| ObjectiveTo detect the expression of high mobility group protein 1(high mobility group box 1, HMGB1) level of human cutaneous squamous cell carcinoma(CSCC) cell line cells SCC13.To explore migration effect of HMGB1 in SCC13 cells in vitro.To explore the effects of HMGB1 on MAPK signal pathway and PI3 K / Akt signal pathway of SCC13 cells and to explore the mechanism of HMGB1-induced migration in SCC13 cells.This study will be of great significance in the seeking the drug which can inhibit the metastasis of CSCC.Methods1. SCC13 cells and A431 cells were regular cultured in vitro, before the trial the cells cultured in serum-free medium for 24 hours, we analysis and compare the levels of HMGB1 in supernatant of samples by Western Blot and enzyme-linked immunosorbent assay(enzyme- linked immunosorbent assay, ELISA). 2. HMGB1-induced migration was tested in different treating time or in condition of different concentrations of HMGB1 in SCC13 cells in vitro by transwell chemokines experiment:(1)The indicated times(3 h, 6 h, 12 h) with the same concentration HMGB1(100 ng/ml) pretreated with SCC13 cells, crystal violet stain under the microscope after taking pictures, statistical polycarbonate film the lower the number of cells, each sample random count 5 horizons(200 x), average;(2) Take SCC13 cells which were regular cultured as control, treated the samples with different concentration of HMGB1(10 ng/ml, 30 ng/ml and 100 ng/ml) respectively under the same treating time(12 h), the same method of counting cells.Each group repeat 3 times. 3. PI3K/AKT and MAPK signaling pathways related proteins: P- AKT, P- p85 PI3 K, P- p42/44 MAPK, P- p38 lightning MAPK were determined by Western Blot.Set up the experimental groups:(1) blank groups: SCC13 cells were regular cultured in vitro;(2) experimental group: SCC13 cells treated with 100 ng/ml HMGB for 24 hours;(3) control group: SCC13 cells treated with 100μM glycyrrhizin(GR), HMGB1 inhibitor for 24 hours;(4) control group: SCC13 cells treated with 100 ng/ml HMGB and 100μM GR for 24 h.Results1. HMGB1 levels in cell supernatants were SCC13 significantly higher than A431 cells, the levels of HMGB1 in the supernatant was detected by Western blot. The relative HMGB1 level(4.8) of SCC13 cells was significantly higher than that in A431 cells(1.2), the difference is statistically significant(P < 0.01); HMGB1 level in SCC13 cell supernatant(483ng/ml) determined by ELISA was also higher that in A431 cells(99ng/ml) markedly. 2. the SCC13 cells were treated with HMGB1(100 ng/ml) for different lengths of time(3, 6 and 12 h),. A significantly higher level of cell migration was evident following incubation for 3 h(P<0.05), however,the highest level was at the 12-h timepoint(P<0.0001).SCC13 cells treated with different concentrations of HMGB1 for 12 h. A significantly higher level of cell migration was evident at 100 ng/ml compared with the blank control group(P < 0.0001). 3. Compared with the control group, an increased expression level of phosphorylated P38, P42/44, AKT and PI3KHMGB1 was evident in HMGB1 group.It suggest that HMGB1 can promote the migration of SCC13 cells via up-regulation of the phosphorylation of p38 MAPK and P42/44 MAPK in MAPK pathway and that of p85 PI3 K and AKT in PI3K/AKT pathway.Conclusion1. SCC13 cells secrete higher HMGB1 levels compared with A431 cells. 2. HMGB1 regulates the migration potential of SCC13 cells in a time- and dose-dependent manner. 3. HMGB1 can induce migration of SCC13 cells by activation of MAPK pathway and PI3K/AKT pathway via an increased level of phosphorylated P38, P42/44, AKT and PI3K. It suggests that HMGB1 can be used as a target for the treatment of skin squamous cell carcinoma metastasis. |
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