MicroRNA‑126‑3p inhibits the proliferation, migration, invasion, and angiogenesis of triple‑negative breast cancer cells by targeting RGS3
- Authors:
- Published online on: July 26, 2019 https://doi.org/10.3892/or.2019.7251
- Pages: 1569-1579
Abstract
Introduction
Breast cancer (BC) is the most commonly diagnosed, and the second most common cause of cancer-related deaths in women (1). There were ~2.1 million new diagnoses of female BC cases worldwide in 2018, accounting for almost 1 in 4 cancer deaths (2). Triple-negative breast cancer (TNBC), one of the BC subtypes has been revealed to be immunonegative for estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2. TNBC is a heterogeneous type of tumor characterized by several clinical features including its aggressive nature, higher rates of relapse and shorter overall survival in comparison with other subtypes of BC (3). In addition, TNBC more often metastasizes to the lungs, bone, liver, and brain than any other of the subtypes of BC (4–6). Although the biomarkers of human epidermal growth factor receptor 2, progesterone receptor and estrogen receptor have provided significant prognostic value for TNBC patients (7), development of new biomarkers is still necessary to obtain valuable information concerning diagnosis and treatment, and to further exploit effective therapeutic strategies and improve survival.
MicroRNAs (miRNAs), composed of 19–24 nucleotides, play crucial roles in a variety of biological processes, including cell proliferation, differentiation, migration, invasion, and apoptosis (8–10). Dysregulation of miRNA expression often appears in many cancers such as lung cancer, BC and cervical cancer, and is directly associated with tumor initiation, progression, and metastases (11–14). Human miR-126-3p is an endothelial-specific miRNA, encoding gene located in the intron of the EGFL7 gene at chromosome 9q34.3. This miRNA plays an important role during embryonic development and in angiogenesis (15). Previously it was revealed that the expression level of miR-126-3p in many aggressive tumors was significantly downregulated, such as osteosarcoma, colorectal cancer, prostate cancer as well as in other cancers (16–20), suggesting miR-126-3p to be a novel biomarker for cancer treatment.
Blood supply supports tumor growth and distant dissemination. It has caused the widespread concern of how angiogenesis occurs and functions, since vasculogenic mimicry (VM) was firstly demonstrated as a phenomenon of highly aggressive and metastatic melanoma cells forming highly patterned vascular channels in vitro in 1999. VM could be composed of a basement membrane in the absence of endothelial cells and fibroblasts (21), supported further by numerous underlying molecular pathway studies (22). Among these mechanisms, miRNAs may be involved. For example, vascular integrity may be affected by miR-126-3p, resulting in a disorganized tumor vasculature (23). Increasing evidence has indicated that the alternative microcirculation pathway was established by vasculogenic mimicry (VM) channels which exert a fatal effect in the early growth, metastasis and relapse of invasive BC (24–26). Hence, in the present study, the relationship between miR-126 and vascular mimicry was investigated, which may provide the theoretical basis for anti-vascular therapy in TNBC.
As a G-protein signaling regulator, regulator of G-protein signaling (RGS3) is characterized by a homologous sequence of a core RGS domain and switches off receptor-activated cellular signal transduction (27,28). RGS3 is associated with tumor cell proliferation and migration (29,30). In addition, ingenuity pathway analysis indicates that the RGS3 mRNA interacts with miR-126 (31,32). Previous studies further revealed that there is a direct 3′-untranslated region binding site between RGS3 mRNA and miRNA-126, indicating that overexpressed RGS3 regulated by miRNA-126 through post-transcriptional modulation, is significantly associated with a poor prognosis of cancer patients (33).
Therefore, to explore new biomarkers for TNBC treatment, miR-126 expression in human MDA-MB-231 and HCC1937 cell lines was firstly investigated. Then, the effect of miR-126 expression on proliferation, migration, invasion, and angiogenesis of human TNBC was determined. Subsequently, RGS3 as the potential target gene of miR-126 was analyzed and validated by bioinformatics and luciferase activity assay. Finally, how miR-126-3p regulates target-gene expression was also investigated and the underlying mechanisms of TNBC progression were elucidated.
Materials and methods
Cell culture
The TNBC cell lines, MDA-MB-231 and HCC1937, and normal breast cell line MCF-10A were obtained from the Cell Resource Center of the Shanghai Institutes for Biological Sciences. The base medium used in the present study was L-15 Medium (cat. no. JCML1514500; Nalgene) for MDA-MB-231 cells, RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc.) for HCC1937 cells, and the complete growth medium was supplemented with 10% fetal bovine serum, 1% penicillin (100 U/ml) and 1% streptomycin (100 U/ml). MCF-10A cells were cultured in complete growth medium consisting of 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 5% (v/v) horse serum, 10 µg/ml insulin, 100 ng/ml cholera toxin, 20 ng/ml recombinant human epidermal growth factor, 0.5 µg/ml hydrocortisone and 1 unit/ml penicillin/streptomycin. The cells were all incubated in a humidified incubator at 37°C with 5% CO2.
Cell transfection
miR-126-3p mimic, miR-126-3p inhibitor, negative control of miR-126-3p (miRNA-NC) which is defined as the cells transfected with the plasmids without miR-126-3p mimic or inhibitor, and RGS3-specific small interfering RNA (siRNA) were all synthesized by GenePharma Co., Ltd. According to the manufacturer's protocols, Lipofectamine 2000 (cat. no. 52887; Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect the synthesized miRNAs aforementioned or expression-ready RGS3 ORF construct and NC construct into the investigated MDA-MB-231 and HCC1937 cells. After 48 h of transfection, the cells were collected for the following experiments.
Cell counting kit-8 (CCK-8) assay
Cell proliferation was assessed by a Cell Counting Kit-8 (Dojindo Molecular Technologies). Cells were seeded at a density of 1×105 cells/well to a 96-well plate. Forty-eight hours after transfection, CCK-8 fluids were added to the cells with a final concentration of 10% and maintained at 37°C for 4 h. The optical density was assessed at 450 nm using a Microplate Reader (Bio-Rad Laboratories, Inc.).
Migration and invasion assays
The Transwell assay kit (product no. 356234; Corning Incorporated) was used to evaluate the invasion of the transfected cells. These cells supplemented with DMEM were seeded to the upper chamber. DMEM supplemented with FBS was added to the lower chamber. The cells were maintained at 37°C for 48 h and were then fixed by methanol. Non-migrated cells on the upper surface of the membrane were removed by a cotton swab. The migrated cells on the lower side of the membrane were stained using 0.1% crystal violet for 5 min at room temperature, and imaged. ImageJ v1.52 software (National Institutes of Health) was used to count the number of invading cells. The invasion assay was performed in accordance with migration assay except that the Transwell membrane was pre-coated with Matrigel (BD Biosciences).
Colony formation
Cells transfected with synthetic miRNAs were seeded to a 6-well plate at a density of 500 cells/well and incubated in DMEM with 10% FBS at 37°C for 2 weeks. Then, the cells were fixed with methanol, washed with PBS and then stained with 0.1% crystal violet solution (Beyotime Institute of Biotechnology). The number of colonies with >50 cells were counted.
VM formation assays
To assess the effect of miR-126-3p and RGS3 on the VM of TNBC cells, 2×104 transfected cells overexpressing plasmid or a control Matrigel (100 µl) were placed on a 48-well plate, and incubated at 37°C. The number of tubes (complete circular structures) in each well were captured and counted using an inverted microscope (Nikon Corp.). The three readings of each well were averaged to be the final reading.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from the cultured cells using Trizol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and quantified using Ultra-micro UV analyzer Q6000UV (Quawell Technology, Inc.). The sequence of stem-loop structures used in the present study was: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGCATTAT-3′. The sequence of microRNA-126-3p was 5′-CGTACCGTGAGTAATAATGCG-3′. The underlined part was the combined part of the stem-loop and target sequence. Thus, the first cDNA strand specific to miRNA-126-3p was synthesized from total RNA using Bestar™ qPCR RT Kit (cat. no. 2220; DBI Bioscience) on a PCR Amplifier (Product no. K960; Hangzhou Jingge Scientific Instrument Co., Ltd.). Real-time PCR was conducted using Stratagene Mx3000P (Agilent Technologies, Inc.) and by applying Bestar™ qPCR Master Mix (cat. no. 2043; DBI Bioscience). U6 snRNA was used as an endogenous control and analyzed with the 2−ΔΔCq method (34). The primers are listed in Table I.
Dual luciferase activity assay
The targets of miR-126-3p were predicted by bioinformatics analysis using algorithm TargetScan (http://www.targetscan.org/vert_72/), and the TargetScan score was investigated for has-miR-126-3p binding to RGS3 in miRDB (http://mirdb.org/cgi-bin/search.cgi). Then, the wild 3′UTR fragment of RGS3 carrying the putative miR-126-binding site was amplified and sub-cloned into the psiCHECK-2 vector (Bioneer Co., Ltd.) to construct pMiR-wild-type (pMiR-WT). The isolated plasmid was sequenced and mutated using Directed Mutagenesis System (Invitrogen; Thermo Fisher scientific, Inc.), to produce pMiR-mutant-plasmid (pMiR-Mut) with seven point mutations: CTGCCC C(CtoA) G(GtoT) G(GtoA) T(TtoC) A(AtoT) C(CtoG) G(GtoA) AGGGGGC. The sites of the mutation in RGS3 sequence are C in 152438, G in 152439, G in 152440, T in 152441, A in 152442, C in 152443 and G in 152444, which is based on the sequence provided in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/nuccore/NG_029512.1?from=4999&to=158013&report=genbank). Sunsequently, the manufacturer's instructions were followed and Lipofectamine 2000 (cat. no. 52887; Invitrogen; Thermo fisher Scientific, Inc.) was used to transfect plasmids containing the 3′UTR fragment (pMiR-WT) or mutant (pMiR-Mut) respectively into MDB-MA-231 and HCC1937 cells to express miRNA-NC or pcDNA3.0-NC or miR-126-3p mimic. Forty-eight hours after transfection, the cells were lysed and a reporter assay was performed using the Dual-luciferase assay system (Promega Corp.).
Statistical analysis
All the experiments were repeated three times. The results were expressed as the mean ± standard deviation (SD). A Student's t-test was used to analyze the differences between two groups. One-way analysis of variance and Tukey's post hoc test were used to analyze the differences among three or more groups. GraphPad Prism 6 (GraphPad Software, Inc.) was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.
Results
Overexpression of miR-126-3p inhibits proliferation, migration, invasion, and angiogenesis of TNBC cell lines whereas silencing of miR-126-3p promotes these processes
The endogenous expression of miR-126-3p in MCF-10A, MDA-MB-231 and HCC1937 cells are presented in Fig. 1A, indicating that the expression of miR-126-3p in TNBC cells was significantly lower than that in normal breast cells (P<0.001, respectively). To investigate the effect of miR-126-3p on human TNBC cells, the MDA-MB-231 and HCC1937 cells were transfected with miR-126-3p mimic or miR-126-3p inhibitor. Non-transfected cells were used as the control group and cells transfected with miRNA-NC were used as the negative control. The expression of miR-126-3p in the miR-126-3p mimic group was increased by 2.2-fold in MDA-MB-231 cells and 2.4-fold in HCC1937 cells, respectively, compared with that in the miRNA-NC groups (P<0.001). The expression level of miR-126-3p in miR-126-3p inhibitor group was decreased by 2.3-fold in MDA-MB-231 cells and 3.8-fold in HCC1937 cells respectively compared with that in miRNA-NC group (P<0.001), indicating that overexpressing or silencing of miR-126-3p were achieved by transfection of miR-126-3p mimic or inhibitor respectively (Fig. 1A). The viability of MDA-MB-231 and HCC1937 cells was significantly increased at the 72-h time-point by miR-126-3p inhibition compared with that in the miRNA-NC group (2.47±0.09 vs. 1.57±0.03 in MDA-MB-231 cells and 3.66±0.05 vs. 2.81±0.05 in HCC1937 cells, P<0.001, normalized to levels at 24 h, respectively), and was decreased at the 72-h time-point by miR-126-3p overexpression compared with that in the miRNA-NC group (1.28±0.05 vs. 1.57±0.03 in MDA-MB-231 cells and 2.22±0.06 vs. 2.81±0.05 in HCC1937 cells, P<0.05, normalized to levels at 24 h, respectively, Fig. 1B). The migration of MDA-MB-231 and HCC1937 cells was significantly enhanced by miR-126-3p inhibition compared with that in the miRNA-NC group (138.33±1.53 vs. 68±6.56 in MDA-MB-231 cells and 81.67±1.53 vs. 40.33±2.52 in HCC1937 cells, P<0.001, respectively), and was reduced by miR-126-3p overexpression compared with that in the miRNA-NC group (37.33±4.04 vs. 68±6.56 in MDA-MB-231 cells and 14.33±1.53 vs. 40.33±2.52 in HCC1937 cells, P<0.001, respectively, Fig. 1C and D). Similarly, MDA-MB-231 cell invasion was significantly enhanced by silencing of miR-126-3p compared with that in the miRNA-NC group (132±2 vs. 72.67±5.69, P<0.001), and was decreased by overexpression of miR-126-3p compared with that in the miRNA-NC group (32±1.73 vs. 72.67±5.69, P<0.001, Fig. 1C and E). The number of colonies exhibited a 50% reduction in MDA-MB-231 cells overexpressing miR-126-3p and a 36% enhancement in MDA-MB-231 cells with miR-126-3p silencing when compared to those in the miRNA-NC respectively (63.67±4.04 and 172.67±8.50 vs. 126.67±5.86, P<0.001, respectively, Fig. 1C and F). The VM assay revealed that tube formation was reduced 70% in the group of MDA-MB-231 cells overexpressing miR-126-3p when compared to that in the miRNA-NC group (P<0.001) while it was increased 3.7-fold in MDA-MB-231 cells with miR-126-3p silencing when compared to the miRNA-NC group (P<0.001) (Fig. 1G and H), indicating that tube formation capacity was inhibited by the miR-126-3p mimic and enhanced by the miR-126-3p inhibitor. There was no difference between the control group and miRNA-NC group in terms of cell viability, migration, invasion, colony formation, and tube formation.
RGS3 is a target gene of miR-126-3p
The TargetScan algorithm predicted that miR-126-3p binded to 3′UTR of RGS3, and the TargetScan score was 67 for has-miR-126-3p binding to RGS3 in miRDB. The DNA sequencing result of pRGS3-Mut finally confirmed the same mutation sites as human pRGS3-WT from the GenBank sequence, which validated the recombinant pcDNA3.0-RGS3 plasmid. Therefore, the pRGS3-WT or pRGS3-Mut with mimic control or miR-126-3p mimic were co-transfected into MDA-MB-231 and HCC1937 cells, and luciferase activity was detected. Luciferase activity in cells transfected with pRGS3-WT and miR-126-3p mimic was reduced 2.2-fold compared with that in cells transfected with pRGS3-WT and mimic control (P<0.001) (Fig. S1), however, the difference between co-transfection with pRGS3-Mut and the miR-126-3p mimic was not significant.
Ectopic expression of RGS3 affects cell proliferation, migration, invasion, and angiogenesis
The endogenous expression of RGS3 in MCF-10A, MDA-MB-231 and HCC1937 cells presented in Fig. 2A, revealed that the expression of RGS3 in TNBC cells was significantly higher than that in normal breast cells (P<0.001, respectively). pcDNA3.0-NC, pcDNA3.0-RGS3 alone or accompanied by si-RGS3 were respectively transfected into MDA-MB-231 and HCC1937 cells. Non-transfected cells were used as the control group. The expression of RGS3 in the pcDNA3.0-RGS3 group was increased by 3.1-fold in MDA-MB-231 cells and 3.4-fold in HCC1937 cells compared with that in the pcDNA3.0-NC groups (P<0.001, respectively). The expression level of RGS3 in the pcDNA3.0+si-RGS3 group was decreased by 2.8-fold in MDA-MB-231 cells and 3.6-fold in HCC1937 cells compared with that in the pcDNA3.0-NC group (P<0.001, respectively), indicating that overexpression or silencing of RGS3 were achieved by transfection of pcDNA3.0-RGS3 or pcDNA3.0+si-RGS3, respectively (Fig. 2A). The viability of MDA-MB-231 cells and HCC1937 cells was significanlty increased at the 72-h time-point in the transfected pcDNA3.0-RGS3 group compared with that in the pcDNA3.0-NC group (2.52±0.05 vs. 1.74±0.05 in MDA-MB-231 cells and 3.80±0.03 vs. 2.35±0.04 in HCC1937 cells, P<0.001, normalized to levels at 24 h, respectively), and was decreased at the 72-h time-point in the transfected pcDNA3.0+si-RGS3 group compared with that in the pcDNA3.0-NC group (1.42±0.07 vs. 1.74±0.05 in MDA-MB-231 cells and 1.66±0.05 vs. 2.35±0.04 in HCC1937 cells, P<0.05, normalized to levels at 24 h, respectively, Fig. 2B). In Fig. 2C-E, migration, and invasion of the MDA-MB-231 cell transfected by pcDNA3.0-RGS3 were enhanced compared with those in the pcDNA3.0-NC group (100 and 77% increase in cell migration and invasion, respectively; P<0.001). In addition, MDA-MB-231 cell migration and invasion were attenuated by pcDNA3.0+si-RGS3 in comparison with the pcDNA3.0-NC group (45% and 52% decline in cell migration and invasion, respectively; P<0.001). The number of colonies presented a 37% increase in the pcDNA3.0-RGS3 MDA-MB-231 cells and a 44% abatement in the pcDNA3.0+si-RGS3 MDA-MB-231 cells when compared to pcDNA3.0-NC respectively (P<0.001, Fig. 2C and F). Tube formation presented in the VM assay was increased 3.2-fold in the pcDNA3.0-RGS3 MDA-MB-231 cells when compared to that in the pcDNA3.0-NC group (P<0.001), while it was reduced 61% in the si-RGS3 MDA-MB-231 cells when compared to the pcDNA3.0-NC group (P<0.05) (Fig. 2G and H). There was no difference between the control group and the pcDNA3.0-NC group regarding cell viability, migration, invasion, colony formation, and tube formation.
Restoration of RGS3 reverses the inhibitory effects of miR-126-3p in TNBC cells
Two groups of miR-126-3p mimic alone or with pcDNA3.0-RGS3 were set up to further validate whether the restoration of RGS3 can reverse the inhibitory effects of miR-126-3p in TNBC cells. Results in Fig. 3A-D revealed that the viability, migration, and invasion of MDA-MB-231 cells were all increased by 1.5- (at the 72-h, time-point normalized to the levels at 24 h), 3.0- and 3.2-fold respectively by co-transfection of miR-126-3p mimic and pcDNA3.0-RGS3 compared with cells only transfected with miR-126-3p mimic (P<0.001). Similarly, the viability and migration of HCC1937 cells were all increased by 1.4- (at the 72-h time-point, normalized to levels at 24 h) and 3.3-fold respectively by co-transfection of miR-126-3p mimic and pcDNA3.0-RGS3 compared with cells only transfected with miR-126-3p mimic (P<0.001). Moreover, there was a 1.5-fold enhancement of the number of colonies in MDA-MB-231 cells co-transfected with miR-126-3p mimic and pcDNA3.0-RGS3 when compared to that in the cells transfected with the miR-126-3p mimic (P<0.001, Fig. 3B and E). In the VM assay, tube formation was increased by 6.5-fold in the MDA-MB-231 cells co-transfected with the miR-126-3p mimic and pcDNA3.0-RGS3 compared with that in the cells transfected with the miR-126-3p mimic (P<0.001, Fig. 3F-G).
Discussion
Increasing evidence has revealed that specific microRNA clusters may play an important role in the biology of TNBC, and may be of clinical relevance in TNBC, knowledge which should assist disease prognostication and therapy (35). The present study revealed that miR-126-3p was decreased in TNBC cells and miR-126-3p overexpression inhibited cell proliferation, migration, and invasion. RGS3 was predicted to be a target gene of miR-126-3p by the TargetScan algorithm and validated by luciferase activity assay. Furthermore, RGS3 silencing reversed the effect of miR-126-3p knockdown. Finally, it was determined that miR-126-3p knockdown activated VM, which could be reversed by silencing of RGS3.
Multiple miRNAs are associated with the metastasis and prognosis of BC (36–39). For instance, overexpression of miR-30c-2-3p inhibited the migration and invasion of BC cells, while low or underexpression of miR-30c-2-3p was correlated with poor overall survival (40). An increasing number of studies has confirmed that miRNA expression profiles are dysregulated in TNBC patients compared to healthy controls. For example, miR-190a, miR-136-5p, miR-126-5p, miR-135b-5p and miR-182-5p may be associated with the development and progression of TNBC (41). Although miR-126-3p has been reported as an oncogenic miRNA, promoting proliferation in gastric cancer cells (42), it has also been revealed to be a tumor suppressor due to its inhibition in the growth of colon cancer cell lines, HEK293 and MCF-7 cells by targeting p85b and IRS-1, respectively (43,44). However, in a study of miRNA expression profiles of 24 triple-negative breast cancers and 14 adjacent normal tissues using deep sequencing technology, miR-126-3p was revealed to be significantly downregulated (P<0.05; fold change 1.8–2.6) in TNBC (45). Additionally, restoring miR-126-3p in MDA-MB-231 cells reduced cell proliferation and the levels of miR-126-3p in tumor tissues predicted TNBC outcomes (46). Novel prognostic and predictive miRNA targets of TNBC, include a miRNA signature that predicts patient response to anthracycline-based chemotherapy, which may improve clinical management and/or lead to the development of novel therapies (47). Notably, it was surmised that miR-126-3p acts as a tumor-suppressor miRNA in TNBC cancer cells in the present study, which further extends the current knowledge of microRNA regulatory characteristics and network in TNBC.
Notably, previous results indicated that a combination treatment with miRNAs, in particular miR-126-3p, enhanced the activity of specific BC drugs in vitro, even in the most aggressive BC subtype, TNBC (48). In addition, whole transcriptome studies, coupled with Gene Ontology and pathway analysis revealed a notable landscape for miR-126-3p activity in BC, such as involvement in cell cycle regulation of BC, in particular the M phase (48). Whether miR-126-3p overexpression affects apoptosis and the cell cycle in TNBC as well as whether a combination treatment with miR-126-3p affects TNBC cell function are our next goals in future research.
RGS3 was predicted to be a potential target of miR-126-3p by TargetScan algorithm due to their complementary binding which was further validated through a luciferase activity reporter assay. Notably, it was revealed that targeting and combination of transfected exogenous RGS3, which lacks the 3′UTR region for miR-126-3p, could revert the inhibitory effects of miR-126-3p on TNBC cell behaviors, indicating that RGS3 was directly involved in miR-126-3p-induced inhibition of cell behaviors.
VM is important for disease progression of many aggressive tumors such as hepatocellular carcinoma, as well as prostate, ovarian and lung cancer (49–52). VM has been reported to be related to the metastasis and poor prognosis of TNBC (24,53–54). The present study revealed that angiogenesis could be induced by miR-126-3p silencing and suppressed by miR-126-3p overexpression, while it was promoted by overexpression of RGS3 and inhibited by RGS3 silencing. Moreover, the overexpression of RGS3 could recover the inhibitory effect of miR-126-3p on VM of TNBC cells, indicating that RGS3 directly contributed to miR-126-3p-induced VM inhibition processes which provides a new target for anti-angiogenesis therapy. Therefore, it was deduced that miR-126-3p/RGS3 has the potential to be a new biomarker for TNBC treatment.
Collectively, the tumor suppressive role of the miR-126-3p/RGS3 axis on cell proliferation, colony formation, migration, invasion, and VM in TNBC was verified, which may be a potential target for cancer therapy treatment.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present research was supported by the Youth Scientific Research Project of Fujian Provincial Health and Family Planning Commission, PRC (no. 2016-1-91).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
ZH performed the majority of the study and wrote the manuscript. CH conducted the vasculogenic mimicry formation assays and data analysis and BM performed the dual luciferase activity assay and data analysis. QW, XZ and LL helped with the cell function assays and data analysis. CW and DC designed the experiments and edited the manuscript. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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