SHCBP1 contributes to the proliferation and self‑renewal of cervical cancer cells and activation of the NF‑κB signaling pathway through EIF5A
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- Published online on: April 21, 2023 https://doi.org/10.3892/ol.2023.13832
- Article Number: 246
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Copyright: © Deng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Cervical cancer (CC) is the fourth most common cancer and the fourth leading cause of cancer mortality in women (1). In 2020, 604,000 new diagnoses and 342,000 CC-related deaths were estimated worldwide, accounting for ~3.2% of all new cancer diagnoses and 3.4% of all cancer deaths (1). Given the high incidence and mortality of CC, the World Health Organization has assembled the CC Elimination Modelling Consortium to eliminate CC (2,3). Despite increases in human papillomavirus vaccination coverage, and advances in the screening approach of CC in recent years, the burden of CC is heavy in low-income countries (3). Therefore, it remains necessary to elucidate the molecular mechanisms underlying the progression of CC as it would help to identify effective therapeutic strategies.
The SHC binding and spindle associated 1 (SHCBP1) gene is located on chromosome 16q11.2; SHCBP1 is a member of the Src homolog and collagen homolog (Shc) family, and it was first characterized as a protein which bound to the Shc adaptor protein, Shc A (4). SHCBP1 is able to regulate cell proliferation, differentiation and wound inflammation (4,5). Increasing evidence has suggested that SHCBP1 is often dysregulated in cancer, and acts as a key regulator of tumor progression and metastasis in various types of cancer, such as gastric carcinoma (6), synovial sarcoma (7) and non-small cell lung carcinoma (8). Nevertheless, the functional and molecular mechanisms of SHCBP1 in CC remain unexplored.
As a transcription factor, NF-κB has been linked to inflammatory responses in various human diseases (9). In mammals, the NF-κB family is composed of five members, namely p65, c-Rel, RelB, p50 and p52, all of which share an N-terminal DNA-binding/dimerization domain referred to as the REL homology domain (10). NF-κB proteins form homo- and heterodimers that can bind to DNA sequences of target genes and ultimately regulate gene transcription (10). A strong association has been established between the activation of NF-κB and cancer, including CC (11–13). Zhou et al (14) confirmed the positive regulation of NF-κB signaling pathway activation and cell mobility in glioma induced by SHCBP1 by performing gain-/loss-of-function experiments. The link between SHCBP1 and NF-κB signaling in CC remains to be fully elucidated.
Eukaryotic translation initiation factor 5A (EIF5A), a key factor involved in translation elongation, exerts oncogenic roles in a variety of cancer types, including colorectal cancer (15), pancreatic cancer (16) and CC (17,18). EIF5A silencing has been reported to inhibit CC cell proliferation and promote NF-κB inhibitor (IκB) expression in CC cells (17). Furthermore, small interfering RNA (siRNA)-mediated inhibition of EIF5A has been shown to reverse the phosphorylation of p65 in multiple myeloma cells (19), highlighting the modulation of NF-κB activation by EIF5A. A dataset (GSE154307) downloaded from the Gene Expression Omnibus (GEO) database indicates that SHCBP1 short hairpin RNA (shRNA) reduced EIF5A mRNA expression in papillary thyroid cancer cells (20). Therefore, it was hypothesized that SHCBP1 may be involved in the activation of the NF-κB signaling pathway in CC cells via EIF5A.
In the present study, gain- and/or loss-of-function experiments were performed to explore the role of SHCBP1 in the proliferation and stemness of CC cells, and the in vitro activation of the NF-κB signaling pathway. In addition, the molecular mechanism of SHCBP1 in the progression of CC was discussed.
Materials and methods
Screening of differentially expressed genes (DEGs)
The CC microarray datasets (GSE9750, GSE63514 and GSE7803) (21–23) were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Genes with P<0.05 and log2(fold change) ≥1 or ≤-1 were considered to be DEGs between healthy control cervical tissues and CC tissues.
Bioinformatics analysis
The Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer–pku.cn/index.html) database was used to profile the expression level of SHCBP1 in tumor tissues from 31 types of cancer and the corresponding healthy control tissues based on the TCGA and GTEx databases. The mutual DEGs among the CC microarray datasets (GSE9750, GSE63514 and GSE7803) were identified using the online tool Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html). The Database for Annotation, Visualization and Integrated Discovery (https://david.ncifcrf.gov/) was utilized to perform Gene Ontology (GO, http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) pathway enrichment analyses for the mutual DEGs.
Cell culture and transfection
Human CC cell lines were obtained from iCell Bioscience, Inc. (HeLa, cat. no. iCell-h088) and Procell Life Science & Technology Co., Ltd. (SiHa,C-33A and CaSki). HeLa, SiHa and C-33A cells were cultured in minimum essential medium (MEM, Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 10% FBS (Zhejiang Tianhang Biotechnology Co., Ltd.), 100 U/ml penicillin and 0.1 mg/ml streptomycin with 5% CO2 at 37°C. CaSki cells were maintained in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 10% FBS (Zhejiang Tianhang Biotechnology Co., Ltd.), 100 U/ml penicillin and 0.1 mg/ml streptomycin with 5% CO2 at 37°C.
For in vitro functional experiments, plasmids (2.5 µg) expressing shRNAs targeting SHCBP1 or non-targeting shRNAs (negative control, NC) were transfected into SiHa cells and plasmids (2.5 µg) expressing SHCBP1 coding DNA sequence (CDS) or empty vectors were transfected into CaSki cells using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions at 37°C, and the medium was changed after 8 h. The sequences of shRNAs were as follows: SHCBP1 shRNA-1, 5′-GGATTGTATGTGTTTGGTTATTCAAGAGATAACCAAACACATACAATCTTTTT-3′; SHCBP1 shRNA-2, 5′-GGGTTATCAGAAGAATTCTATTCAAGAGATAGAATTCTTCTGATAACCTTTTT-3′; and non-targeting shRNA, 5′-GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTT-3′. The mock control represents the cells without any transfection. The shRNA was inserted into the pRNA-H1.1/Neo vector (cat. no. SD1203; GenScript) between BamHI and HindIII. The SHCBP1 CDS (General Biology (Anhui) Co., Ltd.) was inserted into the pcDNA3.1(+) vector (cat. no. V79020; Invitrogen; Thermo Fisher Scientific, Inc.) between HindIII and XhoI. At 24 h post-transfection, the SiHa cells stably silencing SHCBP1 were selected in complete MEM containing 350 µg/ml geneticin (Beijing Solarbio Science & Technology Co., Ltd.) for 1–2 weeks, and the CaSki cells stably overexpressing SHCBP1 were selected in complete RPMI-1640 medium containing 300 µg/ml geneticin for 1–2 weeks, and the medium was changed every 2–3 days. A large number of cells died after 1- or 2-week geneticin selection, and then the surviving cells were cultured in complete MEM or RPMI-1640 medium containing geneticin (SiHa: 175 µg/ml; CaSki: 150 µg/ml) for 2 weeks.
In addition, stable SHCBP1-overexpressing CaSki cells or blank CaSki cells (without any manipulation of SHCBP1 expression) were transfected with siRNA-targeting EIF5A (75 pmol; General Biology (Anhui) Co., Ltd.) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C, and the medium was changed after 8 h. The sequences of siRNAs were as follows: EIF5A siRNA sense, 5′-GCAAGGAGAUUGAGCAGAATT-3′ and anti-sense 5′-UUCUGCUCAAUCUCCUUGCTT-3′; and non-targeting siRNA sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and anti-sense, 5′-ACGUGACACGUUCGGAGAATT-3′. At 48 h post-transfection, the cells were used for subsequent experiments.
RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted from the four CC cell lines with TRIpure reagent (BioTeke Corporation). First-strand cDNA was synthesized using BeyoRT II M-MLV reverse transcriptase (Beyotime Institute of Biotechnology) with dNTPs, 5× buffer, Rnase inhibitor (BioTeke Corporation), oligo (dT)15 and random primers at 25°C for 10 min, at 42°C for 50 min and at 80°C for 10 min. Relative mRNA expression was determined by RT-qPCR on an Exicycler™ 96 Real-Time PCR system (Bioneer Corporation) using SYBR Green 2× Taq PCR Master Mix (Beijing Solarbio Science & Technology Co., Ltd.) and gene-specific primers (General Biology (Anhui) Co., Ltd.). The thermocycling conditions were listed as follows: 94°C for 5 min (initial denaturation), 40 cycles of 94°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec, followed by a single final extension for 2.5 min at 72°C. GAPDH was used to normalize gene expression. The relative expression levels of mRNAs were calculated using the 2−ΔΔCq method (24). The sequences of the gene-specific primers used in RT-qPCR are listed in Table I.
Western blotting
Protein was extracted from the four CC cell lines with RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 1 mM phenylmethanesulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.). The concentration of protein was measured using BCA protein assay kits (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocols. Protein samples (10–20 µg/lane) were separated by SDS-PAGE on 10 or 14% gels and transferred to PVDF membranes (MilliporeSigma). The membranes were blocked with 5% BSA (Biosharp Life Sciences) at room temperature for 1 h, followed by incubation with anti-SHCBP1 (dilution, 1:500; cat no. 12672-1-AP, Proteintech Group, Inc.), anti-cyclin D1 (dilution, 1:500; cat no. A19038, ABclonal Biotech Co., Ltd.), anti-p21 (dilution, 1:500; cat no. A19094, ABclonal Biotech Co., Ltd.), anti-phosphorylated (p)-IκBα (dilution, 1:500; Ser-32/Ser-36, cat no. AF2002, Affinity Biosciences, Ltd.), anti-IκBα (dilution, 1:1,000; cat no. AF5002, Affinity Biosciences, Ltd.), anti-p-p65 (dilution, 1:400; Ser-536, cat no. AF2006, Affinity Biosciences, Ltd.), anti-p65 (dilution, 1:1,000; cat no. AF5006, Affinity Biosciences, Ltd.), anti-EIF5A (dilution, 1:1,000; cat no. 11309-1-AP, Proteintech Group, Inc.), and anti-GAPDH (dilution, 1:10,000; cat no. 60004-1-Ig, Proteintech Group, Inc.) antibodies overnight at 4°C. The following day, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (dilution, 1:3,000; cat no. SE134, Beijing Solarbio Science & Technology Co., Ltd.) or goat anti-mouse IgG (dilution, 1:3,000; cat no. SE131, Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at 37°C. The immunoreactive protein bands were visualized using an ECL kit (Beijing Solarbio Science & Technology Co., Ltd.). GAPDH was used as a loading control to normalize protein expression.
Cell proliferation assays
MTT assays were used to assess cell proliferation. In brief, CC cells were seeded into 96-well culture plates at a density of 4×103 cells/well and cultured for 0, 12, 24, 36 and 48 h. Cell proliferation was measured using MTT assay kits (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. DMSO (Nanjing KeyGen Biotech Co., Ltd.) was used to dissolve formazan and the optical density was measured at 490 nm.
Immunofluorescence assays (IFAs)
SiHa and CaSki cells were grown on glass coverslips (1×105 cells per well) in 24-well plates, fixed in 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd.) for 15 min at room temperature and permeabilized with 0.1% Triton X-100 (Beyotime Institute of Biotechnology) for 30 min at room temperature. After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at room temperature, the coverslips were incubated with anti-ki67 (dilution, 1:200; cat no. A11390, ABclonal Biotech Co., Ltd.) or anti-p65 antibodies (dilution, 1:200; cat no. A19653, ABclonal Biotech Co., Ltd.) at 4°C overnight, followed by incubation with the Alexa Fluor™ 555-conjugated goat anti-rabbit (dilution, 1:200; cat no. A27039, Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Cell nuclei were stained with DAPI (Shanghai Aladdin Biochemical Technology Co., Ltd.) at room temperature for 5 min and observed under a fluorescence microscope. Total p65 and nuclear p65 levels were quantified. Relative p65 nuclear localization was expressed as the ratio of the fluorescence density of nuclear p65 staining to the fluorescence density of total p65 cellular staining.
Cell cycle analysis
Cell cycle progression was examined using cell cycle analysis kits (cat no. BL114A, Biosharp Life Sciences). SiHa and CaSki cells (5×105 cells) were collected by centrifugation (106 × g for 5 min) at 4°C and fixed with ice-cold 70% ethanol for 2 h at 4°C. The cell pellets were washed with PBS twice and the staining buffer, PI (20×) and RNase A (50×) (cat no. BL114A, Biosharp Life Sciences) were added. After incubation at 37°C for 30 min, the cells were analyzed by a flow cytometer (NovoCyte 2040R; ACEA Bioscience, Inc.; Agilent Technologies, Inc.) using the NovoExpress software (version 1.4.1; ACEA Bioscience, Inc.; Agilent Technologies, Inc.).
Sphere formation assays
CC cells were seeded into 6-well ultra-low plates at a density of 3×103 cells/ml and cultured in stem cell medium for 2 weeks with 5% CO2 at 37°C. The medium was changed every 2 days. The stem cell medium was DMEM/F12 (Procell Life Science & Technology Co., Ltd.) supplemented with 1% B-27 (Gibco; Thermo Fisher Scientific, Inc.), 1% N-2 (Gibco; Thermo Fisher Scientific, Inc.), 20 ng/ml EGF (Sino Biological Inc.) and 10 ng/ml FGF (Sino Biological Inc.). Spheres were observed under an inverted phase contrast microscope and analyzed using Image-Pro Plus software (version 6.0, Media Cybernetics, Inc.). Spherical cell clusters were considered to be spheres when their diameter was >75 µm. The spheres were formed based on the cancer stem cell (CSC)-like properties of cancer cells.
Statistical analysis
Statistical analysis was performed using GraphPad Prism (version 8.0.2, GraphPad Software; Dotmatics). Results are presented as the mean ± SD from three independent experiments. One-way and two-way ANOVA followed by Tukey or Sidak tests, unpaired t-test and Mann Whitney U test were used to analyze statistical significance. P<0.05 was considered to indicate a statistically significant difference.
Results
Identification of DEGs in CC
The GSE9750, GSE63514 and GSE7803 datasets indicated 2,176, 1,841 and 796 DEGs in CC compared with the healthy control cervical tissues, respectively (Fig. 1A-C). The Venn diagram of DEGs in the three datasets indicated a total of 316 mutual DEGs (Fig. 1D). The function of the mutual DEGs was examined by performing GO (Fig. 1E) and KEGG pathway (Fig. 1F) enrichment analyses. GO analysis suggested that the mutual DEGs were mainly linked to ‘chromosome segregation’ [biological process (BP)], ‘DNA replication’ (BP), ‘nuclear division’ (BP), ‘chromosomal region’ [cell component (CC)], ‘spindle’ (CC), ‘DNA replication preinitiation complex’ (CC), ‘single-stranded DNA helicase activity’ [molecular function (MF)], ‘ATP-dependent activity, acting on DNA’ (MF) and ‘catalytic activity, acting on DNA’ (MF). The three most enriched pathways in the KEGG analysis were ‘cell cycle’, ‘DNA replication’ and ‘cellular senescence’.
Upregulation of SHCBP1 mRNA levels in CC
The mRNA expression level of SHCBP1 was upregulated in CC compared with the healthy control cervix tissues in the GSE9750, GSE63514 and GSE7803 datasets, and the expression of SHCBP1 in the tumor and healthy control cervical tissues in the three datasets was shown in Fig. 2A. The Gene Expression Profiling Interactive Analysis database showed that SHCBP1 expression was significantly upregulated in 21 human cancer types, particularly lymphoid neoplasm diffuse large B-cell lymphoma and cervical squamous cell carcinoma and endocervical adenocarcinoma (Fig. 2B). The highest median value of SHCBP1 expression was observed in the healthy control for acute myeloid leukemia, which indicated a high level of SHCBP1 mRNA in bone marrow (Fig. 2B). There was not a statistically significant difference between the bone marrow from the healthy control and acute myeloid leukemia patients (Fig. 2B).
Pro-proliferative and pro-stemness role of SHCBP1 in CC cells in vitro
The basal expression levels of SHCBP1 in HeLa, SiHa, C-33A and CaSki CC cell lines were determined by RT-qPCR and western blotting and the results demonstrated a markedly higher expression of SHCBP1 in SiHa cells and a markedly lower expression of SHCBP1 in CaSki cells than that in other CC cell lines (Fig. 3A). Stable SHCBP1-overexpressing CaSki cells and stable SHCBP1-silenced SiHa cells were successfully established (Fig. 3B). MTT assays demonstrated that SHCBP1 overexpression promoted but SHCBP1 knockdown inhibited cell proliferation in vitro (Fig. 3C). Proteins involved in cell cycle progression were detected by western blotting. SHCBP1 overexpression promoted cyclin D1 expression and inhibited p21 expression in CaSki cells, yet SHCBP1 knockdown induced an opposite effect on SiHa cells (Fig. 3D). Flow cytometric analysis suggested that SHCBP1 overexpression enhanced entry into the S phase but SHCBP1 knockdown induced arrest at the G1 phase of the cell cycle (Fig. 3E). In addition, SHCBP1 overexpression decreased the number of cells at the G2 phase, which might be the result of the extreme increases in cell density induced by SHCBP1 overexpression (Fig. 3E). IFA of ki67 showed increased ki67 expression induced by SHCBP1 overexpression and decreased ki67 expression induced by SHCBP1 knockdown, which indicated the pro-proliferative roles of SHCBP1 (Fig. 3F). Subsequently, the effect of SHCBP1 on the CSC-like properties of CC cells was investigated. The mRNA expression levels of stemness-related markers, including CD44, CD133, NANOG and OCT4, were analyzed, and the results demonstrated that SHCBP1 overexpression significantly increased the mRNA expression levels of these markers (Fig. 4A). Furthermore, SHCBP1 overexpression tended to upregulate the sphere-forming ability of CC cells, although there was no statistically significant difference between the empty vector and the SHCBP1 OE group (Fig. 4B). Taken together, SHCBP1 exerted a key role in maintaining the malignant phenotype and self-renewal of CC cells in vitro.
Activation of the NF-κB signaling pathway by SHCBP1 in CC cells
Activation of the NF-κB pathway serves a key role in cancer (25). The phosphorylation of IκBα and p65 was assessed by western blotting. SHCBP1 overexpression increased p-IκBα (Ser-32/Ser-36) and p-p65 (Ser-536) expressions but decreased total IκBα expression, however, SHCBP1 knockdown exerted the opposite effect (Fig. 5A). IFA for p65 demonstrated that a significantly higher number of positive signals of p65 was detected in the nucleus after SHCBP1 overexpression; however, SHCBP1 knockdown significantly reduced nuclear signals of p65 (Fig. 5B). These findings suggested that SHCBP1 was involved in the activation of the NF-κB signaling pathway in CC cells.
SHCBP1 depends on EIF5A to exert pro-proliferative and self-renewal effects and activate the NF-κB signaling pathway in CC cells
EIF5A exerts oncogenic roles in CC (17,18). A GEO dataset (GSE154307) indicates downregulated EIF5A mRNA expression by SHCBP1 shRNA in papillary thyroid cancer cells (20). The results of the present study demonstrated that EIF5A mRNA expression was significantly promoted by SHCBP1 overexpression (Fig. S1A), yet significantly inhibited by SHCBP1 knockdown (Fig. S1B). To investigate whether EIF5A mediated the biological function of SHCBP1 in CC cells, untransfected CaSki cells and stable SHCBP1-overexpressing CaSki cells were transiently transfected with EIF5A siRNA. The knockdown efficiency of EIF5A was verified in untransfected CaSki cells and stable SHCBP1-overexpressing CaSki cells by RT-qPCR and western blotting (Fig. 6A, B and E). The stemness-related markers including NANOG and OCT4 were confirmed to be regulated by EIF5A (26). EIF5A siRNA inhibited the SHCBP1-induced increases in NANOG and OCT4 expression (Fig. 6B). In addition, EIF5A siRNA reversed the pro-proliferative effects (Fig. 6C, D) and the activation of the NF-κB signaling pathway induced by SHCBP1 overexpression (Fig. 6E). These results confirmed that SHCBP1 enhanced the malignant phenotype and self-renewal of CC cells, and activated the NF-κB signaling pathway in vitro via EIF5A. The potential mechanism of SHCBP1 in CC cells is shown in Fig. 6F. Based on the findings of the present study, it was hypothesized that SHCBP1 increased the nuclear translocation of p65 via EIF5A, thereby enhancing the NF-κB-mediated gene transcription and promoting cell proliferation and cell stemness.
Discussion
Unchecked proliferation is a fundamental hallmark of cancer cells (27). The accurate transition from the G1 phase to the S phase of the cell cycle is crucial for the control of cell proliferation, and its disorganization results in uncontrolled cell proliferation (28). SHCBP1 was first identified in proliferating cells and it was absent in quiescent tissues and growth-arrested cells (4). SHCBP1 mRNA is expressed during entry into S phase and its expression was higher in cells treated with a stimulator of cell cycle progression (4). These evidence indicate the important role of SHCBP1 in regulating cell proliferation and cell cycle progression (4). The present study revealed that SHCBP1 increased CC cell proliferation, ki67 expression and cell cycle transition from the G1 phase to the S phase of the cell cycle. The pro-proliferative effect of SHCBP1 has also been observed in cell lines from other types of cancer (20,29–32), highlighting the important role of SHCBP1 in cancer cell proliferation. Therefore, SHCBP1 inhibition may be a potential strategy for the control of tumor progression.
CSCs, a key population of tumor cells, are pluripotent, self-renewing and they contribute to tumor initiation, metastasis, recurrence and resistance to therapies (33). Cumulative reports have highlighted the importance of CSCs in CC (34–36). SHCBP1 knockdown induced inhibition of stem cell-like properties of non-small cell lung carcinoma (8) and papillary thyroid carcinoma (20) cells, including generation of tumor cell spheres and the expression of stemness-related genes and proteins. In line with previous reports (8,20), the present study suggested that SHCBP1 activated CSC-related genes and promoted the self-renewal of CC cells. These findings further indicated the significance of SHCBP1 in CC.
The NF-κB signaling pathway is one of the important pathways involved in cell cycle regulation (37). NF-κB can activate the transcription of cell cycle-related genes, including cyclin D1, cyclin E1, CDK2, CDK2 and Myc, thereby modulating cell cycle distribution (38–41). The NF-κB pathway also contributes to maintaining CSC-like properties in multiple types of cancer (42–44). Overexpression of p65 enhances the expression of CSC markers, such as CD44 and CD133, in CC cells (45). Bay-11-7082, an inhibitor of NF-κB, has been demonstrated to reduce CD44 expression in breast cancer cells (46) and NANOG and SOX2 expression in CD44-positive and myeloid differentiation primary response 88-positive ovarian cancer cells (47). Knockdown of p65 and/or RelB decreases CD133-positive skin cancer cells, and their self-renewal abilities (48). The present study demonstrated that SHCBP1 activated the NF-κB signaling pathway in CC cells. Given the importance of NF-κB in cell proliferation and stemness maintenance in cancer cells, it is hypothesized that the pro-proliferative and pro-stemness effects of SHCBP1 may be attributed to its induction of NF-κB activation.
The positive regulation of NF-κB activation by EIF5A has been indicated in previous studies (17,19). The inhibitory effect of SHCBP1 shRNA transfection on EIF5A mRNA was confirmed in the present study. Similar effects have been observed in papillary thyroid cancer cells (20), and it was hypothesized that EIF5A may mediate the biological function of SHCBP1 and the regulation of the NF-κB signaling pathway by SHCBP1 in CC. The findings of the present study confirm this hypothesis. The regulatory mechanism of SHCBP1 on EIF5A remains unclear, which is one of the limitations of the present study. SHCBP1 could enhance the interaction between CREB-binding protein (CBP) and β-catenin, thereby activating the transcription of genes driven by β-catenin (49). The interaction between CBP and KLF5 is of importance for KLF5 transactivation activities (50). There are KLF5 binding sites at the promoter of EIF5A. KLF5 has been confirmed to bind to EIFA5 and activate its transcription (51). Based on previous studies (49–51), it was hypothesized that the regulation of EIF5A mRNA by SHCBP1 may be associated with the enhanced interaction between CBP and KLF5 triggered by SHCBP1, which needs to be explored in future work. In addition, the effect of SHCBP1 on tumor growth in a xenograft model of CC was not investigated, which may be an additional limitation of the present study.
In summary, the role of SHCBP1 in CC was identified in vitro. Mechanistically, SHCBP1 facilitated CC cell proliferation, stemness and activation of the NF-κB signaling pathway through EIF5A in vitro. The present study suggested a novel molecular mechanism underlying the progression of CC.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This work was supported by the National Natural Science Foundation of China (grant no. 81902633).
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
BYD conceptualized the research project, devised methodology, completed formal analysis and data curation, prepared and wrote the original draft manuscript. ALL supervised the study, performed data analysis and reviewed and edited the manuscript. YZ, YYZ and JF performed experiments and validated the data. YM performed interpretation and visualization of the data. BYD, ALL and YM confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
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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