Y Liu1, Y-L Liu, W Cheng, X-M Yin, B Jiang. 1. Department of Hepatobiliary Surgery, Hunan Provincial People's Hospital, the First Affiliated Hospital of Hunan Normal University, Changsha, China. 13319587618@163.com.
Abstract
OBJECTIVE: To observe the SIRT 3 expression in primary hepatocellular carcinoma (HCC), and then establish the eukaryotic expression vector of SIRT3 to observe the proliferation and apoptosis of pZsGreen-c1-SIRT3 HepG2 cells. Furthermore, we explored the mechanism of SIRT3 in inhibiting HCC. PATIENTS AND METHODS: Immunohistochemistry was used to detect the expression of SIRT3 in the tumor tissue and para-tumor tissue in 32 patients with HCC and the normal liver tissue in 10 patients. The mRNA of SIRT3 from the normal liver tissue was used as a template, reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain the total sequence of SIRT3 gene, and then the gene was cloned and combined with vector pZsGreen-c1, liposome transfection technology was used to transfect the recombined plasmid into HepG2. The cells were divided into three groups: group A (HepG2 cells as a blank control group), group B (pZsGreen-c1 HepG2 cells as an experimental control group) and group C (pZsGreen-c1-SIRT3 HepG-2 cells as an experimental group). MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was used to detect the growth and proliferation of cells in 3 groups; annexinV/PI double staining was used to detect the apoptotic rates of cells in 3 groups. Western blot was used to detect the protein expression of SIRT3, Fas, Bax and P53, and water-soluble tetrazolium salt (WST-1) was used to detect MnSOD content in 3 groups. RESULTS: Immunohistochemistry results showed that SIRT3 in the tumor tissue sample was positive in 19 patients out of 32 HCC patients; however, there was no strong positive case, the positive rate of SIRT3 expression was 59.38% (19/32). SIRT3 in the para-tumor tissue was positive in 31 HCC patients, the positive rate was 96.88% (31/32), and 18 cases were strongly positive; SIRT3 in normal liver tissue was positive in all 10 cases, the positive rate was 100.0% (10/10), and 7 cases were strongly positive. The differences of SIRT3 positive rate and positive score in tumor tissue from para-tumor tissue and normal liver tissue were statistically significant (p<0.05). However, the differences between para-tumor tissue and normal liver tissue were not statistically significant (p>0.05). After pZsGreen-c1-SIRT3 transfection, MTT results showed that the OD values in 3 groups were increased with the time, showing time-dependent manner. At 48 h after culture, the OD values in-group C were significantly different from group A and B, and the inhibitory rates were statistically different (p<0.05). After 48 h, the OD values and inhibitory rates in-group C showed that the cells were obviously inhibited, and the inhibitory rates were increased with the time, showing time-dependent manner. Flow cytometry results showed that the cell numbers of early stage apoptosis and late stage apoptosis were significantly increased in group C, which was significantly higher than group A and B, the differences were statistically significant (p<0.01). Western blot results showed that expression levels of SIRT3, p53, Bax and Fas were not different between group A and B (p>0.05); SIRT3, p53, Bax and Fas in group C were significantly increased, which were statistically higher than group A and B (p<0.01). WST-1 results showed that MnSOD contents were not statistically different between group A and B (p>0.01). MnSOD content in-group C was significantly higher than the other groups, which were statistically significant (p<0.01). CONCLUSIONS: SIRT3 shows low expression or deficiency in HCC tissue, indicating that SIRT3 expression can affect the occurrence and development of HCC. SIRT3 can inhibit the growth and proliferation of HepG2 cells and induce HepG2 cell apoptosis. The mechanism may be related to the up-regulation of MnSOD and p53, the up-regulation of Bax and Fas by MnSOD.
OBJECTIVE: To observe the SIRT 3 expression in primary hepatocellular carcinoma (HCC), and then establish the eukaryotic expression vector of SIRT3 to observe the proliferation and apoptosis of pZsGreen-c1-SIRT3 HepG2 cells. Furthermore, we explored the mechanism of SIRT3 in inhibiting HCC. PATIENTS AND METHODS: Immunohistochemistry was used to detect the expression of SIRT3 in the tumor tissue and para-tumor tissue in 32 patients with HCC and the normal liver tissue in 10 patients. The mRNA of SIRT3 from the normal liver tissue was used as a template, reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain the total sequence of SIRT3 gene, and then the gene was cloned and combined with vector pZsGreen-c1, liposome transfection technology was used to transfect the recombined plasmid into HepG2. The cells were divided into three groups: group A (HepG2 cells as a blank control group), group B (pZsGreen-c1 HepG2 cells as an experimental control group) and group C (pZsGreen-c1-SIRT3 HepG-2 cells as an experimental group). MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was used to detect the growth and proliferation of cells in 3 groups; annexinV/PI double staining was used to detect the apoptotic rates of cells in 3 groups. Western blot was used to detect the protein expression of SIRT3, Fas, Bax and P53, and water-soluble tetrazolium salt (WST-1) was used to detect MnSOD content in 3 groups. RESULTS: Immunohistochemistry results showed that SIRT3 in the tumor tissue sample was positive in 19 patients out of 32 HCC patients; however, there was no strong positive case, the positive rate of SIRT3 expression was 59.38% (19/32). SIRT3 in the para-tumor tissue was positive in 31 HCC patients, the positive rate was 96.88% (31/32), and 18 cases were strongly positive; SIRT3 in normal liver tissue was positive in all 10 cases, the positive rate was 100.0% (10/10), and 7 cases were strongly positive. The differences of SIRT3 positive rate and positive score in tumor tissue from para-tumor tissue and normal liver tissue were statistically significant (p<0.05). However, the differences between para-tumor tissue and normal liver tissue were not statistically significant (p>0.05). After pZsGreen-c1-SIRT3 transfection, MTT results showed that the OD values in 3 groups were increased with the time, showing time-dependent manner. At 48 h after culture, the OD values in-group C were significantly different from group A and B, and the inhibitory rates were statistically different (p<0.05). After 48 h, the OD values and inhibitory rates in-group C showed that the cells were obviously inhibited, and the inhibitory rates were increased with the time, showing time-dependent manner. Flow cytometry results showed that the cell numbers of early stage apoptosis and late stage apoptosis were significantly increased in group C, which was significantly higher than group A and B, the differences were statistically significant (p<0.01). Western blot results showed that expression levels of SIRT3, p53, Bax and Fas were not different between group A and B (p>0.05); SIRT3, p53, Bax and Fas in group C were significantly increased, which were statistically higher than group A and B (p<0.01). WST-1 results showed that MnSOD contents were not statistically different between group A and B (p>0.01). MnSOD content in-group C was significantly higher than the other groups, which were statistically significant (p<0.01). CONCLUSIONS:SIRT3 shows low expression or deficiency in HCC tissue, indicating that SIRT3 expression can affect the occurrence and development of HCC. SIRT3 can inhibit the growth and proliferation of HepG2 cells and induce HepG2 cell apoptosis. The mechanism may be related to the up-regulation of MnSOD and p53, the up-regulation of Bax and Fas by MnSOD.