Vitamin K2 Inhibits The Proliferation Of Human Hepatocellular Carcinoma Cell (HuH-7) And Down-regulates The Expression Of FGFR3

Objective:To confirm the inhibitory effects of Vitamin K2 on the proliferation of human hepatocellular carcinoma cell HuH-7 and the expression of FGFR3,according to the previous results of DNA chip,and to provide convictive experimental bases for further clinical use of Vitamin K2 and the targeted therapy for liver cancer.Methods:(1)The inhibitory effect of Vitamin K2 on the proliferation of HuH-7 was measured by MTT assay.(2)Semi-quantitative RT-PCR(reverse-transcription-polymerase chain reaction)was used to confirm the mRNA levels of FGFR3 and FGFR1 after 96 hours exposure to 0,10 and 30μM of Vitamin K2.(3)Real-time PCR(quantitative polymerase chain reaction)was used to measure the mRNA levels of FGFR3 and FGFR1 after 96 hours exposure to 0,10 and 30μM of Vitamin K2.(4)Western-blot analysis was used to detect the protein level of FGFR3 after 96 hours exposure to 0,10 and 30μM of Vitamin K2.Results:(1)Vitamin K2 had the inhibitory effects on the proliferation of HuH-7 cells.The significant differences of cell numbers were observed after 72 hours and 96 hours in the 10 and 30μM Vitamin K2 groups (P＜0.01).No significant differences of the cell numbers were observed after 48 hours exposure of HuH-7 to 0.01,0.1,1,10 and 30μM Vitamin K2(P＞0.05)and after 72,96 hours in the 0.01,0.1 and 1μM Vitamin K2 groups(P＞0.05).The 30μM Vitamin K2 group showed a time-dependent inhibition on the proliferation of HuH-7 cells.(2)By RT-PCR,the inhibitory effect of Vitamin K2 on the FGFR3 mRNA was in a dose dependent manner.However,no remarkable inhibitory effect of Vitamin K2 on the FGFR1 mRNA was shown.(3)Real-time PCR revealed the significant reductions of the mRNA levels of FGFR3 in the 10 and 30μM Vitamin K2 groups as compared with the control group[0.692±0.087(p=0.0124,＜0.05)at 10μM and 0.385±0.053(p＜0.0001)at 30μM]after 96 hours treatments.The significant difference was demonstrated between 10 and 30μM Vitamin K2 groups(p=0.0236,＜0.05),too.No significant reductions of the mRNA level of FGFR1 was detected in the 10 or 30μM Vitamin K2 groups as compared with the control group[0.938±0.051(p=0.2921,＞0.05)at 10μM group and 0.933±0.096(p=0.5230,＞0.05)at 30μM group].No significant difference was shown between 10 and 30μM Vitamin K2 groups(p=0.9678,＞0.05),too.(4)Significant suppression of the FGFR3 protein expression was revealed in the 10 and 30μM Vitamin K2 groups as compared with the control group(p＜0.01).The significant difference between 10 and 30μM Vitamin K2 groups was shown(p＜0.01),too.Conclusions:(1)Vitamin K2 supresses the proliferation of HuH-7 in dosedependent and time-dependent manners.(2)Vitamin K2 probably inhibits the proliferation of HuH-7 through down-regulating FGFR3 expression.(3)The inhibitory effect of Vitamin K2 on the proliferation of HuH-7 may not be related to FGFR1 gene expression.