| Objective:Cutaneous melanoma is a kind of malignant and aggressive skin tumor that develops from melanocytes with an poor prognosis.As the pathogenesis is still not very clear, there were not any good therapies for it except the traditional surgical method, it is resistant to current chemo-and radiotherapy. Recently, experts in the field of cancer therapy have set off a new upsurge in the study of autophagy which mediated the anticancer therapy. High mobility group protein B1(HMGB1) is an important protein autophagy activator, which is important in the tumorigenesis. In previous studies, it was found that the expression of HMGB1 was increased in many tumor cells, including melanoma, but its extracellular role and signaling pathway is still not very clear in the tumors. Therefore, this study aims to investigate: 1. The influence of extracellular HMGB1 on autophagy in mouse melanoma B16 cells. 2. to determine the MEK/ERK signaling pathway involved in the process of autophagy regulated by HMGB1 in the CB16 cells.Methods: the mouse melanoma B16 cells was treated with drugs and were transfected transiently with sh RNA, then CCK-8 tested the cell viability and the Western blot analysis the molecular markers of the autophagy-related(ATG) proteins LC3-II, LC3-I and autophagic degradation protein p62 and the molecule of MEK1, ERK1 / 2 and p ERK1 / 2 expression associated with the MEK / ERK signaling pathway.to further explore the effect of HMGB1 on autophagy and its related mechanism..1 the mouse melanoma B 16 cells were divided into different groups, the experimental group cells were treated with different levels of HMGB1 for 24 hours,while the control group was not treated with HMGB1.then CCK-8 tested the cell viability, The then CCK-8 tested the cell viability and the western blot detected the expression of autophagy related molecules of LC3-II, LC3-I and p62. 2. To further validate the HMGB1 role in autophagy of B16 mouse melanoma cells, the cells in the experimental group was treated with HMGB1 sh RNA, the control group with negative control sh RNA,Western blotting analysis to detect the expression of HMGB1 and autophagy related protein LC3 II,LC3-I p62 expression changes. 3. Two groups of cells were selected in different ways to deal with the cell. The experimental group was treated with recombinant HMGB1 24 h, and the control group was not treated. Western blotting analysis the autophagy related proteins and the major signaling molecules of the MEK/ERK signaling pathway. 4. the cells were divided into two groups: experimental group was treated with recombinant HMGB1 and 3-MA group and the control group with only HMGB1, Western blotting analysis the autophagy related proteins and the major signaling molecules of the MEK/ERK signaling pathway. 5.with the transfection technique by sh RNA MEK1/2 to knockout the MEK1/2 gene expression of the mouse melanoma B 16 cell and the Western blotting analysis theautophagy related proteins and the major signaling molecules of the MEK/ERK signaling pathway.Results: 1. HMGB1 promotes the proliferation of mouse melanoma B16 cells, and increases with the HMGB1 concentration. compared with the blank control group, the expression levels of autophagy related protein LC3 II and LC3 I in the cells treated by recombinant HMGB1 were significantly higher, while the expression of p62 decreased. 2.the expression levels of autophagy related protein LC3 II and LC3 I in the group that treated with the HMGB1 sh RNA were significantly lower than those in the control group, while the expression of p62 was increased. 3 compared with the control group, the expression of LC3 II and LC3 I, MEK1/2 and p ERK1/2 in the cells treated by HMGB1 was significantly higher than that in the control group. 4 the expression of LC3 II and LC3 I, MEK1/2 and p ERK1/2 in the group that treated with autophagy inhibitor 3-MA was significantly lower than that in the control group. 5. Compared with the control group, the expression of LC3 II, LC3 I, MEK1/2 and p ERK1/2 in the group that treat with MEK1/2 sh RNA was significantly lower than that in the control group.Conclusion: HMGB1 promotes autophagy of mouse B16 melanoma cells by MEK/ERK signaling pathway. |
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