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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.47 No.3 pp.69-80
DOI : https://doi.org/10.17779/KAOMP.2023.47.3.002

Analysis on SOCS-Antigen Protein correlated with Prognostic Factors of Oral Cancer Patients

Mi Heon Ryu1), Min-A Jang2), Hye-Yeon Han3), Uk-Kyu Kim4)*
1)Department of Oral Pathology, School of Dentistry, Education and Research Team for Life Science on Dentistry, Dental and Life Science Institute, Pusan National University
2)Dental and Life Science Institute, Pusan National University
3)Department of Dental Hygiene, Jinju Health College
4)Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University
* Correspondence: Uk-Kyu Kim, D.D.S., Ph.D., Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University, 49, Busandaehak-ro, Mulgeum-eup, Yangsan, 50612, Gyeongsangnam-do, Republic of Korea Tel: +82-55-360-5004 Email: kuksjs@pusan.ac.kr
May 27, 2023 June 2, 2023 June 16, 2023

Abstract


SOCS3, a suppressor of cytokine signaling 3, is known as a negative regulator of various cytokines and a tumor suppressor gene in human tumors. This study aimed to investigate the role of SOCS3 in oral squamous cell carcinoma (OSCC) and its impact on epithelial-mesenchymal transition (EMT) in OSCC cells. Although SOCS3 is recognized as a negative regulator of various cytokines and a tumor suppressor gene in human tumors, its specific effects on OSCC remain poorly understood.



For the assessment of SOCS3 expression in OSCC, the UALCAN website and TCGA data were used to evaluate its expression in head and neck cancer. Additionally, immunohistochemical staining was conducted to determine the SOCS3 expression specifically in OSCC. The findings indicated a significant decrease in SOCS3 expression in tumor tissue compared to that in normal tissues.



To investigate the enhancement of SOCS3 expression in OSCC cancer cell lines, IL6 treatment was administered to MC3 cells. However, no significant differences were observed in cell viability, wound healing assay, and invasion assay. Conversely, the transfection of SOCS3 siRNA into OSCC cells led to a notable increase in cell viability and statistically significant increases in wound healing and invasion assays. These results suggest that SOCS3 plays a crucial role in cell viability and EMT in OSCC, thereby contributing to oral carcinogenesis. Further research is necessary to elucidate the precise role of SOCS3 in OSCC.


SOCS3 , Oral squamous cell carcinoma , Immunohistochemistry , EMT

구강암환자의 예후인자들과 연관된 항원단백인자 SOCS에 대한 분석

유미현1), 장민아2), 한혜연3), 김욱규4)*
1)부산대학교 치의학전문대학원 구강병리학교실, 부산대학교 치의생명과학 교육연구팀, 치의생명과학연구소
2)부산대학교 치의생명과학연구소
3)진주보건대학교 치위생과
4)부산대학교 치의학전문대학원 구강악안면외과학교실, 치의생명과학연구소

초록

    Ⅰ. INTRODUCTION

    Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm originating from the oral squamous epithelium, accounting for over 90% of oral cancers[1]. The main risk factors for OSCC include smoking, alcohol consumption, and viral infections such as HPV, although the exact cause remains unknown [2]. Unfortunately, early diagnosis of OSCC is challenging, often resulting in detection at an advanced stage. OSCC has a 5-year survival rate of 60%-80% if diagnosed early, which drops to less than 50% when diagnosed at an advanced stage [3].

    The treatment for OSCC typically involves surgical intervention, radiation therapy, or a combination of both. Advanced cases, including those affecting the side of the tongue or the floor of the mouth, often require surgical intervention along with radiation therapy and chemotherapy [4]. However, these treatment strategies can also cause damage to normal tissues, presenting a significant drawback. Despite these available treatments, the 5-year survival rate for OSCC has shown little improvement in the recent decades, emphasizing the urgent need for early diagnosis and biomarker development.

    The development and progression of oral cancer are influenced by various genetic and environmental factors. Suppressor of cytokine Signaling 3 (SOCS3) encodes a negative regulator protein involved in immune system regulation and cell growth. Recent reports suggest that SOCS3 also plays a role in suppressing malignant transformation of tumor cells and inducing apoptosis [5]. SOCS3 acts as a negative regulator of the Jak-STAT pathway, promoting cell proliferation and tumor development [6].

    Research on SOCS3 in OSCC patients is limited [7-10]. To analyze the correlation between the expression of SOCS1 and SOCS3 and clinical factors, Trivedi et al. conducted immunohistochemical staining of oral cancer tissues [8]. Christopher et al. proposed the role of SOCS3 as a key regulator associated with inflammation in OSCC [7]. However, the specific effects of the changes in SOCS3 expression on OSCC cells are not well understood. Therefore, this study aims to investigate the expression level of SOCS3 in Korean OSCC patients and determine the impact of SOCS3 expression on OSCC cell lines through in vitro studies.

    Ⅱ. PATIENTS and METHODS

    1. Bioinformatics analysis

    The UALCAN website (https://ualcan.path.uab.edu/index.html) has been utilized in this study to analyze and evaluate the expression of SOCS3 in The Cancer Genome Atlas (TCGA) cancer database. Specifically, we focused on head and neck cancer and examined the expression patterns of SOCS3 in normal tissues (44 samples) and head and neck cancer tissues (520 samples). Furthermore, using Kaplan- Meier survival analysis, the impact of SOCS3 expression was investigated on the survival of head and neck cancer patients. In this study, the head and neck cancer patients were categorized into two groups based on the expression levels of SOCS3: high expression (130 samples) and low expression (389 samples), and then the survival curves were generated based on this categorization.

    2. SOCS3 immunohistochemical (IHC) staining

    For data validation, IHC staining using SOCS3 antibodies was performed on both normal oral mucosal (NOM) tissues and OSCC tissues to evaluate SOCS3 expression. This study was approved by the Institutional Review Boards (IRB) of Pusan National University Hospital and Pusan National and University Dental Hospital (H-1808-017-070 and PNUDH-2017- 004, respectively).

    For IHC staining, 4-μm-thick serial sections of NOM and OSCC tissues were prepared. The slides underwent deparaffinization by immersion in xylene solution, followed by hydration with sequential immersion in 100%, 90%, and 70% concentrations of alcohol. Antigen retrieval was performed using a citric acid-based buffer solution (pH 6.0) for 20 minutes. To block endogenous peroxidase activity, the slides were treated with a peroxidase blocking reagent for 5 minutes.

    Subsequently, the primary antibody, SOCS3 (1:100, NBP2-27116, Novus Biologicals, Littleton, CO, USA), was applied to the slides and allowed to incubate overnight at 4°C. Following this, the slides were treated with REAL EnVision horseradish peroxidase for rabbit/mouse, and color development was achieved using REAL™ DAB+ chromogen with REAL™ substrate buffer. Finally, the slides were counterstained using Mayer's hematoxylin and were covered with a coverslip for mounting.

    3. Evaluation of immunohistochemical staining

    Immunohistochemical staining was evaluated using a semi-quantitative approach. Each slide stained with SOCS3 was examined using an optical microscope for assessment. The proportion of SOCS3-positive cells among the total observed cancer cells on each slide was determined. A scoring system was used, where a score of 0, 1, 2 and 3 indicated no staining, staining in 1% to 50% of cells, staining in 50% to 75% of cells, and staining in more than 75% of cells, respectively.

    4. Cell culture

    SAS, YD15, and MC3 cells were used in this experiment. YD15 cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea; cat. nos. 60504) and were cultured in RPMI1640 medium supplemented with 10% FBS and 1% P/S. SAS cells were cultured in DMEM + Ham's F12 (1:1) medium supplemented with 10% FBS.

    Furthermore, MC3 cells were obtained from the Fourth Military Medical University (Xi'an, China) and were cultured in DMEM + Ham's F12 (1:1) medium supplemented with 10% FBS.

    5. Western blotting

    The cells were harvested after treatment with IL6 or transfection with SOCS3 siRNA and were utilized for protein extraction. The extracted proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a polyacrylamide gel.

    Subsequently, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and blocked using a bovine serum albumin (BSA) solution. The membrane was then incubated overnight at 4°C with the SOCS3 primary antibody (Cell Signaling Technology, Danvers, MA, USA). Following this, a secondary antibody was applied to the membrane. The enzyme bound to the secondary antibody was visualized by adding substrates and measuring the luminescent signal.

    6. Il-6 treatment

    To upregulate the expression of SOCS3, MC3 cells were treated with IL6 at a concentration of 100 ng/ml for 1 hour. Once the increased expression of SOCS3 protein was confirmed by using Western blotting, the subsequent experiments were conducted.

    7. SOCS3 siRNA transfection

    The SAS and YD15 cells were cultured for SOCS3 siRNA transfection. For the transfection, the following sequence of SOCS3 siRNA (Bioneer, Daejeon, Korea) was used:

    • Sense : AGAAGAGCCUAUUACAUCUtt

    • Antisense : AGAUGUAAUAGGCUCUUCUgg

    The SOCS3 siRNA (100 ng/ml) was diluted in Opti-MEM medium along with Lipofectamine 3000 (5μl/ml), as per the manufacturer’s instructions. Together, the diluted siRNA and the transfection reagent were incubated for 1 hour at room temperature allowing complex formation. Subsequently, the cells were incubated with the siRNA complexes for 48 hours in a 6-well plate.

    After transfection, the efficiency of SOCS3 knockdown was assessed through Western Blotting..

    8. Cell viability assay

    Initially, MC3 cells was treated with IL6, while SAS and YD15 cells were transfected with SOCS3 siRNA. Subsequently, these cells were seeded into a 96-well plate at a density of 7 X 103 cells per well and were cultured for 24 and 48 hours, respectively.

    Following that, the CCK-8 kit reagent (Dojindo Molecular Technologies Inc., Kumamoto, Japan) were prepared as per the manufacturer's instructions and were added to the cells in the 96-well plate, replacing the culture medium. After 2 hours, a microplate reader was used to measure the absorbance at 260nm.

    9. Wound healing assay

    For wound healing assay, the MC3 cells were initially treated with IL6 in for 1 hour, or the SAS and YD15 cells were transfected with SOCS3 siRNA for 48 hours. Subsequently, IL6-treated MC3 cells (1.3 X 106 cells) and SOCS3 siRNA-transfected SAS cells or YD15 cells (1.3 X 106 cells) were seeded in a 6-well plate and were cultured until they reach the 80%-90% confluency, ensuring the formation of a uniform monolayer. Once the confluency of SAS and YD15 cells was confirmed, using a sterile pipette tip, a straight scratch was created across the cell monolayer of each plate to ensure a consistent wound width and depth. To monitor the activity of wound healing process, images were then captured at predetermined time intervals (0, 24, and 48 hours). To quantify the rate of wound closure, the distance between the wound edges was measured at different time points. The percentage of wound closure was calculated by comparing the wound width at each time point to the initial wound width. To determine the level of significance, statistical analysis, including t-tests, was performed.

    10. Invasion assay

    For the invasion assay experiment, the SAS and YD15 cells were transfected with SOCS3 siRNA and 2 X 104 cells (MC3 cells) or 3.5 X 104 cells (SAS and YD15 cells) were determined, respectively. These cells were mixed with 500 μL of cell culture media and were seeded onto Matrigel/ECM Gel-coated inserts of the invasion assay kit.

    After incubating the cells at 37°C and 5% CO2 in an incubator for 24 and 48 hours, respectively, all the medium were removed from the Matrigel/ECM Gel-coated inserts and a cotton swab was used to remove non-migrated cells on the upper side of the membrane. Then, the migrated cells were fixed by using 750 μL of cold methanol, were stained with crystal violet, and were allowed to dry. Subsequently, the migrated cells on the lower side of the membrane were counted using a phase contrast microscope.

    11. Statistical analysis

    All statistical analyses were performed using the SPSS software program (version 23.0; IBM SPSS, Chicago, IL, USA), and the graphs were generated using GraphPad Prism (version 9.5; GraphPad Software, San Diego, CA, USA). In vitro analyses were performed at least three times, and the results are presented as mean ± standard deviation (SD). Two-tailed Student's t-tests were used to compare the experimental and control groups, with a statistical significance defined as p < 0.05.

    Ⅲ. RESULTS

    1. Demographic and clinicopathological data

    The SOCS3 mRNA expression was analyzed based on the UALCAN website (https://ualcan.path.uab.edu/index.html), and data were analyzed from the 21 cancer subtype database of The Cancer Genome Atlas (TCGA). The mRNA expression of SOCS3 was found to be generally lower in most cancer tissues compared to that in normal tissues (Fig. 1A).

    Among data from 21 human cancers, the expression patterns of SOCS3 mRNA in head and neck cancer subtypes authors were specifically examined. Data were extracted to examine the expression patterns of SOCS3 mRNA in normal tissues (44 samples) and head and neck cancer tissues (520 samples). The results revealed a significant decrease in SOCS3 mRNA expression in head and neck cancer tissues (n = 520) compared to normal tissues (n = 44) (p < 0.01) (Fig. 1B). These findings were obtained from the TCGA database.

    To investigate the expression of SOCS3 protein in OSCC, immunohistochemical staining using a SOCS3 primary antibody was performed on OSCC tissues (n = 60) and NOM tissues (n = 12). The analysis demonstrated a lower expression of SOCS3 in OSCC tissues compared to NOM tissues (p = 0.0273) (Fig. 1C and 1D).

    Furthermore, the impact of SOCS3 expression on the survival of head and neck cancer patients was analyzed using Kaplan-Meier survival analysis. Based on the expression levels of SOCS3, the head and neck cancer patients were classified into two groups: high expression (130 samples) and low expression (389 samples), as depicted in Fig. 1E. The survival rates showed a statistically significant difference between the two groups (p = 0.01). This analysis utilized the data from the TCGA database of head and neck cancer patients obtained through the cBioportal.

    2. The effect of IL6 treatment on the induction of increased SOCS3 expression in MC3 cells

    To investigate the potential enhancing effect of the SOCS3 gene, IL-6 treatment on MC3 cells at a concentration of 100 ng/ml was performed for 1 hour. Subsequently, cell viability, wound healing, and invasion assays were conducted using these cells.

    In the cell viability assay, the number of MC3 cells cultured for 24 and 48 hours after IL-6 treatment was examined, but no statistically significant difference was observed between the experimental groups (Fig. 2A). Similarly, in the wound healing assay, no statistically significant difference was observed in the extent of wound closure of the MC3 cells at 24 and 48 hours between the control and the experimental groups (Fig. 2B). Additionally, in invasion assay, the number of invaded MC3 cells was assessed, and although a statistically significant increase was not observed in the number of invaded cells, a slight tendency toward a decrease at 48 hours was found (Fig. 2C).

    3. The effect of SOCS3 knockdown by SOCS3 siRNA transfection in SAS and YD15 cells

    To investigate the effects of SOCS3 gene silencing, SOCS3 knockdown was performed using SAS and YD15 cells. Cell viability, wound healing, and invasion assays were conducted using these cells.

    In the case of SAS cells, the cell viability assay revealed a significant increase in the number of SAS cells cultured for 24 and 48 hours after knockdown, indicating enhanced cell viability (Fig. 3A) (p < 0.01). Additionally, in the wound healing assay, the extent of wound closure in SAS cells was significantly increased at 24 and 48 hours, indicating improved wound healing ability (Fig. 3B) (24 hours: p < 0.01, 48 hours: p < 0.05). Furthermore, in the invasion assay, although no statistically significant increase was observed in the number of invaded SAS cells, a noticeable trend of increased invasion at 24 and 48 hours was found (Fig. 3C).

    For YD15 cells, the cell viability assay demonstrated a significant increase in the number of YD15 cells cultured for 24 hours after knockdown (p < 0.01). Although no statistically significant difference was observed after 48 hours of culture, a tendency of increased cell count for YD15 cells was found (Fig. 4A). Moreover, in the wound healing assay, similar to the results of SAS cells, the extent of wound closure in YD15 cells was significantly increased at 24 and 48 hours, indicating enhanced wound healing ability (Fig. 4B) (24 hours: p < 0.01, 48 hours: p < 0.05). In the invasion assay, the number of invaded YD15 cells showed a statistically significant increase at 48 hours, suggesting increased invasion potential (Fig. 4C) (p < 0.05).

    Ⅳ. DISCUSSION

    This study aimed to investigate the impact of SOCS3 expression in oral cancer cell lines through IL-6 treatment- induced increase in SOCS3 expression and SOCS3 knockdown using siRNA. The results of the analysis of SOCS3 expression in various human cancer types using TCGA revealed a decrease in SOCS3 expression in head and neck cancer tissues compared to that in normal tissues. Immunohistochemical staining of SOCS3 expression in Korean oral cancer tissues also showed a statistically significant decrease in SOCS3 expression in oral cancer tissues compared to that in normal oral tissues. Survival curve analysis using TCGA data indicated that patients with high SOCS3 expression had a relatively poorer prognosis (p = 0.01).

    IL-6 is known to regulate the expression of SOCS3 in cancer cells. Previous studies have demonstrated that treatment of cancer cells with IL-6 leads to an increase in SOCS3 expression and cell growth inhibition. For instance, Bluyssen et al. induced SOCS3 expression through IL-6 treatment in an established endothelial cell line (11), suggesting that IL-6-induced SOCS3 expression may have a tumor-suppressive effect. In the present experiment, a similar method was followed by treating MC3 cells with IL-6 at a concentration of 100 ng/ml for 1 hour, confirming an increase in SOCS3 protein expression through Western blotting. However, no statistically significant differences were observed in the cell viability and wound healing assays. Invasion tend to decrease in the invasion assay. Tang et al. reported that cell viability, migration, and invasion decreased when SOCS3 expression was induced in breast cancer stem cells (12). Furthermore, Tomita et al. reported that cell viability decreased when enhancing SOCS3 using a vector (13), which differs from the findings of this experiment. In this study, the method of SOCS3 induction through IL-6 treatment is believed to only have a temporary effect. To obtain more conclusive experimental results, using methods such as SOCS3 vector or CMV-driven plasmid recommended to induce SOCS3 expression.

    Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism that plays a crucial role in cell motility and invasion. (14) The study found significant differences in the wound healing assay and invasion assay of cells with SOCS3 knockdown, indicating that the SOCS3 gene inhibition in oral cancer cells influences their biological behavior. The correlation between SOCS3 and EMT in oral cancer remains incompletely understood. Ji et al. reported changes in the morphology of MHCC97H cells to mesenchymal- spindle-shaped cells and alterations in the expression of proteins such as E-cadherin, vimentin, and α -SMA following SOCS3 siRNA transfection. (15) In colorectal cancer cells (HT290 and SW480), a transformation into a flattened and spread morphology was associated with increased expression of MMP-2 and MMP-9. Conversely, increased SOCS3 expression resulted in decreased MMP-2 and MMP-9 expressions. The findings of this study suggest that SOCS3 silencing in SAS and YD15 cells promotes EMT, showing its significant role in EMT. Further research is needed to understand the detailed mechanisms through which SOCS3 silencing affects EMT.

    The experiment involving the use of SOCS3 siRNA to knockdown SOCS3 showed a statistically significant increase in the cell viability of oral cancer cells. The SOCS3 in cancer cells involves apoptosis and cell cycle transition. (6) Transfection of SOCS3 vector into OSCC cells resulted in increased caspase 3 expression. (9) Additionally, the inhibition of SOCS3 expression in pancreatic cancer cells and non-small cell lung cancer cells led to increased cell viability and reduced apoptosis, consistent with the findings of this experiment. (16, 17) SOCS3 inhibits the JAK/STAT3 pathway, which plays a role in cancer cell survival, differentiation, maturation, and proliferation. Moreover, IL-6 itself can be increased in cancer cells due to inflammation and other causes, leading to excessive activation of the JAK/STAT3 pathway and decreased expression of SOCS3. (18) This creates a positive feedback loop that can promote tumor cell growth and survival. Therefore, SOCS3 is believed to play a key role in cell proliferation in oral cancer cells.

    Studies conducted using oral cancer cells have reported that an increase in SOCS3 expression inhibits tumor cell proliferation and progression. (9) A previous study investigating the expression level of the SOCS3 in head and neck cancer tissues revealed that the loss of SOCS3 expression is an early event in carcinogenesis. Furthermore, a decrease in SOCS3 expression was associated with tumor size and histologic grade of dysplasia. (19) However, contrasting reports also exist regarding the increase in SOCS3 expression in head and neck cancer and oral cancer tissues. Cha et al. reported an increase in the combined expression of MMP1, SOCS3, and ACOX1 genes in head and neck cancer tissues compared to that in normal tissues. (20) Chen et al. also reported an increase in SOCS3 expression in oral cancer tissues (21), and in advanced-stage oral cancer patients, increased SOCS3 expression was associated with an aggressive tumor phenotype (8). Therefore, further large-scale cohort studies and detailed mechanism investigations are necessary to understand the alterations in SOCS3 expression and its underlying mechanisms in oral cancer.

    Ⅴ. CONCLUSION

    In summary, the results of this experiment demonstrate that SOCS3 expression significantly decreased in head and neck cancer tissues and in oral cancer tissues. Additionally, the inhibition of SOCS3 expression increased cell viability, wound healing and invasion ability of SAS and YD15 cells. These findings suggest that the alteration of SOCS3 expression may contribute to oral carcinogenesis and can potentially regulate the process of tumor formation. However, SOCS3 expression in oral cancer varies across different research studies, indicating the need for further large-scale cohort studies and detailed mechanism investigations.

    ACKNOWLEDGMENTS

    This work was supported by a 2-Year Research Grant of Pusan National University.

    CONFLICT OF INTEREST

    The authors have no conflict of interest to declare.

    Figure

    KAOMP-47-3-69_F1.gif

    Analysis of SOCS3 expression between human normal tissues and cancer tissues. (A) SOCS3 expression in 21 types of human cancers was evaluated using the UALCAN database (https://ualcan.path.uab.edu/). (B) SOCS3 mRNA expression in normal tissues (44 samples) and in head and neck cancer tissues. (C) Immunohistochemistry (IHC) staining showed higher expression of SOCS3 protein in NOM tissue compared to OSCC tissue (p = 0.0273). (D) Representative photographs of SOCS3 IHC-stained tissues showing samples with high and low expression in NOM and OSCC tissues. Scale bar: 100 μm. (E) The overall survival analysis was performed by classifying the head and neck cancer patients into two groups based on high SOCS3 expression and low or medium expression.

    KAOMP-47-3-69_F2.gif

    Effect of SOCS3 augmentation in MC3 cell line. (A) After treating MC3 cells with IL-6 at concentrations of 10, 50, and 100 ng/ml for 1 hour, followed by measuring the cell viability assay at 24 and 48 hours, no statistically significant differences were observed in the cell viability assay of MC3 cells. (B) No statistically significant difference was observed when conducting a wound healing assay using MC3 cells treated with IL-6 at a concentration of 100 ng/ml for 1 hour. (C) Although no statistically significant difference was observed when conducting a wound healing assay using MC3 cells treated with IL-6 at a concentration of 100 ng/ml for 1 hour, a trend of decreased invasion cell count was found in the experimental group treated with IL-6.

    KAOMP-47-3-69_F3.gif

    Effect of SOCS3 knockdown on cell viability, wound healing, and invasion ability of SAS cells. (A) The effect of SOCS3 knockdown on the proliferation of SAS cells by CCK-8 assay. (B) The effect of SOCS3 knockdown on the migration of SAS cells by wound healing assay. Scale bar = 100 μm. The representative photographs of the wound healing assay experiments are placed on the left, while the graph depicting the statistical analysis using the t-test is placed on the right. (C) The effect of SOCS3 knockdown on the invasion ability of SAS cells by invasion assay. The representative photographs of the invasion assay experiments are placed on the left, while the graph depicting the statistical analysis using the t-test is placed on the right. (*: p < 0.05, **: p < 0.01)

    KAOMP-47-3-69_F4.gif

    Effect of SOCS3 knockdown on cell viability, wound healing, and invasion ability of YD15 cells. (A) The effect of SOCS3 knockdown on the proliferation of YD15 cells by CCK-8 assay. (B) The effect of SOCS3 knockdown on the migration of YD15 cells by wound healing assay. Scale bar = 100 μm. The representative photographs of the wound healing assay experiments is placed on the left, while the graph depicting the statistical analysis using the t-test are placed on the right. (C) The effect of SOCS3 knockdown on the invasion ability of YD15 cells by invasion assay. The representative photographs of the invasion assay experiments is placed on the left, while the graph depicting the statistical analysis using the t-test is placed on the right. (*: p < 0.05, **: p < 0.01)

    Table

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