AEBP1 inhibition reduces cell growth and PI3K/AKT pathway while less regulates cell mobility in hepatocellular carcinoma

Abstract

Background: Adipocyte enhancer-binding protein 1 (AEBP1) regulates collagen fibrosis, extracellular matrix, and important oncogene pathways, but its regulation on hepatocellular carcinoma (HCC) is not known. This study aimed to investigate the effect of AEBP1 knockdown on HCC cell proliferation, apoptosis, migration, invasion and PI3K/AKT pathway.

Methods: MHCC-97 H and Huh7 cell lines were applied. Negative control or AEBP1 siRNA (siAEBP1) were transfected into cells, and cells without transfection were set as blank control. Quantitative polymerase chain reaction (qPCR), western blot, Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) staining, Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) staining, Transwell invasion, and cell scratch assays were performed.

Results: AEBP1 mRNA and protein expressions were lower after siAEBP1 transfection in MHCC-97 H and Huh7 cells. OD value of CCK-8 and EdU positive cell percentage were decreased, while TUNEL reflected cell apoptosis rate was increased, after siAEBP1 transfection in MHCC-97 H and Huh7 cells. However, invasive cell number and cell migration rate were only reduced after siAEBP1 transfection in Huh7 cells but not in MHCC-97 H cells. Expressions of p-PI3K/PI3K and p-AKT/AKT were downregulated after siAEBP1 transfection in MHCC-97 H and Huh7 cells. Subsequent rescue experiment revealed that th activation of PI3K/AKT pathway by 740Y-P attenuated the effect of siAEBP1 transfection in MHCC-97 H and Huh7 cells.

Conclusion: AEBP1 exhibits the potency to be a target for HCC treatment, reflected by its regulation on HCC proliferation, apoptosis and PI3K/AKT pathway, but its effect on HCC invasion and migration seems limited.

Keywords: AEBP1; Hepatocellular carcinoma; Invasion and migration; PI3K/AKT pathway; Proliferation and apoptosis.

Fig. 1

Transfection efficiency. The expression of AEBP1 mRNA expression (A), western blot images and AEBP1 protein expression (B) after transfection in MHCC-97 H cells. The expression of AEBP1 mRNA expression (C), western blot images and AEBP1 protein expression (D) after transfection in Huh7 cells

Fig. 2

Cell proliferative detection after transfection. OD value by CCK-8 at 0 h, 24 h, 48 h, and 72 h (A), EdU staining images and positive cell percentage (B) after transfection in MHCC-97 H cells. OD value by CCK-8 at 0 h, 24 h, 48 h, and 72 h (C), EdU staining images and positive cell percentage (D) after transfection in Huh7 cells

Fig. 3

Cell apoptotic detection after transfection. TUNEL images and cell apoptosis rate by TUNEL after transfection in MHCC-97 H cells (A) and Huh7 cells (B)

Fig. 4

Cell mobility after transfection. Transwell staining images and invasive cell count (A), cell scratch images and cell migration rate (B) after transfection in MHCC-97 H cells. Transwell staining images and invasive cell count (C), cell scratch images and cell migration rate (D) after transfection in Huh7 cells

Fig. 5

PI3K/AKT pathway after transfection. Expressions of p-PI3K, PI3K, p-AKT, AKT after transfection in MHCC-97 H cells (A) and Huh7 cells (B)

Fig. 6

740Y-P treatment. OD value by CCK-8 at 0 h, 24 h, 48 h, and 72 h after 740Y-P treatment along with or without siAEBP1 transfection in MHCC-97 H cells (A) and Huh7 cells (B)