulation of ADAMTS9-AS1 remarkably impacted patient's prognostic conditions. Meanwhile, qRT-PCR uncovered that level of ADAMTS9-AS1 was prominently low in cancer cell lines. MDA-MB-231 cells (p < 0.05) and MCF-7 cells (p < 0.05) showed the lowest and highest ADAMTS9-AS1 levels, respectively ( Figure 1C).

ADAMTS9-AS1 Restrains Breast Cancer Cell Proliferation and Invasion
After transfection of plasmids oe-NC and oe-ADAMTS9-AS1 was done, the latter exhibited a markedly increase in ADAMTS9-AS1 level, with respect to the former group (Figure 2A). Transfected cells were therefore employed in the succeeding analyses. Cell proliferation was examined through colony formation assay and EdU assay (Figures 2B,C). The results revealed that the cell proliferation was remarkably reduced by overexpressed ADAMTS9-AS1. Meanwhile, the result of Transwell assay uncovered that the overexpressed ADAMTS9-AS1 notably constrained the invasive ability of cells ( Figure 2D). Western blot result revealed that oe-ADAMTS9-AS1 markedly decreased the expression of tumor markers for proliferation and invasion, such as Ki67, PCNA, MMP-2, MMP-9 ( Figure 2E), suggesting that oe-ADAMTS9-AS1 could reduce tumor proliferative, and invasive abilities. ADAMTS9-AS1 was silenced in MCF-7 and the expression was examined afterward, finding a prominent reduction in si-ADAMTS9-AS1-1/si-ADAMTS9-AS1-2 groups ( Figure 2F). The si-ADAMTS9-AS1-1 with the highest interference efficiency was employed hereinafter and was uniformly named as si-ADAMTS9-AS1. Further, the changes of breast cancer proliferative ability (Figures 2G,H) and invasive ability ( Figure 2I) were examined by multiple methods. The results uncovered that cancer cell proliferation and invasion were prominently boosted after the downregulation of ADAMTS9-AS1. Western blot result uncovered that levels of Ki67, MMP-9, MMP-2 and PCNA were notably boosted after the downregulation of ADAMTS9-AS1 ( Figure 2J). All the above, ADAMTS9-AS1 repressed the proliferation and invasion of breast cancer cells. Morphology of cells after ADAMTS9-AS1 silence or overexpression manifested no noticeable differences between groups (Supplementary Figure 2).

ADAMTS9-AS1 Constrains Breast Cancer Cell Proliferation and Invasion via
Regulating JAK STAT Signaling Pathway MCF-7 cells were simultaneously transfected with si-NC and si-ADAMTS9-AS1, and treated with JAK STAT signaling pathway inhibitor INCB018424 (HY-50856, MedchemExpress;Punwani et al., 2012). qRT-PCR result indicated that the expression of ADAMTS9-AS1 in si-ADAMTS9-AS1 + DMSO group was markedly lower than that in si-NC + DMSO group (p < 0.05). While there was no significant difference in ADAMTS9-AS1 expression between the si-ADAMTS9-AS1 + DMSO group and the si-ADAMTS9-AS1 + INCB018424 group (Figure 6A). The expression of JAK STAT signaling pathway-related proteins was detected by western blot. The result suggested that compared with si-NC + DMSO group, the protein expression levels of p-JAK1, p-JAK2, p-STAT1 and p-STAT3 in si-ADAMTS9-AS1 + DMSO group were remarkably increased. Compared with si-ADAMTS9-AS1 + DMSO group, the protein expression levels in si-ADAMTS9-AS1 + INCB018424 group were notably decreased (p < 0.05) ( Figure 6B). In vitro colony formation ( Figure 6C), EdU (Figure 6D), and Transwell ( Figure 6E) assays indicated that compared with the si-NC + DMSO group, the proliferative ability and invasive ability of breast cancer cells in the si-ADAMTS9-AS1 + DMSO group were prominently increased. Compared with the si-ADAMTS9-AS1 + DMSO group, the proliferative and invasive abilities of breast cancer cells in si-ADAMTS9-AS1 + INCB018424 group were markedly decreased (p < 0.05). Western blot result exhibited that compared with si-NC + DMSO group, the expression levels of Ki67, PCNA, MMP-2 and MMP-9 in si-ADAMTS9-AS1 + DMSO group were markedly boosted, indicating increased tumor proliferative activity. Compared with si-ADAMTS9-AS1 + DMSO group, the expression levels of Ki67, PCNA, MMP-2 and MMP-9 in si-ADAMTS9-AS1 + INCB018424 group were prominently constrained, indicating decreased tumor proliferative activity (p < 0.05) ( Figure 6F). In general, ADAMTS9-AS1 could suppress the proliferation and invasion of breast cancer cells by regulating the JAK STAT signaling pathway. Besides, JAK inhibitor did not impact TGFBR2 levels according to detection results (Supplementary Figure 3).

DISCUSSION
Exposed to metastasis and recurrence, patients with breast cancer bear unsatisfied therapy efficacy and survival. Mechanism for invasion and proliferation is therefore urgently warranted for depiction. ADAMTS9-AS1 was noticeably inactivated in breast cancer cells here. ADAMTS9-AS1 is a dysregulated antisense lncRNA in many tumors and is involved in the pathogenesis of a variety of human malignancies (Wang et al., 2016;Xing et al., 2018). For example, in colorectal cancer, lncRNA ADAMTS9-AS1 constrains invasion and migration of colorectal cancer . ADAMTS9-AS1 also represses tumor-relevant prostate cancer behaviors (Zhou et al., 2021). The present paper found that ADAMTS9-AS1 could repress  Frontiers in Cell and Developmental Biology | www.frontiersin.org cancer proliferation and invasion through in vitro functional experiments. Nevertheless, there are reports that are contrary to our research results. For instance, overexpression of ADAMTS9-AS1 aggravates hepatocellular carcinoma (Zhang et al., 2020) and colorectal cancer development (Chen et al., 2020). We speculated that the different roles of ADAMTS9-A1 may be attributed to different cancer types.
To date, lncRNA/miRNA axis has been a limelight in the field of cancer. LncRNA can trap miRNA, regulate mRNA, and modulate progression of various cancers (Ao et al., 2019;Tu et al., 2019). This paper verified miR-301b-3p/ADAMTS9-AS1/TGFBR2 interaction by bioinformatics method, RIP and dual luciferase assay. This paper discovered relevance of miR-301b-3p and poor prognosis of breast cancer patients, and the cancer-promoting effect of si-ADAMTS9-AS1 could be reversed when cells with miR-301b-3p inhibitor at the same time. TGFBR2 was reported as a functional mRNA. For instance, TGFBR2 down-regulation will promote the malignant progression of prostate cancer (Zhou H. et al., 2018). MiR-3139 targets TGFBR2 expression to promote cell migration and invasion in colorectal cancer . Here, inhibiting the expression of TGFBR2 could weaken the tumor suppressor effect of ADAMTS9-AS1, suggesting that TGFBR2 was an inhibitor in breast cancer, which is similar to the results of previous studies.
Subsequently, we conducted enrichment analysis on TGFBR2 and found that it was mainly enriched in the JAK STAT signaling. The STAT3 protein is widely present in a variety of tumor tissue, affecting many biological behaviors of tumor cells, and acting as an oncogene, while JAK is responsible for the activation of STAT (Bournazou and Bromberg, 2013;Panni et al., 2014;Wen et al., 2015). Dysregulation of JAK STAT signaling pertains to breast cancer metastasis along with high risk of recurrence (Khanna et al., 2018). In addition, activated STAT3 triggers characteristics of cancer, including angiogenesis (Chung et al., 2014;Banerjee and Resat, 2016). Herein, in combination with rescue experiment, we found that ADAMTS9-AS1 constrained the activation of the JAK STAT signaling through miR-301b-3p/TGFBR2 axis. Thus, cancer invasion and proliferation were repressed.
The paper confirmed that ADAMTS9-AS1 was inactivated in breast cancer cells. ADAMTS9-AS1 could sequester miR-301b-3p to mediate TGFBR2 protein, thereby affecting the JAK STAT signaling, and repressing tumor progression. ADAMTS9-AS1/miR-301b-3p/TGFBR2 regulatory network may provide a more effective clinical treatment strategy for breast cancer patients. ADAMTS9-AS1 might serve as a promising factor for breast cancer. However, this study also has some limitations. First, we lack tissue samples and in vivo experiments to fully confirm the accuracy of the results. Second, detailed investigations on the genes that make up the lncRNA ADAMTS9-AS1/miR-301b-3p/TGFBR2 axis can further understand the mechanism of lncRNA ADAMTS9-AS1 inhibiting breast cancer progression. In the future, we will add animal experiments, and also pay more attention to the relationship between ADAMTS9-AS1 and other potential target miRNAs. We hope to offer a more perfect basis for therapeutic strategy of breast cancer.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
JC contributed to data analysis and manuscript writing. LC contributed to experiment concrete implementation. WZ acquired the data. RW looked up the literature. XW revised the manuscript. ZC designed the study and wrote the manuscript. All authors read and approved the final manuscript.