Effects of HGF/Met signaling pathway on stem-like phenotype of hepatocellular carcinoma cell line MHCC97-H

Abstract

Objective To investigate the effects of HGF/Met signaling pathway on stem-like phenotype of hepatocellular carcinoma cell line MHCC97-H.Methods The hepatocellular carcinoma cell lines Huh7 and MHCC97-H were cultured in vitro.Cell lines with high expression of Met were selected and divided into the blank group (cells untreated),HGF stimulation group (cells treated with 50 μg/L of HGF),HGF inhibition group (cells treated with 50 μg/L of HGF + 1 mol/L of PHA665752) and epidermal growth factor/fibroblast growth factor (EGF/FGF) stimulation group (cells treated with 50 μg/L of HGF + 20 μg/L of EGF + 20 μg/L of FGF).The protein expressions of Met and phospho-Met (p-Met) in the blank group,the HGF stimulation group and the HGF inhibition group were detected by Western blot.The cell morphological alteration of the blank group,the HGF stimulation group and the HGF inhibition group was observed under the light microscope at 24 hours after the treatment.Different sphere-formation ability was detected by sphere-formation assay under serum-free condition.The expression change of induced pluripotent stem cells related genes was analyzed by Real-Time polymerase chain reaction when MHCC97-H cells had been treated with HGF for different hours (2,4,8,16,24 hours).The measurement data were presented as.x ± s.All data were analyzed using the one-way analysis of variance,repeated measurement and LSD-t test.Results The results of immunofluorescence showed that MHCC97-H cells possessed high expression of Met when compared with Huh7 cells.In the blank group,HGF stimulation group and the HGF inhibition group,the protein expressions of Met were 0.44 ± 0.04,0.37 ± 0.03,0.41 ± 0.04,with no significant difference between the 3 groups (F =2.31,P ＞ 0.05),and the protein expressions of p-Met in the 3 groups were 0.020 ±0.010,0.070 ± 0.020,0.010 ± 0.000,with significant difference between the 3 groups (F =34.11,P ＜ 0.05).The protein expression of p-Met in the HGF stimulation group was significantly higher than that in the blank group and the HGF inhibition group (t =3.87,5.20,P ＜ 0.05).MHCC97-H cells in the HGF stimulation group were partly spindle-shaped which were similar to mesenchymal phenotype when cells had been treated with HGF for 24 hours,and PHA665752 could prevent this morphological alteration.MHCC97-H cells in the HGF inhibition group were like epithelium cells,which were similar to those in the blank group.The numbers of the spheres of the blank group,HGF stimulation group,HGF inhibition group and the EGF/FGF stimulation group were 0,35.0 ± 6.3,4.3 ± 1.5 and 54.3 ± 2.5,with significant difference between the 4 groups (F =511.28,P ＜ 0.05).The number of spheres of the HGF stimulation group was greater than that of the blank group and the HGF inhibition group (t =9.62,8.21,P ＜0.05),while lesser than that of the EGF/FGF stimulation group (t =4.93,P ＜ 0.05).In the blank group and the HGF stimulation groups (2,4,8,16,24 h),the expressions of C-myc mRNA were 1.00 ±0.11,1.68 ±0.09,1.08 ±0.24,1.18 ±0.13,0.78 ±0.14,1.06 ±0.04; the expressions of Klf4 mRNA were 1.00±0.20,1.43 ±0.16,0.87 ±0.28,1.19 ±0.29,2.17 ±0.43,1.41 ± 0.06; the expressions of Oct4 mRNA were 1.00 ±0.12,0.54 ±0.12,0.69 ±0.19,1.08 ±0.12,1.62 ±0.30,0.97 ± 0.1 1,with significant difference (F =13.64,8.52,13.56,P ＜ 0.05).The expression of C-myc mRNA in the HGF stimulation group (HGF stimulation for 2 hours) was 1.68 times higher than that of the blank group (t =8.29,P＜0.05).The expression of K1f4 mRNA of the HGF stimulation group (HGF stimulation for 16 hours) was 2.17 times higher than that of the blank group (t =4.27,P ＜ 0.05).The expression of Oct4 mRNA of the HGF stimulation group (HGF stimulation for 16 hours) was 1.62 times higher than that of the blank group (t =3.32,P ＜ 0.05).Conclusion HGF/Met signaling pathway might prompt the stem-like phenotype of MHCC97-H liver cancer cells through upregulation of pluripotent stem cells related genes. Key words: Liver neoplasms; Hepatic growth factor; Met; Pluripotent stem cells