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. 2022 Jan 31:12:678025.
doi: 10.3389/fonc.2022.678025. eCollection 2022.

ASF1B: A Possible Prognostic Marker, Therapeutic Target, and Predictor of Immunotherapy in Male Thyroid Carcinoma

Weigang Qiu  1 , Xinquan Wu  1 , Haihong Shi  1 , Bingyang Liu  1 , Liqiong Li  1 , Wenyi Wu  1 , Jianqing Lin  1
Affiliations

ASF1B: A Possible Prognostic Marker, Therapeutic Target, and Predictor of Immunotherapy in Male Thyroid Carcinoma

Weigang Qiu et al. Front Oncol. .

Abstract

Background: Thyroid carcinoma (TC) is the most common malignant endocrine tumor worldwide. Several studies have documented that male patients with TC have a higher rate of metastasis and disease recurrence than female patients. However, the mechanism underlying this observation is not completely clear. The goal of our research was to investigate the potential key candidate genes and pathways related to TC progression in male patients at the molecular level.

Methods: A total of 320 samples were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Hub genes were screened out using weighted gene coexpression network analysis (WGCNA) and a protein-protein interaction (PPI) network analysis. Survival analysis was used to identify hub genes associated with disease-free survival (DFS) rates. Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression (ESTIMATE) data were used to assess the relationship between hub genes and immune cell infiltration. The molecular mechanism and biological functions of hub genes were explored using RT-qPCR, Western blot, Cell Counting Kit-8 Assay, flow cytometry, Transwell assays, and scratch assays.

Results: Forty-seven hub genes were identified, and the survival analysis demonstrated that anti-silencing function 1B (ASF1B) was the sole independent risk factor for poor DFS in male TC patients. Possible associations between the results from the ESTIMATE analysis showed that the ASF1B expression level was related to the ESTIMATE score, immune score, and T-cell regulatory (Treg) infiltration level. Through in vitro cell function experiments, we verified that knockdown of ASF1B inhibited KTC-1 cell proliferation, promoted cell apoptosis, and blocked cell cycle. The silencing of ASF1B reduced protein kinase B (AKT), phospho-AKT (p-AKT), and forkhead box p3 (FOXP3) in KTC-1 cells. Moreover, FOXP3 overexpression markedly restored the cell migration, invasion, and proliferation abilities repressed by ASF1B knockdown.

Conclusions: Our results indicate that ASF1B can be considered a prognostic marker, therapeutic target, and predictor of immunotherapy response in male thyroid cancer patients. However, further in-depth studies are required to validate this finding.

Keywords: ASF1B; FOXP3; Treg; hub genes; male thyroid cancer; tumor immunity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Flowchart of the study process.
Figure 2
Figure 2
Volcano plots of differentially expressed genes (DEGs) between tumor tissues and normal tissues.
Figure 3
Figure 3
Heatmaps of differentially expressed genes. The top 20 dysregulated (10 upregulated and 10 downregulated) DEGs and ASF1B are shown in a heatmap.
Figure 4
Figure 4
Sample clustering indicated that no outliers were present that required removal from the subsequent analysis.
Figure 5
Figure 5
Heatmap of module-trait relationships. Each row corresponds to a module, and each column corresponds to a trait. The progressively increase in blue and red saturation indicated a high Pearson’s correlation coefficient. The numbers in the squares indicate Pearson’s correlation coefficients.
Figure 6
Figure 6
Network of the 47 hub genes and the 13 modules containing these hub genes. 47 hub genes: ACTA1, ACTC1, ACTN2, ACTN3, AMBP, ART3, ASF1B, ATP2B2, B3GNT3, BUB1, CADM2, CEP55, CKM, CRX, CXCL10, CXCR5, DLGAP5, DPP6, FGF10, FOXP3, GABRB2, HPGDS, IL4I1, IQGAP3, KCNAB3, KCNQ3, KIF20A, KIF4A, LAMB3, LINGO2, MELK, MKI67, MRAP, MYBPC1, MYH7, MYL2, NGEF, PBK, RBFOX3, SCN10A, SEMA3D, SKA1, SLC1A7, TMPRSS4, TNNT3, TPX2, XPNPEP2.
Figure 7
Figure 7
Kaplan-Meier curve and receiver operating characteristic (ROC) curve analysis of disease-free survival (DFS) in male thyroid carcinoma patients based on ASF1B expression levels. (A) Patients were divided into low- and high-expression groups according to the median value of ASF1B expression. (B) Patients were divided into low- and high-expression groups according to the best cutoff points (2.96) determined by the ROC curve analysis. (C) Area under the ROC curve (AUC) of ASF1B expression in male thyroid carcinoma patient tumors.
Figure 8
Figure 8
The association between the ASF1B expression level and immune cell infiltration. Relationship between the ESTIMATE score and ASF1B expression level (A), between the immune score and ASF1B expression level (B), and between T regulatory cells (Tregs) and the ASF1B expression level (C).
Figure 9
Figure 9
Violin plot showing that ASFB1 expression was higher in male TC patient tumor tissues than in normal thyroid tissues (independent samples t-test, ****P<0.01).
Figure 10
Figure 10
ASF1B could promote proliferation and inhibit apoptosis of male TC cells and accelerate cell cycle. (A) The knockdown efficiency of ASFB1 in KTC-1 cells was measured by real-time PCR. sh-ASF1B-3 (sh3) showed the best silencing effect and was used for the subsequent experiments, *P < 0.05. (B) Cell proliferation were detected by CCK-8 assays in KTC-1 cells, *P < 0.05. Detection of cell cycle (C) and cell apoptosis (D) in KTC-1 cells after transfection with sh-NC or sh-ASF1B by flow cytometry, respectively, *P < 0.05.
Figure 11
Figure 11
Knockdown of ASF1B inhibits male TC cell metastasis in intro. (A) Cell invasiveness was determined by Transwell assay in KTC-1 cells after transfection with sh-NC or sh-ASF1B, *P < 0.05. (B) Cell migration activity was performed by wound healing assay in KTC-1 cells after transfection with sh-NC or ASF1B, *P < 0.05.
Figure 12
Figure 12
ASF1B targets p-AKT, AKT, and FOXP3, and rescue assays confirm that FOXP3 is an important downstream effector of ASF1B. Western blot analyses (A) and relative protein level test (B) indicated that ASF1B silencing decreased p-AKT, AKT, and FOXP3 protein levels, *p < 0.05. (C) RT-qPCR confirmed the overexpression of FOXP3 in FOXP3 overexpressing plasmid-transfected group, *p < 0.05. (D) Cell proliferation was detected by CCK-8 assays for three separate treatments, *p < 0.05. (D) The effect of FOXP3 overexpression on in ASF1B-silenced KTC-1 cell proliferation was determined by CCK-8 assay, *p < 0.05. (F, G) Cell migration activity was performed by wound healing assay in ASF1B-silenced KTC-1 cells after transfection with FOXP3-overexpressing plasmid or empty plasmid, *p < 0.05.
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