The correlation between the high expression of C/EBPγ and the proliferation and migration of liver cancer cells / 中华检验医学杂志

ABSTRACT

Objective:To investigate the levels of C/EBPγ mRNA in liver cancer patients and the effect and mechanism of C/EBPγ on proliferation and migration of HepG2 cells.Methods:This experiment was performed in Department of Clinical Laboratory of Shanghai Songjiang District Central Hospital in vitro from October of 2018 to April of 2019. The pLKO.1 was the control group and shg1/shg2 were interference groups. The transcriptional levels of C/EBPγ in liver cancer were analyzed through Oncomine database and real-time PCR. HepG2 and Hep3B cells were cultivated in DMEM media with 10% FBS at 37℃ and 5% CO 2. Adenovirus vector shC/EBPγ-pLKO.1 was constructed and infected 293T cell combined with packaging carrier pMD2G, pMDLG/REE and pRSV/Rev. The empty vector pLKO.1 served as control. The virus supernatant was collected at 12 h and infected HepG2 cells four times. Puromycin were added at the concentration of 2 μg/mL and cells were selectively cultured for 48-72 h. The puromycin concentration was changed into 1 μg/mL after the selection. Growth curves were used to detect cell proliferation. ROS levels were determined by using DFH-DA assays. The oxidative stress related genes transcription changes were detected by using real-time PCR. The migration of HepG2 cells was assayed through scratch assay. NAC (1 mmol/L) were added to cells against oxidative stress. Results:The mRNA levels of C/EBPγ were much higher in liver cancer patients and cells than normal controls. The proliferation rates of C/EBPγ interference groups at 4 d (shg1: 1.93±0.70, shg2: 1.28±0.40) and 6 d (shg1: 3.05±0.90, shg2: 1.31±0.60) were lower than control groups (4 d: 6.18±0.80, 6 d: 22.41±2.56) significantly ( F=1 507.971, P<0.05) . Reactive oxygen species (ROS) levels were elevated in C/EBPγ interference groups and the transcription level of oxidative stress related genes (HPRT1, NQO1-tv2 and NQO1-tv4) was increased at 12 h and 24 h when the cells were cultured without serum. The scratch assays showed that non-healing area in C/EBPγ interference groups (shg1: 174 922.58±1 239 376.00,shg2: 1 374 656.00±248 882.79) was larger than control groups (66 690.82±1 278 954.00) ( F=39.871, P<0.05) . When the antioxidant NAP was added, the proliferation rates were elevated at 6 h and the ROS levels were decreased at 0.5 h, 1 h and 3 h ( F=7.587, 4.657, 3.903, P<0.05) . Conclusions:C/EBPγ mRNA levels were increased in liver cancer. When C/EBPγ was disturbed in HepG2 cells, the proliferation and migration were decreased, the ROS levels and oxidative stress related genes were elevated significantly, which indicated that C/EBPγ promoted proliferation and migration through reducing ROS levels in liver cancer.