Regulatory factor X-5/SCL/TAL1 interruption site axis promotes aerobic glycolysis and hepatocellular carcinoma cell stemness

Abstract

The incidence and development of various tumors, such as hepatocellular carcinoma (HCC), are linked to tumor stem cells. Although research has revealed how important SCL/TAL1 interruption site (STIL) is in many human tumors, the impact of STIL on HCC stem cells is poorly understood. This study aimed to examine the regulatory mechanisms and the function of STIL in the stemness of HCC tumor cells. Bioinformatics analysis was applied to determine the STIL and regulatory factor X-5 (RFX5) expression in HCC tissues. Immunohistochemistry (IHC) was used to detect the expression of STIL and RFX5 in HCC tissues. Quantitative real-time polymerase chain reaction was utilized to measure the STIL and RFX5 expression levels in HCC cells. The viability of the cells was assessed by the Cell Counting Kit-8 assay. The sphere formation assay was used to evaluate the sphere-forming capacity. The expression levels of the stem cell markers SOX2, Oct-4, CD133, CD44, the glycolysis-related proteins LDHA, HK2, AKT, p-AKT, and β-catenin were assessed by Western blot. Lactate production, oxygen consumption rate, and extracellular acidification rate were measured to assess the glycolytic capacity of HCC cells. Chromatin immunoprecipitation and dual-luciferase experiments were performed to validate the connection between RFX5 and STIL. Bioinformatics analysis determined that STIL exhibited high expression in HCC tissues and was enriched in the glycolysis pathway. In addition, the expression of glycolysis marker genes was positively correlated with STIL expression. Cell experiments verified that the activation of the glycolysis pathway by overexpression of STIL promoted stemness in HCC. Molecular experiments also revealed the binding relationship between STIL and RFX5. IHC detected high expression of STIL and RFX5 in HCC tissues. Cell functional experiments revealed that RFX5 could influence the HCC cells stemness by activating the STIL transcription via the glycolysis pathway. This study identified a novel role for the RFX5/STIL axis in HCC progression, which may offer treatment targets for HCC.

Keywords: RFX5; STIL; glycolysis; hepatocellular carcinoma; stemness.

FIGURE 1

The upregulation of SCL/TAL1 interruption site (STIL) in hepatocellular carcinoma (HCC). (A) Analysis of STIL expression in HCC tissues using The Cancer Genome Atlas database. (B) Immunohistochemistry analysis of STIL expression in HCC tissues. (C) Kaplan–Meier survival curve analysis of the correlation between STIL and HCC patients' prognoses. (D) STIL expression in human normal liver cells and human HCC cells. * indicates p < 0.05.

FIGURE 2

The effect of SCL/TAL1 interruption site (STIL) on the hepatocellular carcinoma (HCC) cell stemness. (A) Correlation analysis of stemness index mRNAsi and STIL. (B, C) Determination of transfection efficiency. (D, E) Determination of cell viability. (F, G) Determination of cell sphere formation ability. (H) Determination of SOX2, Oct‐4, CD133, and CD44 expression. * indicates p < 0.05. OD: optical density.

FIGURE 3

Regulatory mechanism of SCL/TAL1 interruption site (STIL) in regulating stemness of hepatocellular carcinoma (HCC) cells. (A) Enrichment pathway analysis of STIL. (B) Correlation analysis of STIL and glycolysis pathway markers. (C) Determination of cell viability. (D–F) Detection of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in HCC cells. (G) Detection of the lactate production in HCC cells. (H) Detection of AKT, p‐AKT, β‐catenin, HK2, and LDHA expression. (I) Determination of cell sphere formation ability. (J) Detection of SOX2, Oct‐4, CD133, and CD44 expression. * indicates p < 0.05. 2‐DG, 2‐deoxy‐D‐glucose; FCCP, carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone.

FIGURE 4

The regulatory role of RFX5 on SCL/TAL1 interruption site (STIL). (A) UpSet plot of predicted transcription factors and differentially expressed genes. (B) Scatter plot of the correlation between RFX5 and STIL. (C) The binding sites of STIL and RFX5. (D) Analysis of RFX5 expression in hepatocellular carcinoma (HCC) tumor tissues and adjacent cancerous tissues. (E) Immunohistochemistry analysis of RFX5 expression in HCC tissues. (F) The RFX5 expression in HCC cells and human normal liver cells. (G, H) Validation of the binding relationship between RFX5 and STIL. * indicates p < 0.05. 2‐DG, 2‐deoxy‐D‐glucose; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone; OCR, oxygen consumption rate.

FIGURE 5

RFX5 activates SCL/TAL1 interruption site (STIL) to regulate aerobic glycolysis and promote hepatocellular carcinoma (HCC) stemness. (A) Quantitative real‐time polymerase chain reaction detection of transfection efficiency. (B) CCK‐8 detection of cell viability. (C–E) Seahorse XFe96 analysis of ECAR and OCR of HCC cells. (F) Determination of lactate production in HCC cells. (G) Western blot (WB) detection of expression of glycolytic metabolism pathway‐related proteins (HK2 and LDHA) AKT, p‐AKT, and β‐catenin. (H) Cell sphere formation assay detection of cell sphere‐forming ability. (I) WB analysis of the expression of stem cell surface markers (SOX2, Oct‐4, CD133, and CD44). * indicates p < 0.05. 2‐DG, 2‐deoxy‐D‐glucose; ECAR, extracellular acidification rate; OCR, oxygen consumption rate.

FIGURE 6

RFX5 activates SCL/TAL1 interruption site (STIL) to promote stemness of hepatocellular carcinoma (HCC) cells in mice through glycolytic pathway. (A, B) Changes in tumor size as well as mass in mice. (C) Immunohistochemistry detection of RFX5 and STIL protein expression in HCC tissues. (D) Western blot analysis of the expression of stem cell surface markers (SOX2, Oct‐4, CD133, and CD44), glycolytic metabolism pathway‐related proteins (HK2 and LDHA), AKT, p‐AKT, and β‐catenin. * indicates p < 0.05. FCCP, carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone.