Transgelin Inhibits the Malignant Progression of Esophageal Squamous Cell Carcinomas by Regulating Epithelial–Mesenchymal Transition

Objective This article investigates the role of Transgelin (TAGLN) in the epithelial–mesenchymal transition (EMT) of esophageal squamous cell carcinomas (ESCC) and its possible mechanism of inhibiting the invasion of these cancers. Methods Tissue specimens and clinical information of patients with ESCC were collected to analyze the relationship between Transgelin expression level and prognosis of patients with ESCC. Transgelin siRNA was used to knock down Transgelin expression. The expression of Transgelin in Eca-109 and KYSE-150 cells was overexpressed by Transgelin-overexpressing plasmid. The effects of Transgelin overexpression and knockdown on the proliferation of Eca-109 and KYSE-150 cells were examined by Transwell chamber, scratch assay, and CCK-8 cell activity assay. RT-PCR and Western blot were used to detect the effect of Transgelin overexpression or knockdown on the mRNA and protein expressions of E-cadherin and Vimentin. TCGA data were used to analyze Transgelin co-expressed genes and further study the GO and KEGG enrichment analysis results under the influence of Transgelin. Results The expression of Transgelin was low in ESCC, and its expression level was positively correlated with the prognosis of patients with ESCC. The targeted Transgelin siRNA and Transgelin-overexpressing plasmid can effectively regulate the expression of Transgelin mRNA and protein in Eca-109 and KYSE-150 cells. After overexpression of Transgelin, the invasion and proliferation abilities of Eca-109 and KYSE-150 cells were significantly decreased compared with those of the control group (P < 0.05). However, Transgelin knockdown could promote the proliferation, migration, and invasion of ESCC cells. The overexpression of Transgelin inhibits EMT in ESCC. With the increase of Transgelin expression in Eca-109 and KYSE-150 cells, the expression of E-cadherin increased, while the expression of Vimentin decreased, and the difference was statistically significant (P < 0.05). Conclusion Transgelin can inhibit the malignant progression of ESCC by inhibiting the occurrence of EMT.


INTRODUCTION
Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor of the digestive tract with poor prognosis and high mortality (1,2). ESCC is the 5th most common malignancy in men and the 8th most common malignancy in women (3). The incidence of ESCC has declined in recent years because of the advances in early cancer screening (4). However, ESCC treatment remains a difficult problem (5). At present, the most effective treatment for ESCC is surgery, chemotherapy, and radiotherapy as adjuvant treatment. There are abundant lymphocytes under the mucosa of the esophagus, so when the tumor encroaches below the mucosa of the esophagus, cancer cells can easily spread along these lymphocytes. In addition, the esophagus does not have a serous layer, which is less resistant to tumor invasion. When the tumor invades the muscle layer of the esophagus, it may invade the surrounding organs. There are more important organs near the esophagus. When the tumor grows and extends forward or backward, it is more difficult to remove it surgically or to treat it with radiation. Thus, finding more targeted therapeutic targets through in-depth research on the pathogenesis of ESCC is an urgent problem to be solved in clinical practice (6)(7)(8). In addition, the molecular mechanisms related to the pathogenesis and invasion of ESCC remain to be further explored (9).
Tumor invasion and metastasis are the main cause of death in cancer patients (10). Therefore, the mechanism of tumor invasion and metastasis is one of the hot issues in current research (11). Epithelial-mesenchymal transition (EMT) is one of the major factors in tumor cell invasion and metastasis (12). EMT is mainly characterized by the deletion of epidermal phenotype and acquisition of interstitial phenotype (13). EMT can not only enhance the proliferation and migration abilities of tumor cells, but also interact with the tumor microenvironment. EMT induces local tissue remodeling and imparts stem cell properties on tumor cells. Therefore, inhibiting the occurrence of EMT may be a new direction of tumor therapy (14).
Transgelin is a 22 kDa protein that is widely found in smooth muscle tissues (15). Transgelin is an actin gelatin that is sensitive to changes in transformation, mutation, and shape (16). However, few studies have investigated its function. Studies have shown that Transgelin gene expression is reduced or deleted in various tumors. In addition, some studies have found that Transgelin can also be used as signal molecules to participate in cell growth and extracellular matrix degradation and is expressed in various tumor tissues to varying degrees.
Transgelin is closely related to the occurrence, development, and invasion of tumors (17)(18)(19). Recent studies have also shown that Transgelin plays an important role in tumor evolution (20). However, few study have investigated the mechanism of Transgelin involvement in ESCC invasion and metastasis through EMT.
The purpose of this study was to observe the effect of Transgelin on the invasion, metastasis, and proliferation of ESCC cell lines (Eca-109 and KYSE-150) by regulating their expression of Transgelin. Meanwhile, the effects of Transgelin on the expression of E-cadherin and Vimentin were observed to investigate the role and molecular mechanism of Transgelin in the occurrence, invasion, and metastasis of EMT in ESCC. This study aims to provide new ideas and effective approaches for the diagnosis and treatment of ESCC and to find new potential therapeutic targets.

Collection and Information of Clinical Samples
The pathological specimens of 74 ESCC patients were obtained from Tianjin Medical University General Hospital. Each specimen included esophageal carcinoma and adjacent normal tissue (distance from cancer tissue >5 cm). None of the patients received chemotherapy, radiotherapy, or other related antitumor therapies before surgery. The final follow-up time was December 31, 2020. In this study, The pathological types of LGIN, HGIN, and esophageal squamous cell carcinoma in the present study were determined by independent diagnosis by two pathologists (21,22). The tumor stage and grade classification were based on the 8th American Joint Committee on Cancer (AJCC). This research was in accordance with the Helsinki Declaration of the World Medical Association. Every patient signed an informed consent form. The experiment was approved by the Ethics Committee of Tianjin Medical University General Hospital. The clinicopathological characteristics of patients are shown in Table 1.

Cell Transfection
Eca-109 and KYSE-150 cells were seeded into 6-well plates at 5 × 10 5 cells/well for transfection. The culture density was 50%-60%. According to the instructions of Lipofectamine 2000 (Life Technologies, Rockville, MD, USA), two different siRNAs against Transgelin and corresponding negative control siRNAs were transfected into Eca-109 and KYSE-150 cells. The final transfection concentration was 100 nmol/L. After 6 h of culture in serum-free medium, the culture was transferred to 10% serum medium for 24 h. Cells were collected for subsequent experiments. Transgelin-overexpressing Transgelin plasmids (Transgelin cDNA ORF Clone, Human, pCMV3-N-HA as carrier, SinoBiological, China) and no-load control plasmids were transfected with the same method. The final transfection concentration was 1mg/L. The experimental cells were divided into the following groups: Vector-NC (empty plasmid), Transgelin (Transgelin-overexpressing plasmid), si-NC (negative control siRNA), and si-Transgelin (Transgelin siRNAs). All cell phenotype experiments are completed within 48 hours after the completion of transfection.

Detection of Cell Activity by CCK-8 Assay
The transfected cells in the growth phase were taken. Cells were seeded into 96-well plates with 5000 cells per well. After conventional culture for 24, 48, and 72 h, 10 mL of CCK-8 solutions (Beyotime, Shanghai, China) was added to each well. After incubation at 37°C for 1 h, the absorbance (A) value of each well at 450 nm was detected with a microplate analyzer (Multiskan EX, Lab systems, Helsinki, Finland), and the cell viability was calculated.

Cell Scratch Test
Before the wound scratch, incubate the cells with serum-free medium overnight to inhibit cell proliferation on migration.
After digestion, the cells were collected and seeded into 6-well plates with 2 × 10 5 cells/well for culture. When the fusion degree of cell culture reached 100%, the cell center was scratched linearly. Next, 1× phosphate buffer solution (PBS) was used to wash the cell suspension. Serum-free DMEM (Life Technologies) was added to each well. The cells were cultured at 37°C in a cell incubator under 95% relative humidity and 5% CO 2 . Changes of cell migration were observed at 0, 12, 24, and 36 h. Each experiment was repeated three times.

Transwell Invasion Test
Matrigel (BD, Biosciences, Franklin Lakes, NJ, USA) was dissolved overnight at 4°C and diluted in complete medium (1:3). About 50 mL of the mixture was added to the upper and lower compartment of each 24-well plate (Millipore, Billerica, MA, USA). Then, the samples were placed into a 37°C cell incubator and set for 30 min to coagulate. Logarithmic growth cells were digested and collected. About 2 × 10 4 cells were washed in 200 mL of serum-free medium. Resuspended cells were added to the upper compartment. About 600 mL of complete medium was added to the lower chamber. The cell culture plates were cultured at 37°C in an incubator under 95% relative humidity and 5% CO 2 . After 24 h, Matrigel and cells in the upper compartment were removed. The submembrane compartment surface was stained with crystal violet and photographed for cell counting. Nikon (Japan) frontal microscope was used to randomly select 10 fields to count the number of cells, and the average value was obtained. Each experiment was repeated three times.

Detection of the Migration Ability of ESCC Cells in Each Group by Transwell Migration Assay
Unlike the invasion assay, the Transwell migration assay did not require Matrigel-embedded chambers for 1 h before cell inoculation. The remaining steps are the same as those in the Transwell invasion experiment.

Western Blot
The total cell protein was extracted and quantified by BCA method. The sample quantity was 40 mg. The sample was separated by 10% SDS-PAGE. The membrane was transferred to the PVDF membrane by using the wet method (1 kDa/min), and the primary antibody was added after 1 h with TBST sealant containing 5% skim milk powder. Primary antibody was added and incubated overnight at 4°C. Anti-TAGLN/Transgelin The semiquantitative analysis of the absorbance of the strips was carried out with ImageJ software. The ratio of the absorbance value of the target protein band to the absorbance value of the internal reference GAPDH and b-actin protein band was used to represent the relative amount of the target protein.

Immunohistochemical Staining
All specimens were fixed in 10% neutral formaldehyde. Paraffin embedded, 4 mm continuous section. Xylene dewaxing, gradient ethanol hydration. Antigen thermal repair with 0.01 mol/L sodium citrate buffer. Endogenous peroxidase was sealed at 37°C for 20 min. Primary antibody (Transgelin, 1:200, Abcam; CD31, 1:1000, Abcam) was added and incubated overnight at 4°C. The secondary antibody was added and incubated at 37°C for 30 min. Horseradish peroxidase was added and incubated at 37°C for 20 min. DAB staining, hematoxylin redyeing, gradient ethanol dehydration. Xylene transparent, neutral gum sealed sheet, light microscope detection and photography. The staining results were graded according to the staining intensity and percentage of positive cells. The percentages of positive cells <5%, 5%-25%, 26%-50%, 51%-75%, and >75% were 0, 1, 2, 3, and 4, respectively. The cell staining intensity was scored as follows: 0 for non-staining, 1 for light yellow, 2 for brownish yellow, and 3 for yellowish brown. The positive intensity is the product of two scores: 0-2 is negative, 3-5 is weakly positive, 6-8 is positive, and 9-12 is strongly positive. The results were determined by two pathologists, and the average value was taken.

CancerSEA Analysis
CancerSEA (http://biocc.hrbmu.edu.cn/CancerSEA/home.jsp) is a comprehensive database that provides the single-cell functional states of cancer cells at different sites. In this study, we investigated the correlation between Transgelin gene and different functional states of tumors and different cancer types through this website. The average correlation between Transgelin and functional status in different cancers, including invasion, differentiation, metastasis, cell cycle, stemness, and proliferation, was analyzed through this website.

Transgelin Survival Analysis
The data were constructed using the Kaplan-Meier Plotter (https:// kmplot.com/analysis/) database based on the GEO, EGA, and TCGA public database of gene chip and RNA-seq. The effects of 54,675 genes on survival in 21 types of cancer were assessed. The Kaplan-Meier Plotter database integrates gene expression information and clinical prognostic value for meta-analysis and survival-related molecular marker research, discovery, and validation. This website was used to analyze the prognostic value of Transgelin in different tumor types. In the analysis, the Kaplan-Meier Plotter database divided patients into two groups on the basis of different quantiles of Transgelin expression. The Kaplan-Meier survival chart was used to compare two cohorts and calculate the HR, 95% CI, and log rank P values.

Protein-Protein Interaction (PPI) Network Analysis
The study of the interaction network between proteins is helpful to mine the core regulatory genes. In this study, STRING (https://string-db.org/) was used to analyze the PPI network relationship of Transgelin interacting proteins. The STRING database searches for known and predicted interactions between proteins. The database can be applied to 2031 species, containing 9.6 million proteins and 13.8 million protein interactions. In addition to experimental data, mined results from PubMed abstracts, and integrated data from other databases, the STRING database also contains the predicted results using bioinformatics methods.

LinkedOmics Analysis
In this study, the LinkedOmics database was used to analyze the co-expressed genes of Transgelin and their participation in GO and KEGG enrichment analysis. Login LinkedOmics Database (http://linkedomics.org/login.php), the first to register to obtain access to the database. Then, screening conditions were selected successively in the database retrieval interface, and data related to Transgelin gene expression level and prognosis in ESCC were downloaded.

Statistical Analysis
GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis. The measurement data were expressed as mean ± standard deviation. Two-tailed unpaired t-test was used for the comparison between two groups. One-way ANOVA was used for the comparison between multiple groups, and Tukey's test was used for further pairwise comparison. The Pearson correlation coefficient was used for correlation analysis. The Kaplan-Meier method was used to calculate the relationship between Transgelin expression and survival prognosis of tumor patients. Statistical significance was considered at P < 0.05.

Expression of Transgelin Was Low in ESCC, and Its Expression Level Was Positively Correlated With the Prognosis of ESCC
We analyzed the average correlation between Transgelin and functional status, including invasion, differentiation, metastasis, cell cycle, stemness, and proliferation, in different cancers through the CancerSEA database. The analysis results show that the expression level of Transgelin is negatively correlated with invasion, differentiation, metastasis, cell cycle, stemness, and proliferation ( Figures 1A, B). These results indicated that the high expression of Transgelin could reduce the invasion and metastasis of tumors. Furthermore, we analyzed the distribution of Transgelin expression in tumors and the t-SNE diagram of all individual cells, in which the color represented the expression level of the input gene. The analytical results showed that cells  with low Transgelin expression were clustered together ( Figure 1C). The expression results of Transgelin in ESCC and paracancerous tissues in the GEPIA Database (http://gepia. cancer-pku.cn/index.html) are shown in Figure 1D. The results showed that the expression level of Transgelin in ESCC tissues was lower than that in normal esophageal tissues in the dataset of ESCC collected by TCGA (P < 0.05). Through the above database mining information, we found that the expression of Transgelin was decreased in ESCC tissues. To further clarify the relationship between Transgelin expression and prognosis of ESCC, we analyzed the correlation between Transgelin expression and prognosis in patients with ESCC in the Kaplan-Meier Plotter database. Kaplan-Meier analysis showed that the expression level of Transgelin gene was correlated with the survival prognosis of ESCC patients. Patients with high Transgelin expression had a lower overall mortality, while patients with low Transgelin expression had poorer prognosis, and the difference was statistically significant ( Figure 1E, P < 0.05). Prognostic analysis of Transgelin in breast cancer, liver hepatocellular carcinoma, and sarcoma also showed that patients with high Transgelin expression had a long survival time (Supplementary Figure 1).

Overexpression of Transgelin Inhibits the Proliferation, Migration, and Invasion of ESCC Cells
The qRT-PCR results showed that Transgelin mRNA expression level in Eca-109 and KYSE-150 cells transfected with Transgelinoverexpressing plasmid was significantly higher than that in the empty plasmid group, and the difference was statistically significant (  The results showed that Transgelin inhibition significantly reduced the expression of E-cadherin in Eca-109 and KYSE-150 cells. By contrast, the expression level of Vimentin was significantly increased (Figures 5A, B). Furthermore, the protein   Figure S4).

Expression of Transgelin Decreased Gradually With the Progression of ESCC
The expression of Transgelin in different genders, ages, invasion ranges, and clinical stages is shown in Table 1. Statistical analysis showed that the expression of Transgelin in ESCC was not significantly correlated with age, gender, and invasion range of patients (P > 0.05), but was significantly correlated with lymph node metastasis, distant metastasis, and clinical stage ( Table 1, P < 0.05). We further detected the expression changes of Transgelin in normal esophageal tissues, LGIN, HGIN, and tumor by immunohistochemistry ( Figure 6A). The results  showed that the expression of TGLN expression is lower in tumors compared to normal, LGIN and HGIN tissues. The expression level of Transgelin protein in these four tissues showed a continuous declining trend. However, no statistically significant difference was observed in the expression of LGIN in normal esophageal mucosa tissues ( Figure 6B, P > 0.05). To further clarify the relationship between Transgelin expression and prognosis of ESCC, we followed up the patients using the information collected by the research team. Kaplan-Meier analysis showed that the expression level of Transgelin gene was correlated with the survival prognosis of ESCC patients. Patients with high Transgelin expression had a longer overall survival, whereas those with low Transgelin expression had a poorer prognosis, and the difference was statistically significant ( Figure 6C, P < 0.05).

Transgelin Co-Expressed Genes and GO and KEGG Analysis
In this study, the public dataset from LinkedOmics database was used to further analyze the expression of Transgelin gene in ESCC and its clinical significance. First, we analyzed the co-expressed genes of Transgelin. Figure 7A shows a volcanic map information of Transgelin co-expressed genes. Figures 7B, C show heat maps of genes positively and negatively correlated with Transgelin expression, respectively. Through further analysis, we found that Transgelin co-expression was correlated with CNN1, DACT3, MRVI1, and PRELP ( Figure 7D). The results of Transgelin interaction protein network analysis showed that Transgelin was at the center of the interaction. This finding indicates that Transgelin plays an important role (Supplementary Figure 1). Furthermore, we used GO and KEGG analysis functions in LinkedOmics database to enrich Transgelin co-expressed genes.
The results of GO enrichment analysis showed that the enriched biological processes mainly included developmental process, metabolic process, cell proliferation, and cell communication. Moreover, the affected molecular functions mainly included protein binding, ion binding, nucleotide binding, transporter activity, and transferase activity ( Figures 7E-G). In addition, the pathways affected by Transgelin co-expressed genes mainly included the cGMP-PKG signaling pathway, Wnt signaling pathway, and TGF-b signaling pathway ( Figure 7H).

DISCUSSION
Early invasion and metastasis of cancer cells are one of the main causes of death in most patients with ESCC (23). Therefore, investigating the invasion and metastasis mechanism of ESCC is important for its treatment and prognosis (24). Transgelin, also known as SM-22 alpha, is a conserved protein that is mainly expressed in smooth muscles (25,26). Transgelin expression is decreased in lung and breast cancer (27,28). Shields et al. (29) showed that the changes of Transgelin might be more sensitive than CEA in the early malignant changes of colorectal mucosa. They found that the decreased expression of Transgelin was also associated with the activation In this study, the expression of Transgelin in cancer tissues, LGIN, HGIN, and normal esophageal mucosa tissues was further detected by immunohistochemistry. The results showed that the expression of Transgelin protein in ESCC tissues was lower than that in LGIN, HGIN, and normal esophageal mucosa tissues (P < 0.05). This finding suggested that Transgelin may play a role in inhibiting the occurrence of ESCC, and detecting its expression level may provide reference for the early diagnosis of ESCC. Transgelin is associated not only with tumor genesis, but also with tumor invasion and metastasis potential. In this study, the relationship between Transgelin expression and clinical pathological data of patients was analyzed ( Table 1). The results showed that the expression of Transgelin in ESCC was not significantly correlated with the gender, degree of tumor differentiation, tumor location, and tumor diameter of patients (P > 0.05). However, it was significantly correlated with age, T grade, lymphatic, invasion, and AJCC stage (P < 0.05). This finding suggested that Transgelin may play an important role in the invasion and metastasis of ESCC. The detection of Transgelin expression level in ESCC tissues may have certain reference value for malignant degree evaluation and prognosis judgment of ESCC.
It was found that TAGLN expression was significantly reduced in bladder, breast, and renal cell carcinoma tissues compared with matched normal tissues (32,33). In this study, Transgelin was found to be less expressed in esophageal squamous cell carcinoma tissues compared with adjacent normal tissues. Studies have shown that Transgelin is regulated by TGF-b/Smad3. The low expression of Transgelin may be caused by the low expression of TGF-b under the effect of esophageal squamous cell carcinoma tumor microenvironment. Fukuchi et al. found that low expression of TGF-b was an adverse prognostic factor in patients with esophageal squamous cell carcinoma (34). This result also supports our hypothesis and conclusion. In addition, activation of Ras-MEK-ERK-MYC signaling pathway can antagonize TGFb and inhibit the expression of Transgelin (29). Ras-MEK-ERK-MYC signaling pathway was highly expressed in esophageal squamous cell carcinoma (35,36). This may also be the reason for the low expression of Transgelin in esophageal squamous cell carcinoma.
In this study, we found that Transgelin could inhibit the invasion and metastasis of ESCC by regulating the occurrence of EMT in cell experiments. EMT is the transformation of polar epithelial cells into cells that can move freely between the stroma of cells. In this process, cells acquire the ability to infiltrate and migrate (37). EMT is one of the main mechanisms of tumor invasion and metastasis. The disappearance of the polarity of the above skin cells and the acquisition of interstitial characteristics are important features (38). E-cadherin is a commonly used epithelial marker, and Vimentin is a stromal marker. We used qRT-PCR and Western blot to detect the expression of EMT markers in ESCC cells and analyzed the correlation between Transgelin and EMT markers. The results showed that the overexpression of Transgelin in ESCC cells upregulated the expression of E-cadherin. Meanwhile, the expression of Vimentin was inhibited. The results of this study further showed that the downregulation of Transgelin expression was not related to the location of ESCC, but was significantly related to the stage and grade of tumor. The lower the tumor grade, the weaker the expression intensity, which is also similar to the results of previous studies (39,40). Transgelin may be involved in cell differentiation by binding to actin to stabilize the cytoskeleton. Yang (43). In prostate cancer cells, Transgelin inhibits the binding of androgen receptor coactivators to androgen receptors. This in turn inhibits the proliferation and migration of prostate cancer cells (41). In bladder cancer, TAGLN affects cell colony formation, cell migration, and invasion by regulating invadopodia formation and epithelialmesenchymal transformation (30). In bladder cancer, TAGLN can affect the upregulation of TGF-b-stimulated Slug and MMP14, and thus play a role in regulating EMT (30). In this study, through bioinformatics analysis, it was found that Transgelin had a significant positive co-expression correlation with CNN1 ( Figure 7D). CNN1 has been reported to inhibit EMT. Liu et al. found that CNN1 regulates the DKK1/Wnt/b−catenin/c−myc signaling pathway by activating TIMP2 to inhibit the invasion, migration and EMT (44).
Esophageal squamous cell carcinoma is still a complex disease of the digestive system. Current treatments are limited and the prognosis is poor. Therefore, it is necessary to conduct further studies on the occurrence, development and metastasis of esophageal squamous cell carcinoma. Transgelin is mainly involved in the remodeling of the actin skeleton. The biological function of Transgelin in tumors is controversial. The role of Transgelin in esophageal squamous cell carcinoma has not been reported. Especially, the expression pattern, function and mechanism of TAGLN in esophageal squamous cell carcinoma are not clear. We found that TAGLN was low expressed in esophageal squamous cell carcinoma and was associated with prognostic characteristics. Transgelin expression was also associated with stage, grade, and overall survival of esophageal squamous cell carcinoma. We found that TAGLN inhibited the proliferation, invasion and migration of esophageal squamous cell carcinoma cells by inhibiting EMT. Our study shows that TAGLN can inhibit the malignant progression of esophageal squamous cell carcinoma. Transgelin may be a promising biomarker and a new target for follow-up therapy.

CONCLUSION
Transgelin plays an important role in inhibiting the occurrence, invasion, and metastasis of ESCC. It can provide important reference for early diagnosis, molecular targeted therapy, and prognosis judgment of ESCC. The occurrence of EMT is a complex process, in which multiple genes, proteins, and microenvironment interact together. Transgelin may inhibit the invasion and metastasis of ESCC by regulating EMT.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
WZ and BW designed the study. BY, WZ, QC, CW, SS, LZ, and ZZ performed the experiments and analyzed the data. BY, QC, and WZ performed the data analysis. BY, QC, and WZ wrote the initial draft of the paper, with contributions from all authors. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
The authors would like to thank the National Natural Science