Abstract
Objective To investigate the effects of nintedanib on apoptosis and autophagy of hepatocellular carcinoma cells,and to explore the role of non-receptor type protein tyrosine phosphatase 1(SHP-1)and signal transducer and activator of transcription3(STAT3).Methods Hep3B cells were divided into Hep3B group(normal culture),nintedanib group(treated with 10 μmol·L-1nintedanib for 48 h),nintedanib+si-NC group(based on the nintedanib group,transfected with si-NC before nintedanib treatment),and nintedanib+si-SHP-1 group(based on the nintedanib group,transfected with si-SHP-1 before nintedanib treatment).Reverse transcription real-time fluorescence quantitative polymerase chain reaction was used to detect SHP-1 messenger RNA(mRNA)levels.Western blot was used to detect the protein levels of SHP-1,B-cell lymphoma-2(Bcl-2),inhibitor of apoptosis protein-1(IAP-1),Survivin,Beclin-1,phosphorylated Beclin-1(p-Beclin-1),STAT3,phosphorylated STAT3(p-STAT3),Janus kinase 2(JAK2)and phosphorylated JAK2(p-JAK2)in cells.Methyl thiazolyl tetrazolium assay was used to detect the cell viability of human liver cancer cells Hep3B after treatment with different concentrations of nintedanib.Immunofluorescence was used to detect the relative expression levels of SHP-1 and microtubule-associated protein 1 light chain 3(LC3)in cells.TdT mediated dUTP nick end labeling assay was used to detect the apoptosis of Hep3B cells in each group.Results The relative expression levels of SHP-1 mRNA in THLE-2,PLC5,HuH7,Hep3B and SK-Hep1 cells were 1.00±0.16,0.45±0.08,0.58±0.09,0.29±0.04 and 0.34±0.06,respectively;the relative expression levels of SHP-1 protein were 1.00±0.13,0.71±0.09,0.83±0.12,0.56±0.07 and 0.79±0.08,respectively.Compared with the THLE-2 group,the differences in the relative expression levels of SHP-1 mRNA and protein in PLC5,HuH7,Hep3B,and SK-Hep1 groups were statistically significant(all P＜0.05).The cell viability of human liver cancer cells Hep3B treated with 0,1,5,10,15 and 20 μmol·L-1 nintedanib were(100.00±2.39)％,(92.17±16.85)％,(84.63±15.21)％,(59.02±10.74)％,(36.98±7.53)％and(29.34±5.67)％,respectively.Except for 1 μmol·L-1 group,the differences in Hep3 B cell viability between other nintedanib treatment groups and 0 μmol·L-1 group were statistically significant(all P＜0.05).The relative expression levels of SHP-1 mRNA in Hep3B group,nintedanib group,nintedanib+si-NC group,and nintedanib+si-SHP-1 group cells were 1.00±0.19,3.06±0.57,2.84±0.52 and 1.58±0.26,respectively;the relative expression levels of SHP-1 protein were 1.00±0.16,3.07±0.54,3.48±0.63 and 1.32±0.19,respectively;the apoptosis rates were(19.34±3.16)％,(62.85±10.93)％,(67.49±11.78)％and(24.72±4.29)％,respectively;the relative expression levels of Bcl-2 were 1.00±0.12,0.56±0.07,0.63±0.08 and 0.84±0.10,respectively;the relative expression levels of IAP-1 were 1.00±0.15,0.38±0.06,0.44±0.07 and 0.71±0.12,respectively;the relative expression levels of Survivin were 1.00±0.14,0.62±0.09,0.56±0.08 and 0.83±0.11,respectively;the levels of p-Beclin-1/Beclin-1 were 1.00±0.18,2.37±0.41,2.25±0.39 and 1.48±0.26,respectively;the levels of LC3 were 1.00±0.21,3.74±0.69,3.15±0.60 and 1.52±0.28,respectively;the levels of p-STAT3/STAT3 were 1.00±0.11,0.43±0.05,0.49±0.08 and 0.88±0.10,respectively;the levels of p-JAK2/JAK2 were 1.00±0.15,0.54±0.07,0.48±0.07 and 0.79±0.13,respectively.There were statistically significant differences in the above indicators between nintedanib group and Hep3B group,and between nintedanib+si-shp-1 group and nintedanib+Si NC group(all P＜0.05).Conclusion Nintedanib can induce apoptosis and autophagy of hepatocellular carcinoma cells,which may be realized through SHP-1 mediated STAT3 signaling pathway.关键词
尼达尼布/肝癌/凋亡/自噬/非受体蛋白酪氨酸磷酸酶1Key words
nintedanib/hepatocellular carcinoma/apoptosis/autophagy/non-receptor type protein tyrosine phosphatase 1分类
医药卫生
