Effect And Molecular Mechanisms Of HMGB1 Expression Inhibition On Biological Function Of Gastric Carcinoma Cell

BackgroundIn 1973, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and termed high-mobility group(HMG) proteins. High-mobility group box 1(HMGB1) the most abundant and well-studied HMG protein. HMGB1 plays an important role in multiple human diseases, especially cancer and inflammatory diseases. HMGB1, not only inside of the cell as a DNA chaperone, chromosome guardian, autophagy sustainer, and protector from apoptotic cell death, but also outside the cell as cytokine, chemokine, and growth factor, orchestrates the inflammatory and immune response. HMGB1 dysfunction is associated with each hallmark of cancer and contributes to cancer occurrence and development. All of these features make HMGB1 become an important molecular target in cancer research. However, until now there has been little information about the biological role and molecular mechanism of HMGB1 in gastric carcinoma. ObjectiveTo examine the effects of lentiviral-mediated HMGB1-sh RNAi on the biological function of GC cells and to analysis the potential downstream molecules with gene microarray. Western blot were conducted to validate the expression of potential downstream molecules. MethodsFour specific small interfering RNAs of HMGB1(HMGB1-sh RNAs) were designed. Then the lentiviral vectors were constructed named KD1, KD2, KD3 and KD4. Four lentiviral vectors were transfected into SGC-7901 cells with Lipofectamine 2000. RT-PCR and Western blot were performed to determine the effects of KDs on HMGB1 expression. The biological behavior of GC cells were analyzed with the following methods :Proliferation assessed by MTT assay and Brd U assay. Clone tested by Colony formation Assay. Cell cycle and apoptosis demonstrated by flow cytometry. Migration ability determined by Transwell assay.The downstream gene expression was investigated by Gene microarray technique in HMGB1 Knock-down group and Negative control group.The downstream signal pathway was selected and given further analysis. ResultsRT-PCR showed that KD1, KD2 and KD4 significantly inhibited HMGB1 m RNA expression(>75%), KD2 has highest inhibition efficiency. Western blot also confirmed that HMGB1 protein expression was significantly inhibited in KD2 cells compared with Negative Control(NC) and Control(CON). MTT assay and Brd U assay demonstrated cell proliferation was significantly inhibited in KD2 group compared with that in NC and CON group(P<0.05). The colony formation assay show that number of clone in KD2 group was less than that in NC cells(P<0.05). Transwell assay showed that cell migration were significantly inhibited in KD2 compared with CON and NC(P<0.05). The apoptosis rate of KD2 significantly increased comparing with NC(P<0.05). Flow cytometry showed that more cells stayed in S stage in KD2 than NC(P<0.05).The gene microarray analysis showed that 778 genes expression was up-regulated and 587 genes expression was down-regulated though inhibiting HMGB1 expression in GC cells. Bioinformatic analysis showed HMGB1 may plays its biological role by regulating the expression of RAC1、IL1A、TGFBR2、AKT2、DUSP5、MAPK9(JNK2) in MAPK signaling pathway of gastric cancer cells. ConclusionsThe HMGB1-RNAi interfering lentiviral vectors were constructed successfully. With it we got the stable HMGB1 low-expression GC cells. Downregulation of HMGB1 could inhibit cell growth, colony formation and migration ability, but increase cell apoptosis. HMGB1 may play its biological function by regulating MAPK signaling pathway through RAC1、IL1A、TGFBR2、AKT2、DUSP5、MAPK9(JNK2) in GC.