Down-regulation Of GADD45A Expression Enhances Chemosensitivity By The Inhibition Of DNA Repair Activity And Induction Of Apoptosis Via MAPK Pathway In Melanoma

Background: Melanoma is an extreme malignant skin cancer with high recurrence,high metastasis and considerable drug resistance.So far,the most effective means of cancer treatment is surgery,radiotherapy,chemotherapy and immunotherapy.Chemotherapy is a kind of systemic treatment,chemical drugs can throughout the body organs and tissues by circulation of blood.As a result,chemotherapy is the main treatment of high metastatic malignant melanoma.Cisplatin is a kind of platinum chemotherapy drugs commonly used to treat cancer,it can cause cell DNA damage,induce apoptosis and inhibit the growth of cell proliferation,thus killing the cancer cells.But the tumor cells can induce chemotherapy drug resistance by inhibiting drugs resorption and the enhancing of DNA damage repair.For chemotherapy drug resistance of DNA damage repair mechanism is unclear,find new effective targets for enhancing tumor cells to chemotherapy sensitivity is of great significance.GADD45A(Growth Arrest and DNA damage-inducible 45 A)is the key to the DNA damage repair genes,plays an important role in cell apoptosis and cell cycle regulation.Usually,damage factors affect the induction of GADD45 A by DNA damage repair pathways.Therefore,GADD45 A gene plays an important role in tumor cells resistance.Objective:This research mainly study the role of GADD45 chemotherapy drug resistance mechanism by silencing GADD45 A DNA damage repair gene expression in melanoma,and can be used as a new targets to enhanced melanoma chemotherapy sensitivity,so achieving the effect of cure.Methods:1.The mrna expression of 84 DNA damage repair genes in melanoma a375 cells was detected by qpcrarray.2.A375 cells were transfected with si RNA,and the cell model of GADD45 A down-expression was obtained by using small RNA interference technology.3.Human melanoma A375 cells were treated with cisplatin.The expression of GADD45 A was analyzed using Western blot and Real-time q PCR.4.Cell survival and proliferation were evaluated using MTT assay and colony formation.5.The cell cycle and apoptosis were detected by flow cytometry.6.Using comet assay and immunofluorescence assay to detect the expression of DNA damage and DNA damage related protein gamma H2 AX in A375 cells under the cisplatin treatment.Results:1.The PCR array results showed that the expression level of m RNA gene in 84 kinds of DNA repair genes was increased by more than two times,and the expression level of GADD45 A gene was increased by more than 5 times after was activated in A375 cells.2.Results showed that the expression of GADD45 A was increased in a dose-and time-dependent manner.3.Using interference RNA technique,the cell model of GADD45 A expression was successfully obtained.4.Cell survival and proliferation were evaluated using MTT assay and colony formation.5.Flow cytometry revealed that suppression of GADD45 A by si RNA reduced cisplatin-induced G2/M arrest and enhanced apoptosis in A375 cells,resulting in the reduction of cisplatin resistance.6.Further,DNA damage cells were observed using comet assay and ?H2AX immunofluorescence assay.We revealed that GADD45 A combined with Cdc25 A and suppressed the expression of Cdc25 A,resulting in G2/M arrest.Use of a MEK inhibitor showed that GADD45 A was regulated by the MAPK pathway.Conclusion: Our data suggest that GADD45 A plays important roles in the induction of cisplatin resistance in human melanoma cancer.GADD45 A may serve as a novel molecular target for the therapy of malignant melanoma cancer in the future.