# Bioinformatics Analysis IdentifiesASCL1 as the Key Transcription Factor in Hepatocellular Carcinoma Progression

Authors: Hong-yan Zhang; Rui-qing Zong; Fei-xiang Wu; Yi-ran Li
Journal: Disease Markers
 (2023)

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## Abstract

Object. To identify and explore the key transcription factors in hepatocellular carcinoma (HCC) progression. Methods. Differentially transcription factors (DETFs) were identified from differentially expressed genes (DEGs) in GSE62232 and transcription factors. Then, they were analyzed by regulatory networks, prognostic risk model, and overall survival analyses to identify the key DETF. Combined with the regulatory networks and binding site analysis, the target mRNA of key DETF was determined, and its prognostic value in HCC was evaluated by survival, clinical characteristics analyses, and experiments. Finally, the expressions and functions of the key DETF on the DEmRNAs were investigated in HCC cells. Results. Through multiple bioinformatics analyses, ASCL1 was identified as the key DETF, and SLC6A13 was predicted to be its target mRNA with the common binding site of CCAGCAACTGGCC, both downregulated in HCC. In survival analysis, high SLC6A13 was related to better HCC prognosis, and SLC6A13 was differentially expressed in HCC patients with clinical characteristics. Furthermore, cell experiments showed the mRNA expressions of ASCL1 and SLC6A13 were both reduced in HCC, and their overexpressions suppressed the growth, invasion, and migration of HCC cells. Besides, over-ASCL1 could upregulate SLC6A13 expression in HCC cells. Conclusion. This study identifies two suppressor genes in HCC progression, ASCL1 and SLC6A13, and the key transcription factor ASCL1 suppresses HCC progression by targeting SLC6A13 mRNA. They are both potential treatment targets and prognostic biomarkers for HCC patients, which provides new clues for HCC research.


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## Body

## 1. Introduction

Hepatocellular carcinoma (HCC) usually occurs in the liver, including primary and secondary liver cancer [1]. Among them, primary liver cancer contains HCC, intrahepatic cholangiocarcinoma, and mixed liver cancer [2, 3]. In 2020, the World Health Organization (WHO) issued the global cancer ranking data, showing that there were over 900,000 new HCC cases worldwide and 830,000 deaths that year, making it become the sixth largest cancer in the world [4]. The pathogenesis of HCC is complex and multifaceted. Currently, its risk factors contain hepatitis C virus, hepatitis B virus, etc. [5, 6]. Liver transplantation is currently the best treatment for HCC. However, the number of donors available is limited, and it is only available in 30-40% of patients with HCC. Consequently, most patients are only suitable for topical or palliative care [7].Currently, researchers often use microarray technology to explore potential biomarkers of diseases to find better treatment targets [8]. Zhou et al. analyzed the sample data to explore the upregulations of DTL, CDK1, CCNB1, and others in HCC, which might be related to its pathogenesis [9]. Through transcriptome chip analysis, Wang et al. determined that the Wnt signal transduction mechanism mediated by INcTCF7 was related to the tumor proliferation and self-renewal of HCC stem cells [10]. However, these potential biomarkers have not been applied in clinical practice. Consequently, it is still necessary to explore promising molecular markers for HCC patients.Transcription factors (TFs), such asTCF4, RUNX1, HINFP, KDM2B, MAF, and JUN, are potential drivers of various tumors, and they regulate biological activities during tumorigenesis by targeting downstream target genes [11, 12]. Currently, more and more studies have confirmed that TFs combined with downstream target genes can trigger the viral infection of HCC and the growth of tumor cells, which affects the clinical treatment and prognosis of HCC in many aspects. For example, multiple studies have demonstrated that the FOX family can induce the pathogenesis of HCC by activating or inhibiting the expression of various tumor-related molecules [13]. By binding to the CD133 P1 promoter, ikaros inhibits the tumorigenicity and self-renewal capacity of CD133(+), thereby hindering the development of HCC [14]. Accordingly, it is feasible to analyze the mechanism of TFs and downstream target messenger RNAs (mRNAs) on the pathogenesis of HCC.Although studies in recent years have revealed potential targets that affect the occurrence and development of HCC, it is necessary to further analyze the potential key mechanisms affecting the prognosis and survival of HCC. Herein, we plan to combine TFs and microarray data to study the underlying mechanism and potential genes with clinical values in HCC, which further improves the clinical treatment and the prognostic effect for patients.


## 2. Material and Methods

### 2.1. Microarray Data

The GSE62232 microarray dataset was from the GEO database. The dataset had 10 nontumor liver tissues (control group) and 81 HCC tissues (case group), which were used as the basis for follow-up research. We screened the differentially expressed genes (DEGs) in 91 samples through the limma package of the R software, under the premise ofP<0.01, foldchangeFC>2 as the standard for upregulation and FC<0.5 for downregulation. The final results were displayed through the volcano maps. After that, based on the cluster profiler package of the R software, the enrichment of the upregulated and downregulated DEGs in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed, respectively. Under the premise of the threshold P<0.05, the top 15 enrichment items were displayed.


### 2.2. DETFs from TFs and DEGs in the GSE62232 Dataset

TFs are able to connect with specific nucleotide sequences in an upstream gene as proteins, and these proteins can regulate the transcription of its downstream genes. This time, we determined the overlapping genes of DEGs and TFs as DETFs in GSE62232 and identified the potential mechanism in HCC. Then, the coexpressed networks for these DETFs were constructed.


### 2.3. Least Absolute Shrinkage and Selection Operator (LASSO) Regression Analysis

To investigate the impact of key TFs on the prognosis of HCC tumor samples, we constructed a prognostic model through LASSO regression to draw the relationship between partial likelihood deviation andlogλ. 370 HCC tumor samples from The Cancer Genome Atlas (TCGA) database were evaluated by risk score and divided into 185 high-risk and 185 low-risk groups. The survival status in each sample and the expression levels of DETFs were also demonstrated. Then, the survival difference between different groups was compared based on the log-rank test Kaplan-Meier (KM) survival analysis; the hazard ratio (HR) was calculated. Next, the receiver operating characteristic (ROC) analysis was performed to judge the accuracy of the prediction model. Among them, the larger the Area Under Curve (AUC) value, the smaller the log-rank P value, indicating better prediction result.


### 2.4. The Overall Survival (OS) Analysis on the DETFs

To explore the effect of different expression levels of DETFs on the probability of OS in HCC patients, we downloaded HCC samples from TCGA database, plotted the relevant KM survival curves, and used log-rank to calculate the relevantP value. The results with statistical significance were demonstrated.


### 2.5. DEG Identification in TCGA-HCC Samples with High and LowASCL1 Expression

Based on the above findings and previous research,ASCL1 was identified as the research object for the following analysis. Then, the upregulated and downregulated DEGs in TCGA-HCC samples with high and low ASCL1 expression were screened through the limma package of the R software, under the premise of P<0.01, FC>1.3 as the standard for upregulation and FC<0.77 for downregulation. The final results were displayed through the volcano and heatmaps. Moreover, these DEGs were analyzed by KEGG pathway analysis to explore their biological functions.


### 2.6. JASPAR and Ensembl Genome Databases

Combining the regulatory networks, we speculated thatSLC6A13 was its target mRNA from the coexpressed genes in the regulatory network of ASCL1. Then, whether ASCL1 had a binding site on the SLC6A13 promoter was investigated with the help of the JASPAR and Ensembl genome databases. Next, the Spearman correlation analysis on ASCL1 and SLC6A13, and the expressions of ASCL1 and SLC6A13 in HCC normal and tumor tissues were performed through TCGA database.


### 2.7. Survival Analysis on the Key Target mRNA in HCC

To study the relation betweenSLC6A13 and HCC prognosis, based on the HCC samples in TCGA, we verified the effect of different SLC6A13 expressions on the patients’ OS, Progression-Free Survival (PFS), Relapsed-Free Survival (RFS), and Disease-Specific Survival (DSS) and computed the relevant AUC values through ROC curve prediction.


### 2.8. The Relation Analysis betweenSLC6A13 and HCC Clinical Characteristics

The normal and tumor tissues of HCC were downloaded from TCGA database, and Kruskal-Wallis one-way ANOVA was applied to analyze the levels ofSLC6A13 in patients with different clinical parameters, including T, N, M, and G stages, histological subtypes, and TP53 mutation status.


### 2.9. Cell Culture and Transfection

Human liver normal cells (L-02) and 3 HCC cell lines (Huh7, SNU-387, and MHCC-97H) were purchased from Shanghai Institutes for Biological Science, China, and then placed in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS). Next, 100μ/ml of penicillin and 100 μg/ml of streptomycin (Gibco) were added to the medium and maintained in an environment containing 5% CO2 and 37°C. Subsequently, vectors for overexpressing ASCL1 and SLC6A13 were obtained from Gene-Pharma Co., Ltd. (Shanghai, China), with overnormal cells (NC) as a control. Lipofectamine 2000 was used to transfect over-ASCL1 and over-SLC6A13 into HCC cells.


### 2.10. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay

Total RNA was extracted from cells based on Trizol reagent, followed by reverse transcription of RNA into complementary DNA (cDNA) using PrimeScript™ RT Master Mix (TAKARA, Dalian. China). qRT-PCR was conducted by FastStart Universal SYBR-Green Master Mix, and the relative mRNA and protein levels ofASCL1 and SLC6A13 were detected by 2–ΔΔCt.


### 2.11. Cell Proliferation

In the cell proliferation experiment, the transfected cells were first added to a 96-well plate, and 10μl of Cell Counting Kit-8 (CCK-8) solution was dropped into each well plate and cultured at 37°C in a 5% CO2 environment for a period of time (0 h, 24 h, 48 h, 72 h, and 96 h). After that, the optical density (OD) values of cells at 450 nm were detected by a microplate reader at different time periods.


### 2.12. Cell Migration and Invasion

A certain amount of culture medium was added to the upper and lower chambers of the Transwell, the transfected cells were placed in the upper chamber, and the lower chamber was placed in RPMI-1640 medium containing 10% FBS. For cell migration experiments, no Matrigel was coated to the bottom, while invasion experiments were performed with Matrigel (BD, USA). The upper excess cells were removed by cotton swab, and the left cells were fixed with methanol for 10 minutes and stained with DAPI. Finally, the cell migration and invasion in different fields were observed by fluorescence microscopy.


### 2.13. Statistical Analysis

All data was analyzed using SPSS v.16.0 software, with independent experiments presented as mean standard deviation (SD) and differences between groups examined using Student’st-test. The acquired findings were statistically significant when P<0.05 was used.


## 2.1. Microarray Data

The GSE62232 microarray dataset was from the GEO database. The dataset had 10 nontumor liver tissues (control group) and 81 HCC tissues (case group), which were used as the basis for follow-up research. We screened the differentially expressed genes (DEGs) in 91 samples through the limma package of the R software, under the premise ofP<0.01, foldchangeFC>2 as the standard for upregulation and FC<0.5 for downregulation. The final results were displayed through the volcano maps. After that, based on the cluster profiler package of the R software, the enrichment of the upregulated and downregulated DEGs in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed, respectively. Under the premise of the threshold P<0.05, the top 15 enrichment items were displayed.


## 2.2. DETFs from TFs and DEGs in the GSE62232 Dataset

TFs are able to connect with specific nucleotide sequences in an upstream gene as proteins, and these proteins can regulate the transcription of its downstream genes. This time, we determined the overlapping genes of DEGs and TFs as DETFs in GSE62232 and identified the potential mechanism in HCC. Then, the coexpressed networks for these DETFs were constructed.


## 2.3. Least Absolute Shrinkage and Selection Operator (LASSO) Regression Analysis

To investigate the impact of key TFs on the prognosis of HCC tumor samples, we constructed a prognostic model through LASSO regression to draw the relationship between partial likelihood deviation andlogλ. 370 HCC tumor samples from The Cancer Genome Atlas (TCGA) database were evaluated by risk score and divided into 185 high-risk and 185 low-risk groups. The survival status in each sample and the expression levels of DETFs were also demonstrated. Then, the survival difference between different groups was compared based on the log-rank test Kaplan-Meier (KM) survival analysis; the hazard ratio (HR) was calculated. Next, the receiver operating characteristic (ROC) analysis was performed to judge the accuracy of the prediction model. Among them, the larger the Area Under Curve (AUC) value, the smaller the log-rank P value, indicating better prediction result.


## 2.4. The Overall Survival (OS) Analysis on the DETFs

To explore the effect of different expression levels of DETFs on the probability of OS in HCC patients, we downloaded HCC samples from TCGA database, plotted the relevant KM survival curves, and used log-rank to calculate the relevantP value. The results with statistical significance were demonstrated.


## 2.5. DEG Identification in TCGA-HCC Samples with High and LowASCL1 Expression

Based on the above findings and previous research,ASCL1 was identified as the research object for the following analysis. Then, the upregulated and downregulated DEGs in TCGA-HCC samples with high and low ASCL1 expression were screened through the limma package of the R software, under the premise of P<0.01, FC>1.3 as the standard for upregulation and FC<0.77 for downregulation. The final results were displayed through the volcano and heatmaps. Moreover, these DEGs were analyzed by KEGG pathway analysis to explore their biological functions.


## 2.6. JASPAR and Ensembl Genome Databases

Combining the regulatory networks, we speculated thatSLC6A13 was its target mRNA from the coexpressed genes in the regulatory network of ASCL1. Then, whether ASCL1 had a binding site on the SLC6A13 promoter was investigated with the help of the JASPAR and Ensembl genome databases. Next, the Spearman correlation analysis on ASCL1 and SLC6A13, and the expressions of ASCL1 and SLC6A13 in HCC normal and tumor tissues were performed through TCGA database.


## 2.7. Survival Analysis on the Key Target mRNA in HCC

To study the relation betweenSLC6A13 and HCC prognosis, based on the HCC samples in TCGA, we verified the effect of different SLC6A13 expressions on the patients’ OS, Progression-Free Survival (PFS), Relapsed-Free Survival (RFS), and Disease-Specific Survival (DSS) and computed the relevant AUC values through ROC curve prediction.


## 2.8. The Relation Analysis betweenSLC6A13 and HCC Clinical Characteristics

The normal and tumor tissues of HCC were downloaded from TCGA database, and Kruskal-Wallis one-way ANOVA was applied to analyze the levels ofSLC6A13 in patients with different clinical parameters, including T, N, M, and G stages, histological subtypes, and TP53 mutation status.


## 2.9. Cell Culture and Transfection

Human liver normal cells (L-02) and 3 HCC cell lines (Huh7, SNU-387, and MHCC-97H) were purchased from Shanghai Institutes for Biological Science, China, and then placed in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS). Next, 100μ/ml of penicillin and 100 μg/ml of streptomycin (Gibco) were added to the medium and maintained in an environment containing 5% CO2 and 37°C. Subsequently, vectors for overexpressing ASCL1 and SLC6A13 were obtained from Gene-Pharma Co., Ltd. (Shanghai, China), with overnormal cells (NC) as a control. Lipofectamine 2000 was used to transfect over-ASCL1 and over-SLC6A13 into HCC cells.


## 2.10. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay

Total RNA was extracted from cells based on Trizol reagent, followed by reverse transcription of RNA into complementary DNA (cDNA) using PrimeScript™ RT Master Mix (TAKARA, Dalian. China). qRT-PCR was conducted by FastStart Universal SYBR-Green Master Mix, and the relative mRNA and protein levels ofASCL1 and SLC6A13 were detected by 2–ΔΔCt.


## 2.11. Cell Proliferation

In the cell proliferation experiment, the transfected cells were first added to a 96-well plate, and 10μl of Cell Counting Kit-8 (CCK-8) solution was dropped into each well plate and cultured at 37°C in a 5% CO2 environment for a period of time (0 h, 24 h, 48 h, 72 h, and 96 h). After that, the optical density (OD) values of cells at 450 nm were detected by a microplate reader at different time periods.


## 2.12. Cell Migration and Invasion

A certain amount of culture medium was added to the upper and lower chambers of the Transwell, the transfected cells were placed in the upper chamber, and the lower chamber was placed in RPMI-1640 medium containing 10% FBS. For cell migration experiments, no Matrigel was coated to the bottom, while invasion experiments were performed with Matrigel (BD, USA). The upper excess cells were removed by cotton swab, and the left cells were fixed with methanol for 10 minutes and stained with DAPI. Finally, the cell migration and invasion in different fields were observed by fluorescence microscopy.


## 2.13. Statistical Analysis

All data was analyzed using SPSS v.16.0 software, with independent experiments presented as mean standard deviation (SD) and differences between groups examined using Student’st-test. The acquired findings were statistically significant when P<0.05 was used.


## 3. Results

### 3.1. Identification of DEGs and Regulatory Networks of DETFs

Based on the set screening criteria, we obtained 489 upregulated and 352 downregulated DEGs in total (Figure1(a)). According to the results in Figures 1(b), we could see that the enrichment items of upregulated DEGs in GO included microtubule cytoskeleton organization involved in mitosis, intracellular non-membrane-bounded organelle, kinase binding, DNA replication origin binding, and protein kinase binding. In KEGG, upregulated DEGs were enriched in cell cycle, p53 signaling pathway, and small-cell lung cancer (Figure 1(c)). Moreover, the enrichment items of downregulated DEGs in BP contained cellular amino acid catabolic process, steroid metabolic process, epoxygenase P450 pathway, cellular response to copper ion, mitochondrial matrix, intracellular organelle lumen, membrane attack complex, steroid hydroxylase activity, arachidonic acid epoxygenase activity, etc. (Figure 1(d)). In KEGG, tryptophan metabolism, retinol metabolism, glycine, drug metabolism, serine, and threonine metabolism were related to downregulated DEGs (Figure 1(e)). Based on the overlapping results of TFs and DEGs, we screened a total of 7 DETFs in the GSE62232 dataset, namely, DACH1, FOSB, FOS, ASCL1, EBF1, GTF2H2, and GTF2I (Figure 1(f)). Afterwards, we constructed regulatory networks of these DETFs, respectively, and identified coexpressed genes related to 7 DETFs (Figure 1(g)). Among them, GTF2H2 and ASCL1 had a large number of coexpressed genes. The former included CBFA2T3, CLEC4M, and LPA, and the latter included SLC6A13, SLC19A3, and ARHGEF4.Figure 1
Identification of DEGs and regulatory networks of DETFs. (a) Volcano map of DEGs. (b, c) GO and KEGG analyses on upregulated DEGs. (d, e) GO and KEGG analyses on down-regulated DEGs. (f) Heatmap. Distribution of DETFs in GSE62232 dataset samples. (g) The regulatory networks of 7 DETFs, GTF2H2, DACH1, EBF1, FOS, GTF2I,ASCL1, and FOSB in order.
(a)(b)(c)(d)(e)(f)(g)


### 3.2.ASCL1 Was the Key DETF by Prognostic Risk Model

According to the results in Figures2(a) and 2(b), a total of 6 transcription factors were identified by LASSO regression analysis, namely, DACH1, ASCL1, FOSB, FOS, EBF1, and GTF2H2. We divided HCC samples into the high-risk and low-risk groups and found that the number of surviving patients from the low-risk and high-risk groups gradually decreased, and the expressions of 6 DETFs were also different (Figure 2(c)). In the KM survival curve, we observed the survival rate of the high-risk group (log-rank P=5.02e−06, HR=2.334) was lower than that of the low-risk group (Figure 2(d)). The results of the ROC curve analysis demonstrated the 5-year AUC value of HCC patients was the highest, which was 0.755, indicating that the model had a prediction ability (Figure 2(e)). Based on the KM curves, we analyzed the effect of the 6 DETFs on the probability of OS in HCC patients (Figures 2(f)–2(k)). It was found that HCC patients with high expressions of GTF2H2 (log-rank P=0.011) and DACH1 (log-rank P=0.13) had a poorer OS probability, while those with high expressions of FOSB (log-rank P=0.0024), ASCL1 (log-rank P=0.0029), EBF1 (log-rank P=0.048), and FOS (log-rank P=0.23) had a better probability of OS. Thus, we concluded that the key DEDTF was ASCL1.Figure 2
ASCL1 was the key DETF by prognostic risk model. (a) LASSO coefficient spectrum of transcription factors. (b) The optimal parameter in LASSO regression is 6. (c) Scatter plot. Correlation analysis between risk score and surviving conditions. (d) KM survival curves of high-risk and low-risk groups. (e) ROC curves of 1-, 3-, and 5-year survival rates. OS probability analysis on (f) GTF2H2, (g) FOSB, (h) ASCL1, (i) EBF1, (j) FOS, and (k) DACH1.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


### 3.3. Over-ASCL1 Inhibited the Proliferation, Migration, and Invasion of HCC Cells

Next, it was found through a public database thatASCL1 was downregulated in HCC tumors (Figure 3(a)). To explore the relationship between ASCL1 in HCC cells, we used qRT-PCR to sequentially detect the mRNA levels of ASCL1 in HCC cell lines. The corresponding results showed that ASCL1 was also downexpressed in HCC cell lines, especially SNU-387 and MHCC-97H (Figure 3(b)). In Figure 3(c), ASCL1 were overexpressed in SNU-387 and MHCC-97H for the following study. Through CCK-8 and Transwell experiments, we examined the regulation of over-ASCL1 on HCC cells, respectively. In comparison with the control group, over-ASCL1 significantly suppressed the proliferation, invasion, and migration of SNU-387 and MHCC-97H cells (Figures 3(d)–3(g)). These findings indicated that ASCL1 was a suppressor gene in HCC.Figure 3
Over-ASCL1 inhibited the proliferation, migration and invasion of HCC cells. (a) The expression of ASCL1 in HCC normal and tumor groups. (b) The expression of ASCL1 in HCC cell lines. (c) The overexpression efficiency of ASCL1 mRNA in HCC cells was detected by qRT-PCR. (d, e) The over-ASCL1 regulation of HCC cell proliferation was detected by CCK-8. (f, g) The regulation of HCC cell migration and invasion by over-ASCL1 was detected by Transwell. ∗P<0.05and∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


### 3.4. The Identification of TCGA-DEGs Based onASCL1 Expression in HCC

Further, we screened 237 upregulated and 254 downregulated DEGs from TCGA-HCC samples with high and lowASCL1 expressions, respectively (Figures 4(a) and 4(b)). In KEGG pathway enrichment analysis, the upregulated DEGs were enriched in steroid hormone biosynthesis, PPAR signaling pathway, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, bile secretion, chemical carcinogenesis-DNA adducts, and so on (Figure 4(c)), and the downregulated DEGs were enriched in PI3K-Akt signaling pathway, Coronavirus disease (COVID-19), cytokine-cytokine receptor interaction, bladder cancer, AGE-RAGE signaling pathway in diabetic, complications, proteoglycans in cancer, and others (Figure 4(d)). According the regulatory network of ASCL1, SLC6A13 was speculated to be the target mRNA of ASCL1. Interestingly, SLC6A13 was a downregulated DEGs in TCGA-DEGs with low ASCL1 expression.Figure 4
The identification of TCGA-DEGs based onASCL1 expression in HCC. (a, b) The volcano and heatmaps of TCGA-DEGs from HCC samples with high and low ASCL1 expressions. (c, d) The KEGG pathway analysis on the upregulated and downregulated DEGs.
(a)(b)(c)(d)


### 3.5. The Common Binding Site Was Found betweenASCL1 and SLC6A13

Then, based on the results of JASPAR and Ensembl genome database analysis, the binding site ofASCL1 in SLC6A13 was obtained, CCAGCAACTGGCC, indicating that ASCL1 could regulate the expression of SLC6A13 by binding CCAGCAACTGGCC (Figure 5(a)). Next, we assessed the relation between ASCL1 and SLC6A13 by the Spearman analysis and TCGA database and found that SLC6A13, as a HCC tumor suppressor, was positively related with ASCL1 (Figures 5(b) and 5(c)). This finding was verified in function experiments which showed over-ASCL1 promotes SLC6A13 expression in SNU-387 and MHCC-97H cells (Figures 5(d)).Figure 5
The common binding site was found betweenASCL1 and SLC6A13. (a) The red sequence is the binding site of ASCL1 on the SLC6A13 promoter. (b) The Spearman correlation analysis on ASCL1 and SLC6A13. (c) The expression of SLC6A13 in HCC normal and tumor groups. (d) ASCL1 had a positive relation with SLC6A13. ∗∗∗P<0.001.
(a)(b)(c)(d)


### 3.6.SLC6A13 Could Be a Prognosis Biomarker in HCC Progression

Further, we analyzed the prognostic effect ofSLC6A13 expression on HCC patients through KM curves. Figures 6(a)–6(d) indicate that highly expressed SLC6A13 had a higher probability of OS, PFS, RFS, and DSS. In addition, the AUC value in the ROC curve corresponding to SLC6A13 was 0.694, indicating that this mRNA had a certain predictive value for the prognosis of HCC (Figure 6(e)). Besides, we investigated the expressions of SLC6A13 in different clinical parameters of HCC and found that its expression was significantly different in patients with different clinical characteristics. Among them, SLC6A13 was significantly downregulated after HCC incidence, and its expression levels gradually decreased with the progression of HCC (Figures 6(f)–6(k)).Figure 6
SLC6A13 could be a prognosis biomarker in HCC progression. (a–d) The prognostic value of SLC6A13 in OS, PFS, RFS, and DSS. (e) ROC curve analysis of SLC6A13 in CRC. (f–k) Correlation analysis of the levels of SLC6A13 and clinical parameters of different HCC patients, including T stage, N stage, M stage, G stage, histological subtypes, and TP53 mutation status. ∗P<0.05,∗∗P<0.01,∗∗∗P<0.001,and∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


### 3.7. Over-SLC6A13 Inhibited the Proliferation, Migration, and Invasion of HCC Cells

The functional experiments showed thatSLC6A13 were generally downexpressed in HCC cell lines, especially SNU-387 and MHCC-97H (Figure 7(a)). In Figure 7(b), SLC6A13 was overexpressed in SNU-387 and MHCC-97H cells. In CCK-8, over-SLC6A13 significantly suppressed the cell proliferation (Figures 7(c)–7(d)). In comparison with the control group, over-SLC6A13 significantly suppressed the invasion and migration of SNU-387 and MHCC-97H cells (Figures 7(e) and 7(f)).Figure 7
Over-SLC6A13 inhibited the proliferation, migration, and invasion of HCC cells. (a) The expression of SLC6A13 in HCC cell lines. (b) The overexpression efficiency of SLC6A13 mRNA in HCC cells was detected by qRT-PCR. (c, d) The over-SLC6A13 regulation of HCC cell proliferation was detected by CCK-8. (e, f) The regulation of HCC cell migration and invasion by over-SLC6A13 was detected by Transwell. ∗P<0.05and∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)


## 3.1. Identification of DEGs and Regulatory Networks of DETFs

Based on the set screening criteria, we obtained 489 upregulated and 352 downregulated DEGs in total (Figure1(a)). According to the results in Figures 1(b), we could see that the enrichment items of upregulated DEGs in GO included microtubule cytoskeleton organization involved in mitosis, intracellular non-membrane-bounded organelle, kinase binding, DNA replication origin binding, and protein kinase binding. In KEGG, upregulated DEGs were enriched in cell cycle, p53 signaling pathway, and small-cell lung cancer (Figure 1(c)). Moreover, the enrichment items of downregulated DEGs in BP contained cellular amino acid catabolic process, steroid metabolic process, epoxygenase P450 pathway, cellular response to copper ion, mitochondrial matrix, intracellular organelle lumen, membrane attack complex, steroid hydroxylase activity, arachidonic acid epoxygenase activity, etc. (Figure 1(d)). In KEGG, tryptophan metabolism, retinol metabolism, glycine, drug metabolism, serine, and threonine metabolism were related to downregulated DEGs (Figure 1(e)). Based on the overlapping results of TFs and DEGs, we screened a total of 7 DETFs in the GSE62232 dataset, namely, DACH1, FOSB, FOS, ASCL1, EBF1, GTF2H2, and GTF2I (Figure 1(f)). Afterwards, we constructed regulatory networks of these DETFs, respectively, and identified coexpressed genes related to 7 DETFs (Figure 1(g)). Among them, GTF2H2 and ASCL1 had a large number of coexpressed genes. The former included CBFA2T3, CLEC4M, and LPA, and the latter included SLC6A13, SLC19A3, and ARHGEF4.Figure 1
Identification of DEGs and regulatory networks of DETFs. (a) Volcano map of DEGs. (b, c) GO and KEGG analyses on upregulated DEGs. (d, e) GO and KEGG analyses on down-regulated DEGs. (f) Heatmap. Distribution of DETFs in GSE62232 dataset samples. (g) The regulatory networks of 7 DETFs, GTF2H2, DACH1, EBF1, FOS, GTF2I,ASCL1, and FOSB in order.
(a)(b)(c)(d)(e)(f)(g)


## 3.2.ASCL1 Was the Key DETF by Prognostic Risk Model

According to the results in Figures2(a) and 2(b), a total of 6 transcription factors were identified by LASSO regression analysis, namely, DACH1, ASCL1, FOSB, FOS, EBF1, and GTF2H2. We divided HCC samples into the high-risk and low-risk groups and found that the number of surviving patients from the low-risk and high-risk groups gradually decreased, and the expressions of 6 DETFs were also different (Figure 2(c)). In the KM survival curve, we observed the survival rate of the high-risk group (log-rank P=5.02e−06, HR=2.334) was lower than that of the low-risk group (Figure 2(d)). The results of the ROC curve analysis demonstrated the 5-year AUC value of HCC patients was the highest, which was 0.755, indicating that the model had a prediction ability (Figure 2(e)). Based on the KM curves, we analyzed the effect of the 6 DETFs on the probability of OS in HCC patients (Figures 2(f)–2(k)). It was found that HCC patients with high expressions of GTF2H2 (log-rank P=0.011) and DACH1 (log-rank P=0.13) had a poorer OS probability, while those with high expressions of FOSB (log-rank P=0.0024), ASCL1 (log-rank P=0.0029), EBF1 (log-rank P=0.048), and FOS (log-rank P=0.23) had a better probability of OS. Thus, we concluded that the key DEDTF was ASCL1.Figure 2
ASCL1 was the key DETF by prognostic risk model. (a) LASSO coefficient spectrum of transcription factors. (b) The optimal parameter in LASSO regression is 6. (c) Scatter plot. Correlation analysis between risk score and surviving conditions. (d) KM survival curves of high-risk and low-risk groups. (e) ROC curves of 1-, 3-, and 5-year survival rates. OS probability analysis on (f) GTF2H2, (g) FOSB, (h) ASCL1, (i) EBF1, (j) FOS, and (k) DACH1.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


## 3.3. Over-ASCL1 Inhibited the Proliferation, Migration, and Invasion of HCC Cells

Next, it was found through a public database thatASCL1 was downregulated in HCC tumors (Figure 3(a)). To explore the relationship between ASCL1 in HCC cells, we used qRT-PCR to sequentially detect the mRNA levels of ASCL1 in HCC cell lines. The corresponding results showed that ASCL1 was also downexpressed in HCC cell lines, especially SNU-387 and MHCC-97H (Figure 3(b)). In Figure 3(c), ASCL1 were overexpressed in SNU-387 and MHCC-97H for the following study. Through CCK-8 and Transwell experiments, we examined the regulation of over-ASCL1 on HCC cells, respectively. In comparison with the control group, over-ASCL1 significantly suppressed the proliferation, invasion, and migration of SNU-387 and MHCC-97H cells (Figures 3(d)–3(g)). These findings indicated that ASCL1 was a suppressor gene in HCC.Figure 3
Over-ASCL1 inhibited the proliferation, migration and invasion of HCC cells. (a) The expression of ASCL1 in HCC normal and tumor groups. (b) The expression of ASCL1 in HCC cell lines. (c) The overexpression efficiency of ASCL1 mRNA in HCC cells was detected by qRT-PCR. (d, e) The over-ASCL1 regulation of HCC cell proliferation was detected by CCK-8. (f, g) The regulation of HCC cell migration and invasion by over-ASCL1 was detected by Transwell. ∗P<0.05and∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


## 3.4. The Identification of TCGA-DEGs Based onASCL1 Expression in HCC

Further, we screened 237 upregulated and 254 downregulated DEGs from TCGA-HCC samples with high and lowASCL1 expressions, respectively (Figures 4(a) and 4(b)). In KEGG pathway enrichment analysis, the upregulated DEGs were enriched in steroid hormone biosynthesis, PPAR signaling pathway, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, bile secretion, chemical carcinogenesis-DNA adducts, and so on (Figure 4(c)), and the downregulated DEGs were enriched in PI3K-Akt signaling pathway, Coronavirus disease (COVID-19), cytokine-cytokine receptor interaction, bladder cancer, AGE-RAGE signaling pathway in diabetic, complications, proteoglycans in cancer, and others (Figure 4(d)). According the regulatory network of ASCL1, SLC6A13 was speculated to be the target mRNA of ASCL1. Interestingly, SLC6A13 was a downregulated DEGs in TCGA-DEGs with low ASCL1 expression.Figure 4
The identification of TCGA-DEGs based onASCL1 expression in HCC. (a, b) The volcano and heatmaps of TCGA-DEGs from HCC samples with high and low ASCL1 expressions. (c, d) The KEGG pathway analysis on the upregulated and downregulated DEGs.
(a)(b)(c)(d)


## 3.5. The Common Binding Site Was Found betweenASCL1 and SLC6A13

Then, based on the results of JASPAR and Ensembl genome database analysis, the binding site ofASCL1 in SLC6A13 was obtained, CCAGCAACTGGCC, indicating that ASCL1 could regulate the expression of SLC6A13 by binding CCAGCAACTGGCC (Figure 5(a)). Next, we assessed the relation between ASCL1 and SLC6A13 by the Spearman analysis and TCGA database and found that SLC6A13, as a HCC tumor suppressor, was positively related with ASCL1 (Figures 5(b) and 5(c)). This finding was verified in function experiments which showed over-ASCL1 promotes SLC6A13 expression in SNU-387 and MHCC-97H cells (Figures 5(d)).Figure 5
The common binding site was found betweenASCL1 and SLC6A13. (a) The red sequence is the binding site of ASCL1 on the SLC6A13 promoter. (b) The Spearman correlation analysis on ASCL1 and SLC6A13. (c) The expression of SLC6A13 in HCC normal and tumor groups. (d) ASCL1 had a positive relation with SLC6A13. ∗∗∗P<0.001.
(a)(b)(c)(d)


## 3.6.SLC6A13 Could Be a Prognosis Biomarker in HCC Progression

Further, we analyzed the prognostic effect ofSLC6A13 expression on HCC patients through KM curves. Figures 6(a)–6(d) indicate that highly expressed SLC6A13 had a higher probability of OS, PFS, RFS, and DSS. In addition, the AUC value in the ROC curve corresponding to SLC6A13 was 0.694, indicating that this mRNA had a certain predictive value for the prognosis of HCC (Figure 6(e)). Besides, we investigated the expressions of SLC6A13 in different clinical parameters of HCC and found that its expression was significantly different in patients with different clinical characteristics. Among them, SLC6A13 was significantly downregulated after HCC incidence, and its expression levels gradually decreased with the progression of HCC (Figures 6(f)–6(k)).Figure 6
SLC6A13 could be a prognosis biomarker in HCC progression. (a–d) The prognostic value of SLC6A13 in OS, PFS, RFS, and DSS. (e) ROC curve analysis of SLC6A13 in CRC. (f–k) Correlation analysis of the levels of SLC6A13 and clinical parameters of different HCC patients, including T stage, N stage, M stage, G stage, histological subtypes, and TP53 mutation status. ∗P<0.05,∗∗P<0.01,∗∗∗P<0.001,and∗∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)


## 3.7. Over-SLC6A13 Inhibited the Proliferation, Migration, and Invasion of HCC Cells

The functional experiments showed thatSLC6A13 were generally downexpressed in HCC cell lines, especially SNU-387 and MHCC-97H (Figure 7(a)). In Figure 7(b), SLC6A13 was overexpressed in SNU-387 and MHCC-97H cells. In CCK-8, over-SLC6A13 significantly suppressed the cell proliferation (Figures 7(c)–7(d)). In comparison with the control group, over-SLC6A13 significantly suppressed the invasion and migration of SNU-387 and MHCC-97H cells (Figures 7(e) and 7(f)).Figure 7
Over-SLC6A13 inhibited the proliferation, migration, and invasion of HCC cells. (a) The expression of SLC6A13 in HCC cell lines. (b) The overexpression efficiency of SLC6A13 mRNA in HCC cells was detected by qRT-PCR. (c, d) The over-SLC6A13 regulation of HCC cell proliferation was detected by CCK-8. (e, f) The regulation of HCC cell migration and invasion by over-SLC6A13 was detected by Transwell. ∗P<0.05and∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)


## 4. Discussion

In recent years, the increasing incidence and the mortality rate of HCC have both increased [15]. At present, the main therapies for HCC are liver resection and transplantation, but the prognosis is unsatisfying [16]. Nowadays, the development of bioinformatics and genetics continues to advance, which has further promoted the development of biomarkers and genetic models [17, 18]. These biomarkers and genetic models provide great opportunities for evaluating the prognosis of patients, thereby making the prognosis more rational and individualized [19, 20]. With further in-depth research on HCC, effective prognostic biomarkers for HCC continue to appear, which facilitates the tumor-specific changes related to HCC progression and guides treatment decisions.In this study, we analyzed a total of 489 upregulated and 352 downregulated DEGs from the samples of the GSE62232 dataset. Through enrichment analysis, it was found that these DEGs were mainly enriched in microtubule cytoskeleton organization involved in mitosis, DNA metabolic process, cell cycle, DNA replication, p53 signaling pathway, cellular amino acid catabolic process, epoxygenase P450 pathway, PPAR signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, etc. Among these enrichment items, some have been confirmed to be involved in the occurrence and development of HCC. The cell cycle is a regulatory mechanism in tumor development, and when its regulatory mechanism is disrupted, it can lead to the uncontrolled transformation of normal cell growth into tumor cells [21]. Relevant studies have pointed out that the abnormal regulation mechanism of cell cycle G1 phase is related to the occurrence of HCC, and the cell cycle can affect the development of HCC through the regulation of SPATS2 [22], quercetin [23], MITD1, and other factors. Some reports mentioned that the DNA replication process was associated with mammalian genome stability and carcinogenesis [24]. Liang et al. concluded through bioinformatics analysis of public datasets that cell cycle and DNA replication are both involved in the regulation of HCC [25]. The investigation revealed that the p53 signaling pathway was reported to have a role in maintaining the stability of the genome and was associated with HCC. For example, the results reported by Zhao et al. indicated that xanthohumol could induce the growth and apoptosis of HCC by regulating the NF-κB/p53-apoptosis signaling pathway [26]. In addition, Li et al. confirmed by bioinformatics technology that p53 signaling pathway and cell cycle were also important pathways for HCC progression [27]. PPAR controls many intracellular metabolic processes, and the PPAR signaling pathway can also act as a regulator of liver metabolism and participate in the occurrence and development of HCC [28]. The study by Fengboo et al. showed that in the majority of human HCC, the activation of the MAPK signaling pathway was observed in the presence of wild-type genes for RAS, RAF, and downstream components [29]. In the article, Ganne-Carrie and Nahon propose that IL-17A is a tumor-promoting cytokine that critically regulates inflammatory responses in macrophages and cholesterol synthesis in fatty liver cells in an experimental model of alcohol-induced HCC [30]. The above theories are consistent with the results of our present study, indicating that these enriched terms can be applied in the mechanism study of HCC.TFs are protein molecules that have a unique structure and function in gene regulation, which is essential for a series of key cellular processes and widely used in tumor research. TFs like Tbx-1, CP2, andPLAGL2 have also been confirmed to be involved in the expression of thyroid cancer, colorectal cancer, and gastric cancer [31, 32], respectively, and they might be key targets for cancer treatment. After screening DEGs, we determined the DETFs in the GSE62232 dataset and obtained a total of 7 DETFs. Subsequently, a prognostic model of these TFs was constructed. It was found that 6 DETFs had significant effects on the prognosis of HCC patients, namely DACH1, FOSB, FOS, ASCL1, EBF1, and GTF2H2. The high expressions of GTF2H2 and DACH1 were associated with poor prognosis of patients, while high expressions of FOSB, ASCL1, EBF1, and FOS had a better OS probability. These DETFs all have the potential to be the clinical biomarkers for HCC patients.Previously, these DETFs have been studied by researchers in the progression of HCC.DACH1 regulates gene expression during cell development, and mutations in its expression can lead to abnormal progression of lung adenocarcinoma and endometrial cancer. Cheng et al. demonstrated that knocking down the expression of the SIX1 gene increased the expression of DACH1 [33], thereby activating the expression of p53 and inhibiting the progression of HCC cells in vivo and in vitro. FOSB is a member of the FOS gene family and a regulator of cell proliferation, differentiation, and transformation. Liu et al. analyzed 4 gene expression datasets through correlation bioinformatics and found that FOS and FOSB were TFs associated with HCC, and their mRNA levels were lower in HCC [34]. EBF1 acts as a tumor suppressor in various cancers. Armartmuntree et al. reported in liver fluke infection-associated cholangiocarcinoma (CCA), the expression of EBF1 was inhibited in CCA, which was associated with long-term oxidative stress [35]. Other studies have confirmed that GTF2H2 inhibits the proliferation of HCC cells and promotes its apoptosis, which is a potential inhibitor of hepatocarcinogenesis [36] and GTF2H2 can also affect the growth of hepatoma cells Hep3B by mediating the AKT molecular signaling pathway [37]. The study of Olsen et al. points out that ASCL1 is underexpressed in small-cell lung cancer and ASCL1 deletion suppresses the neural crest stem cell-like state in SOX9+ small cell lung cancer [38]. In addition, other studies have also confirmed that ASCL1 can combine with NKX2-1 and PROX1 complex to regulate NOTCH signaling, cell cycle, and other pathways involved in subtype-specific genes in small cell lung cancer [39]. The genes not introduced here have just few reports yet. Based on what we found and previous research, ASCL1 was identified as the key DETF related to HCC.According to the regulatory network and previous studies ofASCL1, we speculated SLC6A13 as a key target gene by the regulatory network of ASCL1, which is one of the 6 members of the solute carrier family and involved in amino acid trans-plasma membrane import and monocarboxylic acid transport. Long et al. discovered novel biomarkers from blood during Alzheimer’s disease, including SLC6A13, ECH1, and NHLRC2 [40]. Based on the JASPAR and Ensembl genome databases, we identified the common binding sites of ASCL1 and SLC6A13 as CCAGCAACTGGCC. Subsequently, ASCL1 and SLC6A13 were found to be positively related by public database and functional experiments. These findings indicate that which SLC6A13 is a mRNA target of ASCL1 in HCC. In the survival and ROC analysis on SLC6A13, the high expression of SLC6A13 was associated with a better survival rate in HCC patients and had the certain predictive ability for the prognosis of HCC patients. Besides, we verified the expressions of SLC6A13 in HCC samples with different clinical parameters based on public databases and found that the expression of SLC6A13 gradually decreased with the HCC progression. All these results demonstrate the clinical application of SLC6A13 in HCC prognosis in the future.In cell experiments, both ofASCL1 and SLC6A13 had low expressions in HCC groups, and we then evaluated the effect of the key targets ASCL1 and SLC6A13 on the progression of HCC. The corresponding results showed that the upregulated expressions of ASCL1 and SLC6A13 in HCC cells could significantly inhibit the proliferation, migration, and invasion of HCC cells. In addition, over-ASCL1 could upregulate the expression of SLC6A13 in HCC cells. From the above research conclusions, we can conclude ASCL1 and SLC6A13 are the key tumor suppressor factors of HCC, and they might be the treatment targets in the progression of HCC.


## 5. Conclusion

In conclusion, we performed the enrichment analysis of DEGs on the GSE32232 dataset based on bioinformatics methods and obtained 6 DETF with predictive ability in HCC prognosis. Then, the key DETF,ASCL1, and its downstream target SLC6A13 mRNA were identified, both lowly expressed in HCC tumor tissues and inhibiting the development of HCC in vitro. ASCL1 could suppress the progression of HCC by upregulating SLC6A13, and they are also promising treatment targets and prognostic biomarkers in HCC progression. However, there are still some limitations. For example, one of the limitations of the current study is that all the results are preliminary, and the further mechanism of the hub gene should be explored deeply. The further research is needed to be done in the future.



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*Source: 3560340-2023-01-30.xml*