The Potential Significance Of High Mobility Group Box 1 In Hepatocellular Carcinoma Development

Objectives:(1) To identify the expression levels of HMGB1 in liver tissues, serum and peripheral blood mononuclear cell (PBMC) from hepatocellular carcinoma patients. (2) To investigate the effect of high mobility group box-1 protein (HMGB1) on proliferative activity of human hepatoma cell line HepG2 and its potential regulating mechanism. (3) To investigate the effect of specific inhibiting HMGB1 gene expression by small interfering RNAs (siRNA) on the proliferation and apoptosis of human hepatoma cell line HepG2.Methods:(1)Serum and PBMC from healthy control were collected. Similarly, serum, PBMC and liver tissues from HCC patients were also collected. Then, all samples were subjected to Western blot, RT-PCR or immunohistochemical analysis for the expression of HMGB1, respectively. At the same time HE staining was performed to evaluate the feature of liver tissue damage. (2) The cultured HepG2 cells were treated with recombinant HMGB1 (10,50,100ng/ml) or HMGB1-Ab, or both for 24h. Cell proliferation was observed by MTT and trypan blue exclusive analysis. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 protein and mRNA respectively. In situ apoptosis was evaluated by terminal deoxynucleotidyl transferase-deoxyuridine triphosphate nick end labeling (TUNEL) assay. (3) Three specific siRNAs of HMGB1 were designed and synthesized, and transiently transfected into HepG2 cells by Lipofectamine TM 2000.The HMGB1 expression in HepG2 cells was detected by RT-PCR and Western blot after transfection respectively. The proliferation activity in vitro was assessed by MTT assay. Western blot and RT-PCR were used to detect the expression of PCNA and cyclin Dl protein and mRNA respectively. In situ apoptosis was evaluated by TUNEL assay. The concentration of supernatant AFP from the transfected HepG2 cells was detected using chemiluminescence immunoassay (CLIA) method.Results:(1) According to immunohistochemical staining and Western blot analysis, HMGB1 protein levels were significantly overexpressed in carcinoma tissues, as compared to the corresponding non-cancerour tissues from the same HCC patient. Correspondingly, HMGB1 mRNA levels were also elevated in carcinoma tissues, as compared to noncancerous counterparts from the same HCC patient. However, HMGB1 neither protein in serum nor mRNA levels in PBMCs had any difference between HCC patients and healthy controls. (2) Compared with no-treatment, HMGB1 at the concentration of 10ng/ml, 50ng/ml and 100ng/ml obviously increased HepG2 cells proliferation, cyclin Dl and PCNA protein and mRNA expression after treatment for 24 hours respectively (P<0.05). However, when both HMGB1 (10ng/ml) and anti-HMGB1 (20μg/ml) treated HepG2 cells, anti-HMGB1 could significantly inhibit the proliferation effect and cyclin Dl and PCNA mRNA and protein expression of HMGB 1 on HepG2 cells (P<0.05). (3) All of these specific HMGB1-siRNAs (1,2,3) efficiently and specifically inhibited the expression of the HMGB1 gene and the levels of HMGB 1 mRNA were 1.147±0.024,1.014±0.042,0.435±0.055, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in LipofectamineTM2000 alone group (1.411±0.065, P<0.01); Correspondingly, all of these specific HMGB1-siRNAs (1,2,3) could efficiently and specifically inhibit the expression of the HMGB 1 protein and the levels of HMGB1 protein were 0.369±0.035,0.340±0.028, 0.097±0.020, respectively, in HMGB1-siRNAs transfection group, which was significantly lower than that in LipofectamineTM2000 alone group (0.553±0.051, P<0.01). Of the 3 specific HMGB1-siRNAs, HMGB1-siRNA-3 (siRNAH3) had the highest inhibition rate (70-80%). The proliferation of HepG2 cells was markedly inhibited by siRNAH3 transfection. Compared to mock-transfection, seventy-two hours after introduction of siRNAH3 into HepG2 cells, siRNAH3 dramatically suppressed not only the proliferation activity of HepG2 cells but also the expression of PCNA and cyclin D1 mRNA and protein, as well as the secretion of AFP in the culture supernatant (P<0.01). Moreover, siRNAH3 can induce apoptosis by inhibiting HMGB1 expression (P<0.01).Conclusions:(1) It is further identified that HMGB1 levels were significantly elevated in hepatocellular carcinoma tissues. Our data indicate a strong correlation between expression of HMGB1 and HCC development. (2) HMGB1 has potent growth-promoting effect on HepG2 cells proliferation, and anti-HMGB1 can reverse this effect. (3) siRNA targeting HMGB1 mRNA can specifically reduce HMGB1 gene and protein expression. siRNAH3 can effectively suppress the proliferation and induce apoptosis of HepG2 cells by inhibiting HMGB1 expression, providing a novel target for hepatocarcinoma therapy.