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Apolipoprotein M (ApoM) is considered a protective factor that inhibits the occurrence and development of liver cancer, but the specific underlying mechanisms require further investigation. Previous studies have demonstrated that ApoM gene knockout promotes the expression of the transcription factor sterol regulatory element-binding protein 1 (SREBP1; also known as SREBF1) in the livers of mice. SREBF1 is closely associated with factors involved in fatty acid synthesis and has a role in the promotion of tumor progression. The present study initially confirmed that the expression levels of ApoM in cancer tissues were significantly decreased compared with those in normal tissue, while the expression levels of SREBF1 were significantly increased. In addition, ApoM gene knockout significantly increased the expression levels of SREBF1 and the key glycolytic enzyme ATP-dependent 6-phosphofructokinase, liver type (PFKL). Binding site prediction and a dual-luciferase reporter gene assay indicated that SREBF1 regulates the promoter region of PFKL. To the best of our knowledge, the present study was the first to propose the regulation of glycolytic enzyme transcription levels by SREBF1. Furthermore, cell proliferation and Transwell assays demonstrated that ApoM gene knockout increased the expression levels of SREBF1 and further enhanced the activity of the promoter region of PFKL, ultimately promoting the proliferation, migration and invasion of liver cancer cells.
Liver cancer is one of the most common malignant tumors of the digestive system and exhibits characteristics of high-grade malignancy, rapid progression, high recurrence rates and a high probability of metastasis (
As a member of the apolipoprotein family, apolipoprotein M (ApoM) participates in the synthesis of high-density lipoprotein (HDL) and the reverse transport of cholesterol (
Based on the above viewpoints, ApoM is an apolipoprotein that inhibits the occurrence and development of liver cancer, and it is closely related to the body's glucose and lipid metabolism. At present, the metabolic level and tumor metabolic microenvironment are still the focus of cancer related research. But whether ApoM affects the development of liver cancer through glycolipid metabolism remains unclear. Of note, the results of a previous study by our group demonstrated that deficiency of the ApoM gene causes damage to autophagy activity in the liver and eventually leads to lipid accumulation (
Huh-7 cells (BeNa Culture Collection) and Mhcc97h cells (Guangzhou Saiku) were cultured in high-sugar DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Shanghai ExCell Biology, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and were incubated at 37°C in an atmosphere with 5% CO2. As for the reason for choosing Huh7 and Mhcc97h cells, it was observed that SREBF1 was highly expressed in Huh7 and Mhcc97h cells (
The Ensembl database (
Each group of cells (si-NC, si-SREBF1, pcDNA3.1-NC, pcDNA3.1-SREBF1, si-ApoM−/−+si-SREBF1 and si-SREBF1+pcDNA3.1-PFKL) was stained according to the instructions of the EDU Cell Proliferation kit (cat. no. C10310-1; Guangzhou RiboBio Co., Ltd.). Images were obtained using an inverted fluorescence microscope (Olympus Corporation). ImageJ software V1.8.0.112 [National Institutes of Health (NIH)] was used for data analysis.
RIPA lysis buffer (cat. no. BL651A; Biosharp Life Sciences) and PMSF (cat. no. BL507A; Biosharp Life Sciences) were used for tissue and cell protein extraction, and the protein concentration was measured using a NanoDrop® 2000 mini-spectrophotometer (Thermo Fisher Scientific, Inc.). Proteins (60 µg) were separated using SDS-PAGE (10 or 12% gel), transferred to a PVDF membrane (MilliporeSigma) and subsequently blocked with blocking solution (cat. no. P0023B; Beyotime Institute of Biotechnology) for 10 min at room temperature. Subsequently, samples were incubated with the appropriate primary antibody solution overnight at 4°C. The next day, the PVDF membranes were incubated in secondary antibody solution for 2 h at room temperature (both 1:3,000 dilution; cat. nos. BL001A and BL003A; Biosharp Life Sciences). ECL chemiluminescent fluid (cat. no. BL520A; Biosharp Life Sciences) and an imaging system (ShanghaiTanon-5200 Co., Ltd.) were used for exposure. The following antibodies were used: Anti-ApoM (cat. no. A5336; 1:1,000 dilution; ABclonal Biotech Co., Ltd.), anti-PFKL (cat. no. A7708; 1:1,000 dilution; ABclonal Biotech Co., Ltd.) and anti-SREBP1 (cat. no. ab138663; 1:1,000 dilution; Abcam) antibody were used to react with human hepatoma cells, while anti-SREBP1 (cat. no. ab28481; 1:1,000 dilution; Abcam) antibody was used to react with mouse tissue proteins and β-actin (A1978; 1:5,000 dilution; MilliporeSigma). ImageJ software V1.8.0.112 (NIH) was used for statistical analysis.
Following cell transfection with si-NC, si-SREBF1, pcDNA3.1-NC, pcDNA3.1-SREBF1, si-ApoM−/−+si-SREBF1 or si-SREBF1+ pcDNA3.1-PFKL plasmids for 48 h at 37°C, the cell concentration was adjusted to 1×105 cells/ml using serum-free cell culture medium and 200 µl cell suspension was added to the upper chamber for the migration assay (cat. no. 3422; Corning, Inc; PC membrane, 6.5 mm; pore size, 8.0 µm). A total of 600 µl cell culture medium with 20% serum was added to the lower chamber and plates were cultured for 48 h. Cells that passed through the membrane were stained with 4% paraformaldehyde (Phygene Brotechnology Co., Ltd.) and 0.1% crystal violet solution (cat. no. C0121; Beyotime Institute of Biotechnology.) for 10 min at room temperature. Finally, use a cotton swab to wipe the cells that have not crossed the membrane. Migrated cells were counted under a microscope (Olympus Corporation). For the invasion assay, Dilute Matrigel® to 200 µg/ml with PBS, and 50 µl Matrigel® was added to the upper chamber prior to incubation at 37°C for 2 h. The remaining steps were followed in an identical manner to those of the migration assay above. ImageJ software V1.8.0.112 (National Institutes of Health) was used for data analysis.
Following cell transfection with si-NC, si-SREBF1, pcDNA3.1-NC, pcDNA3.1-SREBF1, si-ApoM−/−+ si-SREBF1 or si-SREBF1+pcDNA3.1-PFKL plasmids for 48 h, a 200-µl pipette tip was used to make a linear scratch on the cell monolayers. Cells were subsequently washed three times with PBS and cultured in DMEM. After 48 h of incubation, the width of the gap refilled by the cells was measured and recorded, and the wound-healing rate was calculated. ImageJ software V1.8.0.112 (NIH) was used for data analysis.
The present study was approved by the Experimental Animal Welfare and Ethics Committee of Wannan Medical College (approval no. LLSC-2020-001). According to literature reports, there are more new cases of liver cancer in Chinese males than in females (
The liver issues of WT healthy mice and WT tumor-forming mice were obtained. The levels of LA, ATP, TG, T-CHO, HDL-C and LDL-C were determined in tissues using kits purchased from Beijing Solarbio Science & Technology Co., Ltd. (cat. no. BC2235) and Nanjing Jiancheng Bioengineering Institute (cat. nos. A095-1-1, A110-1-1, A111-1-1, A112-1-1 and A113-1-1), according to the manufacturers' protocols.
Tissues were fixed in 10% formalin for 48 h at room temperature, embedded in paraffin and cut into 4-µM sections. Following deparaffinization and rehydration, the sections were stained using H&E and the morphology was observed under a microscope (Olympus Corporation).
The liver tissues of WT healthy mice and WT tumor-forming mice were embedded in paraffin and the sections were subsequently dissected into thin slices and deparaffinized in xylene. Tissue slides were incubated with SREBF1 antibody (cat. no. ab28481; 1:150 dilution; Abcam) overnight at 4°C. Following primary incubation, the slides were incubated with the secondary antibody (cat. no. GB23303; 1:200 dilution; Servicebio, Co., Ltd.) at 37°C for 50 min. The slides were subsequently incubated with DAB (1:50 dilution; Servicebio, Co., Ltd.) to visualize the staining. Samples were counterstained using hematoxylin solution (Servicebio, Co., Ltd.) for 90 sec and then differentiated using 1% hydrochloric acid alcohol for several seconds at room temperature. The slides were mounted using neutral gum (cat. no. G1403; Servicebio, Co., Ltd.) prior to being placed under a microscope (Olympus Corporation) to observe the expression of SREBF1 protein in each tissue. Image-Pro-Plus 6.0 software (Media Cybernetics, Inc.) was used for data analysis.
R software (version 3.6.3) [DESeq2 (version 1.26.0) 25516281] was used to enter the Level 3 HTSeq-Counts RNASeqV2 data in TCGA (
Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc.) and SPSS 26.0 (IBM Corporation) software. A total of three parallel experiments were set up in each group and these were performed as three repeats. Values are expressed as the mean ± standard deviation. The Kolmogorov-Smirnov test was applied to determine compliance with a normal distribution. Pairwise comparisons between groups in the presence of multiple groups were performed using one-way ANOVA followed by Tukey's post-hoc test. An unpaired Student's t-tests was used to determine significant differences between two groups. P<0.05 was considered to indicate a statistically significant difference.
N-nitrosodiethylamine was used to induce liver cancer in mice. Mice induced by N-nitrosodiethylamine developed up to two tumors in the liver, but more frequently, one tumor was formed. The tumor diameter in mice with liver tumors was 0.3-0.6 cm (experimental period, 5–6 months) (
Results of a previous study by our group demonstrated that ApoM gene knockout in promoted the expression of SREBF1 in the liver (
Results of a previous study by our group demonstrated that ApoM gene knockout significantly increased the proliferation of liver cancer cells (
Metastasis is one of the most important causes of malignancy. Thus, Transwell assays were performed in the present study to detect whether ApoM regulates PFKL through the transcription factor SREBF1 and affects the migration and invasion of liver cancer cells. First, the role of SREBF1 in the development of liver cancer cells was identified (
Results of previous studies have demonstrated that ApoM is commonly associated with liver cancer. Previous reports indicated differential ApoM mRNA levels and ApoM protein mass in liver cancer tissue and adjacent tissues (
Although ApoM has been confirmed to be involved in glucose and lipid metabolism (
The glycolysis pathway contains three key rate-limiting enzymes: Hexokinase, phosphofructokinase (PFK) and pyruvate kinase. Results of previous studies have demonstrated that PFK may act as the most important regulator in the glycolysis pathway, including three PFK isoforms, platelet, liver and muscle isoform (
The present study also aimed to explore the mechanism by which increased ApoM gene expression is negatively correlated with PFKL expression. Results of a previous study demonstrated that ApoM deficiency significantly suppressed the autophagy function in the mouse liver and caused lipid accumulation; furthermore, the expression levels of SREBF1 were significantly increased (
In conclusion, the present study further elucidated the potential mechanisms by which ApoM inhibits the development of liver cancer by regulating glycolysis; however, further investigations are required. Although metabolic enzymes are known to regulate metabolic processes, their non-metabolic activities require further investigation, which may include protein interactions and the crosstalk between different compartments of the signaling pathway. Similarly, this view is also particularly important for SREBF1. The present study confirmed that SREBF1 as a transcription factor may enhance the promoter activity of PFKL and its expression level was affected by ApoM. With regard to the question of how ApoM affects the expression of SREBF1, integration of existing research results provides noteworthy and highly relevant clues. First of all, SREBF1, as the central transcription factor regulating lipid metabolism, mainly regulates the expression of the factors required for fatty acid synthesis (
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
XZ and YB designed and supervised the study. WP performed the data analysis, statistical analysis and completed the manuscript writing. XZ, WZ and XL performed the experiments. XZ and WP performed plausibility checks and confirmed the authenticity of the raw data, which were further edited and later approved for publication by all authors. All authors read and approved the final manuscript.
The use of the C57BL/6J mice required for the experiment was approved by the Experimental Animal Welfare and Ethics Committee of Wannan Medical College (Wuhu, China; approval no. LLSC-2020-001).
Not applicable.
The authors declare that they have no competing interests.
High expression of SREBF1 in N-nitrosodiethylamine-induced mouse hepatocellular carcinoma tissues. (A) Images of livers from mouse models with N-nitrosodiethylamine-induced liver cancer tumors. (B-F) A test kit was utilized to detect the content of (B) HDL-C, (C) LDL-C, (D) T-CHO, (E) TG and (F) ATP in liver cancer tissue of WT mice and liver tissue from healthy mice. (G) H&E staining revealed the morphology of liver tissue in mice prior to and after induction with N-nitrosodiethylamine (scale bar, 100 µm). (H) SREBF1 expression levels were determined using immunohistochemistry (scale bar, 50 µm). (J and K) Western blot analysis was performed to evaluate the expression levels of SREBF1 and ApoM in liver cancer tissue of WT mice and liver tissue from healthy mice. (J) Representative western blots and (K) quantified results. Analysis in each group was performed three times in parallel. *P<0.05, **P<0.03, ***P<0.01 vs. WT. HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; T-CHO, total cholesterol; TG, triglyceride; WT, wildtype; SREBF1, sterol regulatory element-binding protein 1; ApoM, apolipoprotein M.
SREBF1 is able to regulate the expression of PFKL. (A-H) Detection of the expression levels of SREBF1, PFKL and ApoM in Huh-7 and Mhcc97h cells using western blot analysis after knockdown or overexpression of ApoM. (A) Representative western blot image for Huh-7 cells with ApoM knockdown and (B) quantified expression levels. (C) Representative western blot image for Mhcc97h cells with ApoM knockdown and (D) quantified expression levels. (E) Representative western blot image for Huh-7 cells with ApoM overexpression and (F) quantified expression levels. (G) Representative western blot image for Mhcc97h cells with ApoM overexpression and (H) quantified expression levels. (I-P) Detection of the expression levels of SREBF1 and PFKL in Huh-7 and Mhcc97h cells using western blot analysis after knockdown or overexpression of SREBF1. (I) Representative western blot image for Huh-7 cells with SREBF1 knockdown and (J) quantified expression levels. (K) Representative western blot image for Mhcc97h cells with SREBF1 knockdown and (L) quantified expression levels. (M) Representative western blot image for Huh-7 cells with SREBF1 overexpression and (N) quantified expression levels. (O) Representative western blot image for Mhcc97h cells with SREBF1 overexpression and (P) quantified expression levels. (Q-T) Western blot analysis was used to validate the PFKL overexpression model in Huh-7 cells and Mhcc97h cells. (Q) Representative western blot image for Huh-7 cells with PFKL overexpression and (R) quantified expression levels. (S) Representative western blot image for Mhcc97h cells with PFKL overexpression and (T) quantified expression levels. (U) Prediction of the binding site of SREBF1 and PFKL. (V) Results of the luciferase-based gene reporter assay used to detect the promoter activity of PFKL through promoting SREBF1. Each group was set up three times in parallel. *P<0.05, **P<0.03, ***P<0.01 vs. NC group. ApoM, apolipoprotein M; SREBF1, sterol regulatory element-binding protein 1; PFKL, ATP-dependent 6-phosphofructokinase, liver type; NC, negative control; si-, small interfering RNA; Luc, luciferase.
Knockdown of ApoM promotes SREBF1 to regulate PFKL to promote the proliferation of hepatoma cells. An EDU staining assay was used to detect the proliferation activity of (A) Huh-7 cells or (B) Mhcc97h cells in each group (pcDNA3.1-NC, pcDNA3.1-SREBF1, si-NC, si-SREBF1, si-SREBF1+pcDNA3.1-PFKL and si-ApoM+si-SREBF1; scale bar, 100 µm). Statistical analysis of the results for (C) Huh-7 cells or (D) Mhcc97h cells using ImageJ. Each group was set up three times in parallel. ns, no significance; ***P<0.01 vs. NC. NC, negative control; SREBF1, sterol regulatory element-binding protein 1; siRNA, small interfering RNA; PFKL, ATP-dependent 6-phosphofructokinase, liver type; ApoM, apolipoprotein M.
Knockdown of ApoM promotes SREBF1 to regulate PFKL to promote the migration and invasion of hepatoma cells. Huh-7 cells or Mhcc97h cells were transfected with pcDNA3.1-NC, pcDNA3.1-SREBF1, si-NC, si-SREBF1, si-SREBF1 + pcDNA3.1-PFKL or si-ApoM + si-SREBF1. (A-H) Transwell assays. Representative images of Huh-7 cells transgressed through the membrane in the (A) migration and (B) invasion experiment and quantified results for (C) migration and (D) invasion. Representative images of Mhcc97h cells transgressed through the membrane in the (E) migration and (F) invasion experiment and quantified results for (G) migration and (H) invasion (scale bars, 100 µm). (I-L) Wound-healing assay. (I) Representative images of migration of Huh-7 cells and (J) quantified results. (K) Representative images of migration of Mhcc97h cells (scale bars, 200 µm) and (L) quantified results. Each group was set up three times in parallel. ns, no significance; ***P<0.01 vs. NC. NC, negative control; SREBF1, sterol regulatory element-binding protein 1; si-, small interfering RNA; PFKL, ATP-dependent 6-phosphofructokinase, liver type; ApoM, apolipoprotein M.