By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Wiley Online Library will be unavailable on Saturday 7th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 08.00 EDT / 13.00 BST / 17:30 IST / 20.00 SGT and Sunday 8th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 06.00 EDT / 11.00 BST / 15:30 IST / 18.00 SGT for essential maintenance. Apologies for the inconvenience.
MicroRNAs (miRNAs) play an important role in the regulation of a variety of cellular processes, including cell growth, differentiation, apoptosis and carcinogenesis. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in colorectal cancer. The expression of miR-148b was significantly downregulated in 96 pairs of human colorectal cancer tissues (p < 0.0001) and three cell lines (p < 0.01) compared with non-tumor adjacent tissues by quantitative real-time PCR. The results of in situ hybridization highlighted that miR-148b was important in the cancer transformation process. Using statistical analysis, we found that the expression level of miR-148b was associated with tumor size (p = 0.033) in colorectal cancer patients. Moreover, overexpression of miR-148b in HCT-116 and HT-29 cells could inhibit cell proliferation in vitro and suppress tumorigenicity in vivo. Importantly, the result of luciferase activity assay and western blot showed that the cholecystokinin-2 receptor gene (CCK2R) was a target of miR-148b and was downregulated by miR-148b at the translational level. Then, we used siRNA, radioimmunoassay and ELISA to demonstrate that miR-148b might have an effect on cell proliferation by regulating the expression of CCK2R which functioned depending on the gastrin in colorectal cancer. Taken together, our data provides the first evidences that miR-148b acts as a tumor suppressor in colorectal cancer and should be further evaluated as a biomarker and therapeutic tool against colorectal cancer.
Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide.1 The CRC incidence and mortality rates in China have increased rapidly in the past several decades.2 Although the pathogenesis of CRC is well characterized, new molecules that play a role in this process are still being discovered. MicroRNAs (miRNAs) are ∼22 nucleotide noncoding RNA molecules that regulate a variety of cellular processes, including cell differentiation, cell cycle progression and apoptosis.3–6 It has been demonstrated that miRNAs play a significant role in tumorigenesis by downregulating tumor suppressor genes or oncogenes.7, 8 An increasing number of studies have found miRNA related mechanisms in the development of CRC, potential miRNAs as biomarkers in the diagnosis and prognosis of CRC and promising effects with miRNAs in the treatment of cancer at the molecular level.9–12
MiR-148b is located on chromosome 12q13. Our previous studies have revealed that miR-148b is downregulated in gastric cancer.13 Moreover, recent studies have found a downregulation of miR-148b in oral, pancreatic and colon cancer tissues using microarray analysis.14–16 However, the microarray results of Wang et al. did not show an obvious downregulation of miR-148b in six CRC cases.17 So in the present study, we investigated miR-148b expression in 96 cases of CRC by real-time RT-PCR. Importantly, our investigation revealed that miR-148b functioned as a tumor suppressor by inhibiting cell proliferation in vitro and in vivo. Furthermore, we found that the cholecystokinin-2 receptor gene (CCK2R) was a target of miR-148b, and miR-148b might have an effect on cell proliferation by regulating the expression of CCK2R which functioned depending on the gastrin in CRC. These results provide a better understanding of the biological activities of miR-148b in CRC.
Material and Methods
Human tissue samples
Ninety-six pairs of human CRC tissue samples were obtained from patients who had undergone surgical resection at the First Hospital of China Medical University between 2007 and 2008 and were diagnosed with CRC based on histopathological evaluation. The matched non-tumor adjacent tissue (NAT) was obtained from a part of the resected specimens that was farthest from the tumor (>5 cm). The samples were snap-frozen in liquid nitrogen and stored at −80°C. No previous local or systemic treatment had been conducted on these patients before the operation. One section of every sample was stained with hematoxylin-eosin (H&E) and used for histopathological evaluation. The tumor histological grade was assessed according to the World Health Organization criteria and was staged using the TNM staging of the International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) system (2010). The study was approved by the Research Ethics Committee of China Medical University, China. Informed consents were obtained from all patients.
The human CRC cell lines HCT-116, HT-29 and SW-620 were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences(Shanghai, China). HCT-116 and HT-29 were cultured in McCoy's 5a medium (Sigma) and SW-620 was cultured in Leibovitz's L-15 medium (Invitrogen). They were all supplemented with 10% fetal bovine serum at 37°C and 5% CO2.
RNA isolation and real-time RT-PCR
Total RNA from the specimens and cultured cells was isolated using the miRVana RNA isolation kit according to the manufacturer's instructions (Ambion). Poly(A) tail was added to RNA using a Poly(A) Tailing Kit according to the manufacturer's instructions (Ambion). The first-strand cDNA was synthesized using the SuperScript® III First-Strand Synthesis System according to the manufacturer's instructions (Invitrogen). Quantitative PCR was done using EXPRESS SYBR® GreenER™ qPCR Supermix Universal (Invitrogen) and was performed in a Real-time PCR System Rotor-Gene 6000 (QIAGEN). The expression of miR-148b was calculated relative to an endogenous reference (U6 RNA) and relative to the non-tumorous control. Changes in expression were calculated using the ΔΔCt method.18 The value of the relative expression ratio <1.0 was considered as low expression in cancer relative to the nontumorous control whereas the ratio >1.0 was regarded as high expression. Primers used for RT-PCR are indicated in Supporting Information Table S1.
In situ hybridization
In situ detection of miR-148b was performed on paraffin sections using DIG-labeled miRCURY™ Detection probe according to the manufacturer's instructions (Exiqon). Sixteen cases of CRC, adenomatous polyps, hyperplastic (inflammatory) polyps and non-tumor adjacent tissues (NATs) were selected. Sections were deparaffinized and deproteinated, which was followed by prehybridization, hybridization (hybridization temperature = TM probe –21°C), a stringency wash and immunological detection. The sections were then exposed to a streptavidin–peroxidase reaction system and developed with 3,3′-diaminobenzidine (DAB). Slides were counterstained with H&E and analyzed with a Nikon 80i microscope and Nikon NIS-Elements F 2.3 software (Nikon).
RNA oligoribonucleotides and cell transfection
MiR-148b mimics was an RNA duplex designed as described previously.19 The negative control RNA duplex (NC) was nonhomologous to any human genome sequence (Supporting Information Table S2). The anti-miR-148b, with sequence of 5′-ACAAAGUUCUGUGAUGCACUGA-3′, was designed as an inhibitor of miR-148b. The anti–NC, with a sequence of 5′-CAGUACUUUUGUGUAGUACAA-3′, was used as a negative control for anti–miR-148b in the antagonism experiment. The specific siRNA sequence 5′-AAGCGCGTGGTGCGAATGTTG-3′ resides in exon 5 of the human CCK2R gene (Genbank accession no. NM_176875). The control siRNA (si-NC) sequence, 5′-AAGCTTCATAAGGCGCATAGC-3′ is located on chromosome 11 of the mouse and has no homology with the human genome by BLAST comparison.13 All pyrimidine nucleotides were substituted by their 2′-O-methyl analogues to improve RNA stability and purchased from Genepharma (Shanghai, China).
Transfection was performed with Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer's protocol. A final concentration of 50 nM of RNA mimics or 200 nM of inhibitor or 100 nM of siRNA and their respective negative controls were used for each transfection in proliferation and animal experiments. Moreover, each transfection was set up with a blank control without plasmids.
MTT proliferation assay
The capacity for cellular proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Twenty-four hours after RNA transfection, cells (approximately 5 × 103) were seeded into 96-well culture plates for 24, 48, 72 and 96 hr. The cells were then incubated with 20 μL of MTT (5 mg/mL) for 4 hr at 37°C and 150 μL of DMSO was added to solubilize the crystals for 20 min at room temperature. The optical density was determined with a spectrophotometer- Multiskan MK3 (Thermo) at a wavelength of 490 nm.
Twenty-four hours after RNA transfection, equal numbers (approximately 4 × 104) of cells were seeded into six-well plates. The cells were harvested and counted by a trypan blue exclusion method every day after seeding.
Tumorigenicity assays in nude mice
Six-week-old female BALB/c athymic nude mice were subcutaneously injected into the right armpit region with 1.5 × 106 cells in 0.15 mL of PBS. Three groups (each n = 8) of mice were tested. Group 1 (miR-148b mimics group) was injected with HCT-116 cells transfected with miR-148b mimics; group 2 (NC group) was injected with HCT-116 cells transfected with NC; and group 3 (HCT-116 group) was injected with HCT-116 cells alone. The tumor size was measured every 2 or 3 days with calipers. The tumor volume was calculated using the formula: (L × W2)/2, where L is the length and W the width of the tumor. After the mice were sacrificed at 4 weeks, the weights of the tumors were measured. All experimental procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals and were performed according to the institutional ethical guidelines for animal experiments.
The miRNA targets predicted by computer-aided algorithms were obtained from PicTar,20 TargetScan21 and miRBase Targets.22 Then, the overlap of these results were further studied by Expression Analysis Systematic Explorer (EASE) analysis based on the Gene Ontology and KEGG Pathway databases.23
Luciferase activity assay
To construct pGL3-CCK2R-3′UTR, the partial 3′UTR of the CCK2R segment of human CCK2R mRNA (1568-2044nt) containing the putative miR-148b binding sites was amplified by PCR and cloned into the vector pGL3-control (Promega). Then, we used the same method to construct pGL3-DNMT1-3′UTR (5080-5399nt), pGL3-WNT10B-3′UTR (1574-1844nt), pGL3-NOG-3′UTR (1299-1457nt) and pGL3- ROBO1-3′UTR (5903-6340nt). We constructed another two luciferase reporters. One was pGL3-CCK2R-3′UTR-conserved, which contains a putative miR-148b binding site in a conserved region of 3′UTR. The other was pGL3-CCK2R-3′UTR-poorly conserved, which contained a putative miR-148b binding site in a poorly conserved region of 3′UTR. For each binding site, we constructed luciferase reporters pGL3-CCK2R-MUT1 (MUT-1) and pGL3-CCK2R-MUT2 (MUT-2) in which DNA segments with scrambled target sites designed to interfere with seed sequence recognition were cloned into pGL3-control vector serve as control for specificity. In addition, we also constructed a luciferase reporter that had a complete complementary sequence to miR-148b as a positive control. Primers used for vector construction are indicated in Supporting Information Table S3.
Cells were seeded into 24-well plates at 5 × 104 cells per well the day before transfection. Cells were then co-transfected with 400 ng of Firefly luciferase reporter, 70 nM of miR-148b mimics or NC and 40 ng of pRL-TK Renilla luciferase control vector (Promega) using Lipofectamine 2000. Firefly and Renilla luciferase activities were measured consecutively using the Dual-Luciferase Reporter Assay System (Promega) and Centro LB 960 (Berthold) 24 hr after transfection.
Protein extraction and western blot
Total protein from the specimens and the cultured cells was extracted using the total protein extraction kit according to the manufacturer's instructions (KeyGen). Proteins were separated by 8% SDS polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). Unspecific sites were blocked in 5% nonfat milk at room temperature for 1 hr. Antibodies directed against CCK2R (1:200, Abcam) and β-actin(1:5000, Sigma)were used. The proteins were visualized with an ECL kit (Pierce) and MF-Chemi BIS 3.2 Pro (Micro Photonics) with GelCapture Version software. The intensity of protein fragments was quantified using FluorChem 2.01 software (Alpha Innotech). CCK2R protein levels in cancer tissues were presented as fold change normalized to an endogenous reference (β-actin protein) and relative to the matched non-tumor adjacent tissues. Therefore, the fold change of CCK2R protein <1.0 was considered as low expression, whereas the fold change of CCK2R protein >1.0 was regarded as high expression.
Amidated gastrin and progastrin detection
The concentrations of amidated gastrin in culture medium were detected by radioimmunoassay (RIA) using an Iodine [125I] Gastrin Radioimmunoassay Kit (Beijing North Institute of Biological Technology, China) according to the manufacturer's instruction; 200 × 104 HCT-116 cells were resuspended in 0.5 mL deionized water and disrupted by ultrasound. After centrifugation, concentrations of progastrin in the cell lysate were evaluated by ELISA Kits (R&D) according to the manufacturer's instruction.
Data are presented as mean ± SD from at least three separate experiments. Statistical analysis was performed with Student's t-test, non-parametric test (Mann-Whitney U test between two groups and Kruskall-Wallis test for three or more groups). The statistical significance of correlations between the expression of miR-148b and CCK2R protein were calculated by a Chi-square test and Spearman's rank correlation. Statistical analysis was performed using SPSS 16.0 computer software and p < 0.05 was considered statistically significant.
Expression of miR-148b was significantly downregulated in CRC tissues and cancer cell lines compared with non-tumor adjacent tissues
Among 96 patients with CRC, there was a significantly lower expression of miR-148b in CRC tissues in comparison with non-tumor adjacent tissues (p < 0.0001); 76 (79.17%) cases of CRC revealed >50% reduction in the miR-148b level in the tumor tissues relative to the level in their non-tumor adjacent tissues (Fig. 1a). Furthermore, compared with three non-tumor adjacent tissues, miR-148b was downregulated at different degrees in HT-29 (0.042 ± 0.004-fold, p < 0.01), HCT-116 (0.139 ± 0.003-fold, p < 0.01) and SW-620 (0.221 ± 0.069-fold, p < 0.01) cells (Fig. 1b). Moreover, in situ hybridization results of 16 cases of CRC, adenomatous polyps, hyperplastic (inflammatory) polyps and NATs confirmed the expression of miR-148b in these tissues. Furthermore, the expression levels of miR-148b in hyperplastic (inflammatory) polyps and NATs were similar and both were slightly higher than that of adenomatous polyps. And the expression level of miR-148b in CRC tissues was much lower than the other three kinds of tissues (Fig. 1c and Supporting Information Fig. S1).
Low expression of miR-148b was associated with increased tumor size
Using statistical analysis (non-parametric test), we found that the expression level of miR-148b was associated with tumor size (p = 0.033, Mann-Whitney U test) in CRC patients (Table 1). The patients with lower expression levels of miR-148b tended to have larger tumor sizes (>6 cm). In addition, there were no significant differences between the expression level of miR-148b and other clinicopathologic characteristics, including sex, age, tumor location, histologic grade, pT stage, pN stage, clinical stage or lymphatic vessel invasion in CRC.
Table 1. Associations between the expression of miR-148b and clinicopathological features in 96 patients with CRC
MiR-148b inhibits cell proliferation in vitro
First, we detected expression of miR-148b by qRT-PCR 48 hr after transfection of miR-148b mimics, anti-miR-148b, their respective NCs and blank controls (HCT-116 and HT-29). Transfection efficiency was perfect (Supporting Information Fig. S2). As shown in Figures 2a and 2b, the results of MTT showed that the cells which transfected with miR-148b mimics had a growth inhibition compared to matched NC. At the time point of 48, 72 and 96 hr after transfection, the inhibition rates were 8.91%, 10.53% and 13.93% in the HCT-116 cells. And in the HT-29 cells, the inhibition rates were 24.39%, 9.77% and 11.64%, respectively. Growth curves also revealed that the growth ability of HCT-116 cells was reduced by overexpression of miR-148b (Fig. 2c). To provide further evidence that miR-148b was indeed involved in cell growth, we studied the effect of the inhibitor of miR-148b on the HCT-116 cell growth. The proliferation of the cells transfected with anti-miR-148b was increased compared with that of the cells transfected with anti-NC and blank control (Fig. 2d). Taken together, these results suggest that miR-148b was indeed involved in the negative regulation of cell growth.
MiR-148b suppresses tumorigenicity in vivo
HCT-116 cells transfected with miR-148b mimics or NC, or blank control, were injected separately into three groups of nude mice. The tumor volumes were measured every 2 or 3 days until the mice were sacrificed on day 28. Figures 3a and 3b showed that the group with miR-148b mimics formed substantially smaller tumors in nude mice than did the other two groups. The tumor volume at the time of death in mice injected with miR-148b mimics cells was 185.933 ± 185.095 mm3 and the tumor volume of mice injected with NC cells was 328.094 ± 201.655 mm3, whereas the tumor size of mice injected with HCT-116 cells was 394.290 ± 224.186 mm3 (Fig. 3c). The mean tumor weight at the end of the experiment was lower in the miR-148b mimics group than in the NC and blank control group (0.235 ± 0.191 g versus 0.511 ± 0.335 g and 0.542 ± 0.230 g, respectively; p = 0.019 and p = 0.002, respectively; Fig. 3d). These results indicate that inoculation of miR-148 mimics significantly inhibits tumorigenicity of HCT-116 cells in the nude mouse model.
CCK2R is a potential target gene of miR-148b in HCT-116 cells
Using the bioinformatics method, eleven genes were picked out as candidate targets of miR-148b (Supporting Information Table S4). We hypothesized that CCK2R, DNMT1, WNT10B, NOG and ROBO1, whose functions were associated with carcinogenesis or cancer development were putative target genes of miR-148b. As shown in Figures 4a, the luciferase activity of pGL3-CCK2R-3′UTR was much lower in cells with miR-148b mimics than that in cells with NC. We subsequently found the 3′-UTR of CCK2R have two presumed sites in conserved and poorly conserved regions that can bind with the seed region of miR-148b (Fig. 4b). We therefore constructed another two luciferase plasmids, pGL3-CCK2R-3′UTR-conserved and pGL3-CCK2R-3′UTR-poorly conserved. The results revealed that the relative luciferase activity of the pGL3-CCK2R-3′UTR-conserved had a more obvious variation than that in pGL3-CCK2R-3′UTR-poorly conserved. And there was no significant difference of the relative luciferase activity of MUT-1 and MUT-2 reporters in miR-148b-transfected HCT-116 cells compared with NC-transfected HCT-116 cells (Fig. 4a).
To confirm whether CCK2R is a target of miR-148b, we transfected HCT-116 cells with miR-148b mimics, anti-miR-148b and their respective negative controls. Forty-eight hours after transfection, we examined the mRNA level of CCK2R in cells by real-time RT-PCR. However, we did not find any variation (p > 0.05, Supporting Information Fig. S3). Interestingly, the protein level of CCK2R was suppressed by miR-148b mimics and increased by anti-miR-148b at 48 hr after transfection (Fig. 4c). Taken together, our results suggest that CCK2R is a potential target gene in HCT-116 cells and CCK2R is downregulated by miR-148b only at the translational level.
In addition, we measured CCK2R protein levels in 22 pairs of the previously studied 96 CRC tissues and their matched non-tumor adjacent tissues, which had already been verified as expressing miR-148b by qRT-PCR. After we quantified the protein fragments, an obvious inverse correlation was observed between the expression of CCK2R and miR-148b in tissue samples (p < 0.05, Table 2).
Table 2. Correlation between the expression of miR-148b and CCK2R protein in 22 CRC cases
Effects of knockdown of the CCK2R gene on cell proliferation in HCT-116 cells
After transfection with si-CCK2R and si-NC in HCT-116 cells, we found that the cells which transfected with si-CCK2R had an obvious growth inhibition compared to matched si-NC and HCT-116 cells by MTT assay (Fig. 4d). At the time point of 48, 72 and 96 hr after transfection, the inhibition rates were 14.32%, 14.01% and 12.66%, respectively.
Amidated gastrin and progastrin can be detected in HCT-116 cells
Radioimmunoassay (RIA) was performed to determine whether gastrin was secreted into the cultured medium. The concentration of amidated gastrin in the culture MCC medium gradually increased along with the prolonging culture time (Fig. 4e). Furthermore, in 104 HCT-116 cells, 0.87 ± 0.02 pg of progastrin was detected as revealed by ELISA. Transfection of miR-148b mimics into HCT-116 cells has no significant effect on expression levels of progastrin at the protein levels (Fig. 4f).
Cancer is a very complex genetic disease characterized by alterations in genes encoding oncogenic and tumor-suppressor proteins.24 Recently, it has been noted that the expression profiles of miRNAs can be used for classification, diagnosis and prognosis of human malignancies.25–27 In the present study, our results of real-time RT-PCR showed significantly low expression of miR-148b in CRC tissues and cancer cell lines relative to non-tumorous controls. Schetter et al. also found this tendency in CRC tissues by microarray analysis.16 And the results of in situ hybridization highlighted that miR-148b was important in the cancer transformation process. Moreover, we found that low expression of miR-148b was associated with increased tumor size in CRC. Although the size of a colorectal mass was not an independent prognostic factor of survival, it still correlates with the outcome of CRC at some degree.28, 29 In view of the above, we speculate that miR-148b may play an important role in CRC.
As is commonly known, deregulated cell proliferation is a key mechanism for neoplastic progression.30 Our results from the MTT assay and growth curves both indicated that miR-148b produces significant growth inhibition in CRC cells in vitro. This was further supported by the finding that the overexpression of miR-148b could inhibit tumor formation and growth in nude mice. Therefore, miR-148b may be involved in carcinogenesis and the development of CRC as a tumor suppressor gene.
Moreover, miRNAs are known to regulate the expression of genes involved in the control of development, proliferation, apoptosis and stress responses.3–6, 31 Recent studies showed a direct link between miRNAs and human cancers.32, 33 As previously shown, the important cancer genes are regulated by aberrant expressions of miRNAs, such as let-7 and Ras,34 miR-15a-miR-16-1 cluster and Bcl-2,35 miR-27a and prohibitin.36 In the present study, our results highlight that miR-148b interacts with CCK2R and negatively regulates its expression at the translational level. CCK2R is widely distributed throughout the human gastrointestinal tract, pancreas, lung and some neuroendocrine tissues. The main function of CCK2R is to mediate the normal physiological function of gastrin. In CRC, Jin et al. found that inactivation of the CCK2R gene inhibited progastrin-dependent colonic tumor formation in mice.37 Moreover, a recent study also found that disrupting the gastrin–CCK-2 receptor autocrine loop by neutralizing the endogenous gastrin or by knocking down CCK2R expression significantly inhibited cell proliferation in SGC-7901 cells.38 We also used siRNA, radioimmunoassay and ELISA to demonstrate that miR-148b might have an effect on cell proliferation by regulating the expression of CCK2R which functioned depending on the gastrin in CRC. On the other hand, since CCK2R has been localized to the colonic crypts, this may suggest that miR-148b is not well expressed normally in the stem/progenitor zone of the colon. Furthermore, our results revealed that miR-148b mimics and si-CCK2R could inhibit cell proliferation, so we speculate miR-148b mimics could be combined with CCK2R antagonists to prevent or treat colon cancer in the future.
Our results showed that CCK2R was negatively regulated directly by miR-148b depending on the seed sequence recognition of 3′UTR in HCT-116 cells. The 2-8 nt of miRNA known as the “seed region” is suggested to be the most important for recognition.39 Bioinformatics analysis showed that miR-148b has the same “seed sequences” as miR-148a and miR-152. Our previous work revealed that miR-148a and miR-152 were significantly downregulated in gastric and CRCs.40 Therefore, miR-148a, miR-148b and miR-152 may play a similar role in CRC and the relationship among them needs further investigation. In addition, we also found that miR-148b was frequently downregulated in gastric cancer and inhibited cell proliferation by targeting CCK2R.13 So miR-148b may have the same function by regulating the same target gene in different cancers. On the other hand, Duursma et al. studied the target of miR-148 in the protein coding region and found that human miR-148 represses the expression of the DNA methyltransferase 3b (Dnmt3b) gene, which is the primary mediator of establishment and maintenance of DNA methylation in mammals.41 Therefore, miR-148b may regulate the same or different targets in the same or different cells or may regulate different targets depending on different binding regions.
In summary, we found significant low-expression of miR-148b in CRC tissues and cell lines compared with their non-tumor counterparts. Moreover, our data also suggest that miR-148b might have an effect on cell proliferation by regulating the expression of CCK2R which functioned depending on the gastrin in CRC. It is possible that miR-148b may be a potential biomarker and therapeutic tool against CRC.
We thank the Department of Surgical Oncology of First Hospital of China Medical University for providing human colorectal tissue samples. We also thank the College of China Medical University for technical assistance in experiments. The authors declare that they have no financial disclosures or conflict of interest.