The effect of LOXL2 in hepatocellular carcinoma

Abstract

Lysyl oxidase-like 2 (LOXL2) is key in the hepatocellular carcinoma (HCC) tumor microenvironment and metastatic niche formation. However, its effect on proliferation and clinical parameters in HCC require further elucidation. The present study aimed to investigate LOXL2 expression in HCC from in vitro and clinical aspects. The present study constructed LOXL2‑small interfering RNA with a lentiviral vector, investigated the effect of LOXL2 on proliferation using HCC cell lines via a series of assays, including reverse transcription‑quantitative polymerase chain reaction, cell counting, colony formation, assessment of cell cycle and apoptosis using flow cytometry, MTT and BrdU. Furthermore, 80 tissue samples from HCC patients at The First Affiliated Hospital of Dalian Medical University (Dalian, China) from 2007 to 2010. Immunohistochemical staining was used to clinically verify LOXL2 expression. The results of the present study demonstrate that LOXL2 silencing decreased cell numbers, proliferation, colony formations and cell growth, induced cell cycle arrest and increased apoptosis. Clinically, expression levels of LOXL2 was markedly increased in matched adjacent non‑tumor tissue (ANT) samples compared with levels in tumor tissue (TT) samples, and this gradually increased with higher histological grade and more advanced TNM classification in the matched ANT and TT samples. LOXL2 was determined to promote proliferation of HCC and demonstrated to be highly expressed in HCC ANT samples compared with TT samples.

Figure 1

Expression levels of the LOXL2 gene in hepatocellular carcinoma cell lines. LOXL2, lysyl oxidase-like 2.

Figure 2

Comparison of LOXL2 mRNA expression levels in hepatocellular carcinoma cell lines treated with LOXL2-siRNA and controls. ^**P<0.01 vs. control. LOXL2, lysyl oxidase-like 2; siRNA, small interfering RNA.

Figure 3

LOXL2 silencing decreased proliferation of hepatocellular carcinoma cells. (A) The number of cells and fold change in proliferation in HepG2 cells. (B) The number of cells and fold increase in proliferation in SMMC-7721 cells. LOXL2, lysyl oxidase-like 2; siRNA, small interfering RNA.

Figure 4

Effects of LOXL2 silencing on hepatocellular carcinoma cell colony formation. (A) Representative images of the colony formation assay and (B) quantification of colony formation efficiency in HepG2 cells. (C) Representative images of the colony formation assay and (D) quantification of colony formation efficiency in SMMC-7721 cells. ; ^*P<0.05, ^**P<0.01 vs. the control group. LOXL2, lysyl oxidase-like 2; siRNA, small interfering RNA.

Figure 5

Effects of LOXL2 silencing on hepatocellular carcinoma cell cycle distributions. (A) Representative histograms of flow cytometry and (B) histogram of percentage of different cell cycle phases in HepG2 cells. (C) Representative histograms of flow cytometry and (D) histogram of percentages of different cell cycle phases in SMMC-7721 cells. ^**P<0.01 vs. the control group. LOXL2, lysyl oxidase-like 2; siRNA, small interfering RNA.

Figure 6

Effects of LOXL2 silencing on hepatocellular carcinoma cell apoptosis. (A) Representative histograms of flow cytometry and (B) apoptosis rates in the HepG2 cell line. (C) Representative histograms of flow cytometry and (D) apoptosis rates in the SMMC-7721 cell line. ^**P<0.01 vs. the control group. LOXL2, lysyl oxidase-like 2; siRNA, small interfering RNA.

Figure 7

Effects of LOXL2 silencing on hepatocellular carcinoma cell absorbance. (A) MTT assays were performed on the indicated days to determine the absorbance of HepG2 cells. (B) BrdU labeling was performed on the indicated days to demonstrate the absorbance of SMMC-7721 cells. ^*P<0.05 vs. the control group. LOXL2, lysyl oxidase-like 2, siRNA, small interferingRNA; OD, optical density.

Figure 8

Immunohistochemical staining of lysyl oxidase-like 2 in hepatocellular carcinoma tissue. (A) Matched adjacent non-tumor tissue; (B) tumor tissue; (C) normal liver tissue (magnification, ×200).

Figure 9

Changes to LOXL2 expression levels in adjacent non-tumor tissues with different tumor histological grades. ^*P<0.05, ^**P<0.01 vs. grade I samples. LOXL2, lysyl oxidase-like 2; IOD, integral optical density.

Figure 10

Changes to LOXL2 expression levels in tumor tissue with different tumor histological grades. ^**P<0.01 vs. grade I samples. LOXL2, lysyl oxidase-like 2; IOD, integral optical density.

Figure 11

Changes to LOXL2 expression levels in adjacent non-tumor tissues with different TNM classification. LOXL2, lysyl oxidase-like 2; TNM, tumor node metastasis; IOD, integral optical density.

Figure 12

Changes to LOXL2 expression levels in tumor tissue with different TNM classification. LOXL2, lysyl oxidase-like 2; TNM, tumor node metastasis; IOD, integral optical density.