Evaluation of GPC3 expression in PM2.5-treated hepatocellular carcinoma cell line by immunocytochemistry

Abstract

Background: PM2.5 is a mixture of various chemical substances released during the combustion of vehicles, industrial processes, and household fuels. It penetrates into the alveoli and enters the bloodstream. Ruyi Li et al. reported that PM2.5 fine particles in the air enhance the synthesis of Matrix metalloproteinases (MMPs) proteins and may be involved in migration and pathogenesis. In the study by Qian Zhang et al., PM2.5 particles had an effect on the proliferation, invasion, and migration of HCC. Aim: In vitro study of the effect of PM2.5 fine particles on hepatocellular carcinoma (HepG2) Materials and Methods: A human liver cancer cell line (HepG2) was obtained from the Central Scientific Research Laboratory at the Institute of Medical Sciences. HepG2 cells were seeded at a concentration of 1*105 cells/mL in a culture flask and cultured in DMEM medium supplemented with 10% FBS, and 1% antibiotic mix (penicillin, streptomycin) in a humidified atmosphere of 5% CO2 at 37 °C. The cytotoxic effect of PM 2.5 in HepG2 cells was evaluated using an MTT assay. After the MTT assay, HepG2 cells were seeded on cell culture dishes with prepared cover glasses and treated with the selected PM 2.5 dosage. The expression of MMP1 and MMP9 proteins, which are involved in HepG2 cell migration and tumor cell metastasis, was determined using wound healing and immunocytochemical methods, respectively, and compared with the control group. Images were captured under a microscope, and the results were processed using Image J software. Statistical analysis of the results was performed using Microsoft Excel 2010, and graphics were generated using Graphpad Prism 8 software. Differences between groups were calculated by Student’s t test, and significance was considered when the p value was <0.05. Results: By MTT assay, PM2.5 doses of 5 and 15 μg/ml increased cell viability (103.2 and 108.1%), while doses above 25 μg/ml decreased cell viability. In the HepG2 cell line, doses of 5, 15, 25μg/ml of PM2.5 particles were selected based on MTT analysis after 24 and 48 hours of treatment. The percentages of gap closure were 49% and 68% in control group, 53% and 76 % in 5 μg/ml group, 64% and 86% in 15 μg/ml group, 49% and 66% in the 25 μg/ml group at 12 and 24 hours, respectively. The HepG2 cell group treated with 15μg/ml of PM2.5 particles was compared with the control group to evaluate MMP1, MMP9 protein synthesis. By Immunocytochemistry assay, MMP9 protein staining intensity was 12.03% (7.44-18.28%), while MMP1 was 7.86% (3.35-11.57%) in control group, whereas MMP9 protein staining intensity was 31.22% (26.7235.97%), MMP1 was 23.83% (13.40-34.04%) in PM2.5 treated group, which is statistically significant (p<0.001), (p<0.001). Conclusions: Doses of PM2.5 particles collected from the Bokhiin Urgoo area of Ulaanbaatar city, above 25 μg/ml, inhibit the viability of HepG2 cells in vitro. In addition, PM2.5 particles not only increased the migration of the HepG2 cell line in vitro but also enhanced the synthesis of MMP1 and MMP9 proteins, which are involved in migration.