Angiotensin II type 2 receptor promotes apoptosis and inhibits angiogenesis in bladder cancer
- Nana Pei†1, 2,
- Yingying Mao†2,
- Pengfei Wan2,
- Xinglu Chen2,
- Andrew Li3,
- Huiying Chen2,
- Jinlong Li2,
- Renqiang Wan4,
- Yanling Zhang2,
- Hongyan Du2,
- Baihong Chen2,
- Guangyu Jiang1,
- Minghan Xia1,
- Colin Sumners5,
- Guixue Hu6Email author,
- Dongsheng Gu7Email author and
- Hongwei Li2Email author
© The Author(s). 2017
Received: 1 December 2016
Accepted: 19 May 2017
Published: 9 June 2017
Bladder cancer (BCa) is the ninth most common form of cancer in the world. There is a continuing need not only for improving the accuracy of diagnostic markers but also for the development of new treatment strategies. Recent studies have shown that the renin-angiotensin system (RAS), which include the angiotensin type 1 (AT1R), type 2(AT2R), and Mas receptors, play an important role in tumorigenesis and may guide us in meeting those needs.
In this study, we first observed that AT1R and Mas expression levels were significantly upregulated in BCa specimens while AT2R was significantly downregulated. Viral vector mediated overexpression of AT2R induced apoptosis and dramatically suppressed BCa cell proliferation in vitro, suggesting a therapeutic effect. Investigation into the mechanism revealed that the overexpression of AT2R increases the expression levels of caspase-3, caspase-8, and p38 and decreases the expression level of pErk. AT2R overexpression also leads to upregulation of 2 apoptosis-related genes (BCL2A1, TNFSF25) and downregulation of 8 apoptosis-related genes (CASP 6, CASP 9, DFFA, IGF1R, PYCARD, TNF, TNFRSF21, TNFSF10, NAIP) in transduced EJ cells as determined by PCR Array analysis. In vivo, we observed that AT2R overexpression caused significant reduction in xenograft tumors sizes by downregulation VEGF and induction of apoptosis.
Taken together, the data suggest that AT1R, AT2R or Mas could be used as a diagnostic marker of BCa and AT2R is a promising novel target gene for BCa gene therapy.
Bladder cancer (BCa) is a cancer of the genitourinary system that typically originates from cells that line the inside of the bladder . Although BCa can occur at any age, it is typically seen in older adults and in high risk individuals with a history of smoking and occupational exposure to carcinogens [2, 3]. Roughly 70% of BCa cases are non-invasive bladder cancer that can be successfully physically excised, however, the other cases have a risk of progression to muscle invasive bladder cancer and metastasis to distant organs, endangering of the lives of patients. Despite improvements in therapy due to advances in diagnostic and surgical techniques, the majority of deaths caused by BCa result from metastasis that are resistant to conventional therapy [4–6]. Thus, novel treatment strategies for bladder cancer are urgently needed.
The renin-angiotensin system (RAS) consists of renin, angiotensinogen, angiotensin-converting enzyme, and multiple angiotensin peptides. A major regulatory component is angiotensin II (Ang II) which acts through the angiotensin type 1 (AT1R) and type 2 (AT2R) receptors and has been suspected of playing a major role in carcinogenesis [7–10]. In contrast to the well-known harmful activities of AT1R, AT2R is considered to be the protective arm of RAS and often acts in opposition to AT1R . Studies have shown that AT1R antagonists prevent angiogenesis and growth of xenograft models of human BCa [12, 13]. AT2R is known to inhibit cell proliferation and stimulate apoptosis in a variety of cell lines including vascular smooth muscle cells, cardiomyocytes, endothelial cells, prostate cancer cells, and lung cancer cells [14–19]. These findings suggest AT2R as a potential cancer therapeutic and no evidence for AT2R effectiveness in BCa has been documented until now.
In this study, we investigated the therapeutic potential of AT2R in BCa using adenovirus vectors. We first confirmed the role of adenoviral-induced AT2R overexpression on inhibiting proliferation and inducing apoptosis in bladder carcinoma cells. Second, we investigated the role of AT2R overexpression on BCa tumorigenesis in a xenograft murine model. Finally, we explored the mechanism of AT2R on BCa in vitro. This study demonstrates AT2R as a potential therapeutic agent for BCa and may allow us to gain further insight into BCa pathogenesis.
Human bladder cancer cell lines (EJ, UM-UC-3, 5637) were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in RPMI-1640 (Invitrogen) medium supplemented with 10% FBS under 5.0% CO2. Sera and media were purchased from Invitrogen and American Type Culture Collection. HEK 293A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen).
Primary bladder cancer biopsy specimens and normal biopsies were obtained from Nanfang Hospital (Guangzhou, Guangdong, China). The clinical information of patients was previously described . Both tumor and normal tissues were histologically confirmed by H&E (hematoxylin and eosin) staining. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of Nanfang Hospital.
Recombinant adenoviral construction and preparation
Recombinant adenoviral vectors were constructed, prepared, and titrated as previously described . These vectors were: an adenoviral vector containing the enhanced green fluorescent protein gene controlled by a cytomegalovirus promoter (Ad-CMV-eGFP) and an adenoviral vector containing genomic AT2R (G-AT2R) DNA with introns 1 and 2 and the encoding region and enhanced green fluorescent protein gene controlled by cytomegalovirus promoters (Ad-G-AT2R-eGFP).
For viral transduction, bladder cancer cell line cells (5 × 105) were seeded into six-well Nunc tissue culture plates. On the following day, cells were transduced with Ad-G-AT2R-eGFP or the control vector Ad-CMV-eGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA). Transduced cells were used 24 to 48 h later, depending on the specific protocol.
Cells transduced with Ad-G-AT2R-eGFP or Ad-CMV-eGFP for 48 h were washed briefly with Dulbecco’s PBS and then fixed for 10 min at 4 °C with cold methanol. Immunostaining was then done on the fixed cells as detailed previously  using a goat anti-AT2R receptor polyclonal antibody (1:200; Santa Cruz Biotechnology) followed by Alexa Fluor 594 goat anti-rabbit IgG (1:1,000; Invitrogen) as the secondary antibody. AT2R immunoreactivity (red) and green fluorescence (from eGFP) were detected using an Olympus BX41 fluorescence microscope.
Cell proliferation and cytotoxicity assays
Cell proliferation and cytotoxicity were evaluated using a CytoScan WST-1 Cell Proliferation Assay (G-Biosciences). WST-1 reagent was added to the culture medium (1:10 dilution), and absorbance was measured at 450 nm.
Apoptosis of viral vector-transduced cells was measured using a DeadEnd Colorimetric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System (Promega) and One Step TUNEL Apoptosis Assay Kit (Beyotime) as described previously . The number of stained cells that exhibit apoptotic-like morphology was assessed by counting cells from 10 randomly chosen fields per well.
Caspase-3-like protease activity
Caspase-3–like protease activity was assessed using the BD ApoAlert caspase-3 colorimetric assay kit (BD Biosciences) as described by the manufacturer. Transduced and control cells (106) were lysed in the lysis buffer contained in the kit followed by centrifugation (15,000 × g for 10 min at 4 °C). Caspase-3–like activity was assessed in supernatants by following the proteolytic cleavage of the colorimetric substrate Ac-DEVD-pNA. Samples were read at 405 nm in a spectrophotometer using a 100 μL quartz cuvette. DEVD-z-DEVD-fmk, a specific inhibitor of caspase-3, was used to confirm assay specificity.
RNA isolation, reverse transcription, and quantitative real-time RT-PCR
Total RNA was extracted using an RNeasy Mini-Kit (Qiagen) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed on an ABI 7500 real-time PCR system (Applied Biosystems) as described previously . The primers are listed in Table 1. The samples were quantified by the comparative △△CT method by using human GAPDH as the internal standard.
Apoptotic gene expression analysis using real-time PCR array
Genes involved in apoptosis were performed by means of the Human Apoptosis RT2 Profiler PCR Array (PAHS-012Z; SABiosciences, USA) as described previously . Genes with relative fold changes greater than ± 2 were considered as up or downregulated in expression. Genes that yielded a p-value of <0.05 were considered to display statistical significance for the study.
Western blot analysis
Western immunoblots were run as described previously . Primary antibodies and their sources were as follows. Anti-total p38 MAPK, anti-phosphorylated p38 (pp38) MAPK, anti-Erk, anti-phosphorylated Erk, anti-activated caspase 3, anti-activated caspase 8 were from Cell Signaling Technology. Anti-β-actin and the secondary antibodies horseradish peroxidase-conjugated anti-rabbit IgG and anti-rabbit IgG were from Sigma-Aldrich. Anti-goat IgG was from Santa Cruz Biotechnology.
Tumor growth assay
Female BALB/c nude mice aged 4 to 5 weeks were purchased from the Institute of Comparative Medicine and Center of Laboratory Animals of the Southern Medical University (SMU). Animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of SMU. Athymic mice were subjected to s.c. injections of human EJ bladder cancer cells (1.0 × 106) in Matrigel (50:50) into the lower flank to induce tumor growth. After the tumors reached ∼ 50 mm3, the mice were placed into three groups at random. The animals received intratumor injections of Ad-G-AT2R-eGFP (1 × 109 vg/mouse), Ad-CMV-eGFP (1 × 109 vg/mouse) or PBS with multiple target points after measuring the tumor size every 3 days. Each group contained 6 mice and the experiment was repeated 3 times. Intratumor injections were conducted a total of 9 times. Before each injection, the length and width of the tumors were measured by using a Vernier caliper. Following the study the mice were anesthetized and euthanized by decapitation and tumors were dissected. To confirm the transgene expression within the tumor, a series of 7-μM-thick fresh-frozen sections of the samples were made using a microtome at low-temperature and observed under fluorescence microscopy. Tumor volumes were calculated as follows: volume = (D × d2)/2, where D meant the longest diameter and d meant the shortest diameter. Tumors were isolated for Western blot and quantitative real-time RT-PCR analysis, or fixed in 10% buffered formalin and used for histologic and immunohistochemical analysis.
Tumors were fixed in 4% paraformaldehyde for 24 h and incubated in 70% ethanol for 48 h before embedding in paraffin. The embedded tumors were cut into 5-μm-thick sections and stained with H&E to determine morphology. Cell proliferation in the transplanted tumors was analyzed for Ki67 (1:200; Abcam) expression. Apoptosis of viral vector transduced cells in tumors was also measured using a DeadEnd Colorimetric terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) System (1:100; Promega). Visualization was achieved using the EnVision + peroxidase system (Dako). Ki67 immunoreactive cells were expressed as a percentage of the total cell number of examined fields. The apoptosis was assessed by counting the number of the apoptotic cells from 10 random fields per tumor. Counts were done by an individual who was blinded as to the treatment.
SPSS 21.0 software was used for statistical analysis. Data are presented as mean ± standard deviation (SD) from 3 to 6 independent experiments. Statistical differences were evaluated by one-way ANOVA followed by Dunnett’s post hoc test. The criterion for statistical significance was set at P < 0.05.
AT1R, AT2R and Mas expression in human BCa clinical specimens
Endogenous and adenoviral-mediated expression of AT2R in bladder cancer cells
The presence of endogenous AT2R in human bladder cancer cell lines was first assessed using real-time RT-PCR. Endogenous AT2R mRNA expression was minimally detectable in basal bladder cancer cell lines. Ct values for endogenous AT2R mRNA in untreated EJ, UM-UC-3 or 5637 cells were all greater than 35.0, which are defined as within the negative range.
In addition, the fluorescence micrographs presented in Fig. 2b show that incubation of EJ cells with Ad-G-AT2R-eGFP (100 ifu/cell) produced a high level of expression of AT2R immunoreactivity and eGFP at 24 h after viral transduction when compared with EJ cells that had been infected with Ad-CMV-eGFP, which demonstrate only eGFP fluorescence.
Effect of increased AT2R expression on cell proliferation in EJ cells
Analysis via a WST-1 assay of the proliferation of different types of BCa cells revealed that transduction with Ad-G-AT2R-eGFP (100 ifu/cell, 24 h) produced a significant cell growth inhibition when compared with cells transduced with Ad-CMV-eGFP (100 ifu/cell, 24 h) (Fig. 2c).
AT2R overexpression induces apoptosis in bladder cancer cells
Transduction of BCa cells with Ad-G-AT2R-eGFP (100 ifu/cell) for 2 days resulted in a large number of cells that exhibited apoptotic-like morphologic characteristics, when compared with Ad-CMV-eGFP–treated EJ, UM-UC-3 and 5637 human BCa cells (Fig. 2d). The AT2R-expressing BCa cells exhibited irregular-shaped nuclei and a clear boundary between nuclei and cytoplasm when compared with the controls, consistent with our previous report using TUNEL labeling in AT2R-expressing DU145 cells . The apoptotic action following AT2R transduction was confirmed by the finding that incubation of EJ cells with Ad-G-AT2R-eGFP (100 ifu/cell) for 2 days produced a significant increase in TUNEL labeling compared with the Ad-CMV-eGFP (100 ifu/cell)–treated cells (Fig. 2e and f).
AT2R overexpression-induced changes in gene expression in EJ cells
Primers for real-time RT-PCR
Differentially expressed genes in human BCa cells following Ad-G-AT2R-eGFP transduced
Name of gene
Relative fold change
(Gene Bank ID)
BCL2-related protein A1
Caspase 6, apoptosis-related cysteine peptidase
Caspase 9, apoptosis-related cysteine peptidase
DNA fragmentation factor, 45 kDa, alpha polypeptide
Insulin-like growth factor 1 receptor
NLR family, apoptosis inhibitory protein
PYD and CARD domain containing
Tumor necrosis factor
Tumor necrosis factor receptor superfamily, member 21
Tumor necrosis factor receptor superfamily, member 25
Tumor necrosis factor (ligand) superfamily, member 10
AT2R-induced apoptosis is mediated by Caspase-3
AT2R suppressed bladder tumor growth in nude mice
AT2R reduces cell proliferation in EJ xenograft tumors
Tumor sections from mice injected with viral vectors or PBS underwent immunostaining using antibodies to the Ki67 markers of cell proliferation. The results showed that both the staining intensity and the number of hyper-proliferative Ki-67 tumor cells were significantly decreased in the Ad-G-AT2R-eGFP-treated group compared with both control groups (Fig. 6e), suggesting that AT2R reduces cell proliferation in vivo.
AT2R induced apoptosis in vivo
Effect of AT2R on VEGF in bladder tumor xenografts
It was reported that AT2R was associated with increased VEGF secretion at low Ang-II concentrations  and our in vivo study also found that moderately increasing AT2R expression could increase the growth of HCC tumors . To characterize these effects in this experiment, we analyzed the VEGF expression level from the xenograft tumors. Our results shown that VEGF mRNA and protein were significantly reduced in tumors from nude mice administered Ad-G-AT2R-eGFP when compared to tumors from Ad-CMV-eGFP or PBS treated animals (Fig. 7c, d).
In this study, we investigated the effects of adenoviral mediated overexpression of AT2R on BCa cells and xenograft tumor models of BCa. Overexpression of AT2R by Ad-G-AT2R-eGFP transduction significantly reduced the viability of BCa cells and promoted apoptosis. The induction of apoptosis is associated with activation of p38 MAPK and inactivation of ERK MAPK, and is partly dependent upon activation of caspase-8 and caspase-3. Furthermore, the data also indicated that overexpression of AT2R significantly reduces human bladder xenograft growth and tumor angiogenesis.
We first examined the expression of AT1R, AT2R, and Mas in clinical tissue samples of bladder carcinoma. It is widely acknowledged that, apart from AT1R, RAS harbors other receptor subtypes, some of those which mediate actions opposing those of AT1R . AT2R and Mas are the most well characterize of these opposing or protective receptors with the Mas-related G-protein-coupled receptor (MrgD) as a more recently discovered candidate . Most importantly, AT2R and Mas mediate a multitude of strikingly similar tissue protective and regenerative effects including anti-inflammatory, anti-fibrotic, neuroregenerative, vasodilatory, and beneficial metabolic effects [28–30].
Our results indicate that AT1R was upregulated and AT2R was downregulated in bladder carcinoma tissues (Fig. 1). Consistent with our results, a recent study demonstrated that AT1R was upregulated in human bladder cancer and greater expression of AT1R was associated with greater microvessel density . There is also a direct correlation between AT1R expression and tumor stage and liver metastasis and corresponding inverse correlation with AT2R expression in human colorectal cancer .
In our study, Mas was also significantly upregulated in human bladder tissues (Fig. 1), consistent with our previous study showing that Mas was upregulated in human nasopharyngeal carcinoma tissues . Recently, it has also been demonstrated that Mas is increased in colonic adenocarcinomas  and higher expression of Mas is observed in hepatic colorectal metastases when compared to the surrounding liver tissue . To our knowledge, this is the first time that expression levels of AT1R, AT2R and Mas have been assessed in human bladder tissue and our results suggest that AT1R, AT2R, and Mas can be therapeutic targets.
Various sources have demonstrated AT2R overexpression-induced apoptosis in a variety of cell lines [15–19]. Apoptosis is an important mechanism by which cancer therapeutic agents can induce cancer cell death. Here, we show that endogenous AT2R mRNA levels were below the detection limit and Ad-G-AT2R-eGFP induced overexpression of AT2R in all three BCa cell lines produced apoptosis. In a recent study, we have demonstrated AT2R induced prostate cancer cell apoptosis in prostate xenograft tumors . However, our previous study also showed that high levels of AT2R elevated apoptosis in HCC cell lines, while moderate and low level of AT2R did not impact apoptosis . Due to the highly sensitive nature of this AT2R mediated effect, it is important to obtain a more comprehensive and deeper understanding of the signaling and mechanisms underlying AT2R activation.
AT2R-mediated apoptosis undergoes different cellular mechanisms of apoptosis depending on the cell type. In a rat insulinoma cell line such as the INS-1O, for example, overexpression of AT2R induces caspase-8, caspase-9, and caspase-3 cleavage and decreases Bcl-2, pAkt, and pERK expression levels . In intestinal epithelial cells, Ang II signals through AT2R to upregulate GATA-6 expression which in turn upregulates the expression of Bax and eventually leading to apoptosis in these cells . In HL-1 cardiomyocytes, iNOS upregulation following induced AT2R expression seems to be the basis for increased in cardiomyocyte apoptosis . In another case, AT2R signaling stimulates the MAPK tyrosine phosphatase, (MKP)-1, which inhibits MAPK activation and consequently inactivates Bcl-2 and induces apoptosis in proximal tubular cells . Our previous studies suggest that AT2R-mediated apoptosis was mediated by p38 MAPK and caspase-3 and downregulation of Gadd45a, TRAIL-R2, and harakiri Bcl-2-interacting protein (HRK) in prostate cancer cells [17, 23]. In HCC cell lines, we observed activation of p38 MAPK and phosphorylated c-Jun N-terminal kinase (pJNK) . Consistent with the latter, the current experiments indicate that AT2R overexpression induced apoptosis in BCa cells is mediated via an extrinsic cell death signaling pathway that is dependent on activation of p38 MAPK, caspase-8, and caspase-3 and downregulation of Erk/MAPK.
To our knowledge, this is the first study which clearly assesses apoptosis-related gene expression profiles associated with AT2R-induced apoptosis in BCa cells. We observed that forced overexpression of AT2R resulted in significant changes in a large number of genes including both pro-apoptotic genes and anti-apoptotic genes, as determined by PCR array analysis. BCL2A1, which was elevated to 8.57-fold above control values, belongs to the pro-survival BCL2 family and is one of the less extensively studied anti-apoptotic proteins. BCL2A1 has been recently described as an oncogene responsible for resistance to BRAF inhibition in melanoma . A study also showed that elevated BCL2A1 protein prevents apoptosis . In the present study, BCL2A1 was upregulated, suggesting that BCL2A1 may actually oppose the apoptotic effect that is induced by AT2R in BCa.
Moving on to TNF, TNFα is a double-edged sword in tumor development. In most cases, TNFα acts as a promoter rather than a killer in tumor cells and tissues . It should be noted that in our case the expression of TNF was 0.07-fold of control values. This leads to the suspicion that TNF may be a negative regulator in AT2R induced apoptosis in BCa cells. Our results showed that the expression of TNFRSF25 (DR3) was increased, while TNFSF10 (TRAIL) and TNFRSF21 (DR6) were downregulated. Interestingly, TNFSF10 was also downregulated and TNFRSF10B (DR5) was increased in AT2R-mediated apoptosis in prostate cancer cells in our previous study . In addition, others have demonstrated that several cancers are resistant to TRAIL-induced cytotoxicity and increased expression of the DR could overcome human cancer resistance to TRAIL [41, 42]. Overall, it can be said that other factors or other mechanisms may be important regulators of sensitivity to TRAIL-induced apoptosis in these cancer cells. Here, we would like to conclude that TNFSF10 (TRAIL) and DRs were involved in the AT2R induced apoptosis in BCa cells, however the mechanisms or the sensitivity to TRAIL-induced apoptosis in BCa needs to be studied further.
Apoptosis-associated genes like DFFA, PYCARD, NAIP, IGF1R, CASP6 and CASP9 were also up or downregulated in the present study. However, these genes have not been observed to change in our previous study in AT2R-expressing DU145 cells. In all, these results may help to elucidate the complicated mechanisms of AT2R inducing apoptosis in different tissues and cells.
Angiogenesis is a complex process and a large number of factors are involved in tumor angiogenesis. A recent study notes an AT1R antagonist as an angiogenic inhibitor in a xenograft model of bladder cancer  and prostate cancer . There is also evidence showing that AT1R was associated with colorectal tumor VEGF-A secretion and microvessel density of bladder cancer [25, 31], suggesting that AT1R could regulate tumor angiogenesis. Kosugi et al demonstrated that an AT1R antagonist could prevent tumor growth and angiogenesis in xenograft models of human BCa using KU19-19 cells through the suppression of VEGF [12, 13]. In most pathophysiological situations and in some experimental models of angiogenesis , AT2R is thought to oppose the actions of the AT1R subtype. Yet some data indicate that AT2R may be pro-angiogenic  and works in concert with the AT1R subtype to increase VEGF levels and blood vessel formation [46, 47]. These conflicting reports leave open the question of whether AT2R activation has beneficial or deleterious effects on tumor angiogenesis. In our case, we found that AT2R markedly reduced VEGF expression in human bladder tumor xenografts, suggesting that this growth factor is involved in the antiangiogenic response to the receptor. A significant decrease of VEGF mRNA was observed in the tumors from mice treated with Ad-G-AT2R-eGFP when compared with tumors from control animals. VEGF receptors (Flt-1 and Flk-1) were also reduced in the tumors from mice treated with Ad-G-AT2R-eGFP when compared with the controls (Fig. 7). Taken together, these results demonstrate that VEGF downregulation is also responsible for the observed AT2R-mediated reduction in human bladder xenograft growth and tumor angiogenesis.
In summary, we have shown that intratumor administration of adenovirus-based therapeutic gene delivery causes high gene expression in vivo. The administration of Ad-G-AT2R-eGFP then significantly attenuates the growth of bladder carcinoma tumors suggesting the effectiveness of this adenovirus-based gene therapy for bladder cancer treatment. The underlying mechanism for the reduced human bladder xenograft growth and tumor angiogenesis as suggested by the results is AT2R inhibiting proliferation and promoting cell apoptosis of human BCa cells. In all, these data suggest that the AT2R may serve as a novel anti-angiogenic treatment for BCa and a potential target gene for bladder carcinoma gene therapy.
The authors acknowledge Shengyao Wang and Renhe Yan for their technical assistance and help with Western blot analysis and realtime RT-PCR.
This work was supported by National Natural Science Foundation of China Grants 81072113 (H.L.), 81272482(J.L) and 81401920(H.D.), Guangdong Province Science and Technology Project 2013B010404026 (H.L.), the Fundamental Research Funds for the Central Universities 11616328(N.P), as well as by awards 2011DFA33290 and 2011CBA01006 to W.G. to H.D. from the Chinese government.
Conception and design: NP, YM, JL, GH, DG, HL. Development of methodology: NP, YM, PW, YZ, HD, CS, HL. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): RW, XC, HD, GJ, MX. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): NP, YM, PW, JL, AL, HL. Writing, review, and/or revision of the manuscript: NP, YM, AL, CS, GH, DG, HL. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): NP, YM, BC, GH, DG, Study supervision: HL. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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