Role Of HMGB1 In Proliferation And Invasion Of Hepatocellular Carcinoma Cells And Its Mechanism

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignant tumor worldwide, affecting approximately one million people around the world every year. HCC is the third most frequently diagnosed cancer in China, but the second most frequent cause of cancer death. Surgery including liver transplantation is the mainly curative modality for HCC. However, despite resection with curative intent, the clinical course is variable and the long term prognosis is poor with reported5-year survival rates ranging from17%to53%. Therefore, to find the related genes and understand their role and mechanisms would be crucial for prevention and treatment of HCC.High mobility group box1(HMGB1) is a nonhistone chromosomal protein, which is implicated in diverse biological processes including DNA replication, repair, recombination and transcription. During cell damage and inflammation, HMGB1can be released into the microenvironment to participate in extracellular signalling transduction regulating cell growth and differentiation. Recent studies showed that HMGB1was highly expressed in many kinds of cancer cells and tissues, including human gastrointestinal adenocarcinoma, colorectal cancer and breast cancer, which promoted cancer development by enhancing cell proliferation, migration and invasion. These suggested that HMGB1may represent a potential target for the therapeutic intervention in cancer. The serum HMGB1level in HCC was significantly higher than those in chronic hepatitis, liver cirrhosis and healthy control, suggesting that high HMGB1may be a useful marker for evaluating the tumour stage and predicting prognosis in HCC.However, the function and mechanism of HMGB1in tumors is still unclear, especially in HCC. In this study, we tried to figure out the effect of HMGB1on HCCLM3cell proliferation, invasion and the mechanisms involved.1) The specific siRNAs were designed and transfected into HCCLM3cells to knockdown HMGB1, then RNAi efficiency and its effect on the proliferation, apoptosis, cell cycle, migration, invasion of HCCLM3cells were analysed in vitro.2) Stable HMGB1knockdown cells were established, expression of HMGB1was stably suppressed by vector-based transfection of specific short hairpin RNA (pMKO.1) in HCCLM3. The cells were subcutaneously implanted into right-flanks of mice, subsequently the tumor volume were measured every five days to detect the role of HMGB1in HCC cell growth in vivo. 3) We analyzed the alterations of MAPKs and NF-κB pathway, and the expression of p21waf/cip1and MMP2in HMGB1knockdown cells to understanding the mechanisms of HMGBl in HCC cell growth and invasion.The results were as follows.1) Knockdown of HMGBl in HCCLM3cells by specific siRNA caused growth inhibition, apoptosis and S-phase cell cycle arrest, and inhibited cell migration and invasion in vitro.2) Stable knockdown of HMGB1in HCCLM3cells inhibited the growth of it’s xenograft in Balb/c athymic mice.3) HMGB1knockdown reduced the activation of MAPKs, including ERK1/2, p38and SAPK/JNK, as well as the MAPKKs (MEK1/2, SEK1), and the substrates (c-Jun, c-Myc), and down-regulated NF-KB/p65expression and it’s phosphorylation level at Ser536. In contrast, re-exprssion of HMGB1in the knockdown cells enhanced the phosphorylation level of MEK1, p44/p42, p38, c-Myc and NF-KB/p65, except SEK1, SAPK/JNK and c-Jun.4) p21was up-regulated after stable knockdown of HMGB1in HCCLM3cells, while down-regulated by re-exprssion of HMGB1.5) Knockdown HMGB1decreased MMP2activity in HCCLM3, and the activity of MMP2was enhanced by overexpression HMGB1in stable knockdown cells.In conclusion, HMGB1can promote the proliferation and invasion of HCCLM3cell, partly by enhancing MAPKs and NF-κB pathway, down-regulating p21and up-regulating MMP2. The inhibitory effect of HMGB1on p21expression maybe p53independent, while employ MAPKs/c-Myc pathway. c-Myc is a key transcription factor at the end of MAPKs pathway, and also an important negative regulator of p21. HMGBl can activate MAPKs and c-Myc, therefore inhibits the expression of p21. However, the more function and involved mechanisms of HMGB1in HCC growth and development still need further investigation.