Satb1 And Mir-34a Cutaneous Basal Cancer Cells And The Mechanism Research

Every living organism on the surface of the earth is exposed to the ultraviolet (UV) fraction of the sunlight. This electromagnetic energy has both life-giving and life-endangering effects. Superfluous UV radiation can damage DNA and mutagenize several genes which are involved in tumor suppressive, oncogenic, and cell-cycle control signaling pathways and induce the initiation, promotion, and development of the skin cancer. With the destruction of the ozone layer, UV irradiation increases gradually, so the incidence of skin basal cell cancer is on the rise around the world. In view of that there is many disadvantages in the current clinical treatment for skin cancer, we hope to explore the molecular mechanism of skin basal cell cancer induced by UV to provide new clues for the clinical diagnosis and treatment of skin basal cell cancer.Pathways underlining UV-induced DNA damage and cell apoptosis in basal cell carcinomas is centred by the activated antiapoptotic gene Bcl-2and mutated p53tumor-suppressor gene. Previous studies showed that microRNA-34(miR-34a) family to be a direct target of p53, functioning downstream of the p53pathway as tumor suppressors. MiR-34a was identified to represent the status of p53and participate in initiation and progress of cancers. Bcl-2is a key rerulator of apoptosis, and previous work has demonstrated that SATB1is positively correlated with Bcl-2expression and regulate its function. So we hypothesize that miR-34a and SATB1both are involved in the skin basal cell carcinomas induced by UV and the two may have regulatory linkage.We first detect expression of SATB1and miR-34a in human basal cell tumor and control tissues, and in human basal cancer cell model A431cell line using real-time PCR and Immunochemistry staining method. We found that both SATB1and miR-34a expressions are reversely correlated in human basal cell tumor tissues and A431cells, suggesting potential regulatory linkage between SATB1and miR-34a. What more, the significantly increased SATB1expression in the detected tumor tissues suggest that SATB1is related to human basal cell cancer. To detect the influence of SATB1and miR-34a on the pathology of human basal cancer, we performed FCA, MTT and Trans-well assay in geneticly modified A431cells. SATB1and miR-34a were found to affect the invasion, proliferation and apoptosis of A431. Subsequently, to test if S ATB1and miR-34a affect the normal skin basal cell Hacat activity in the respose of UV, we performed comet assay, FCA and Hoechest staining analyses, and found that SATB1can increase the degree of DNA damage and obviously decrease the survival ratio, while miR-34a can decrease the degree of DNA damage caused by UV. To further explore how SATB1and miR-34a may connect to each other, we first performed bioinformatic analysis, which suggests SATB1as a potential target of miR-34a. Further luciferase reporter assay, real-time PCR, Western-blot, ChIP and EMSA assay were performed to reveal the precise regulatory mechanisms. Firstly, we constructed the miR-34a expression plasmid and wild-type or mutant form of SATB13'UTR reporter plasmids. Luciferase activity analysis showed that miR-34a directly target SATB13'UTR. We then overexpressed/inhibited the miR-34a in Hacat cells and used real-time PCR and Western boltting to detect the expression of SATB1.The result showed that miR-34a represses the expression of SATB1on both mRNA and protein levels. Interestingly, using SATB1overexression or knockdown cells, we found that SATB1positively control the expression of miR-34a. Further luciferase reporter assay, CHIP and EMSA analyses confirmed the binding of SATB1at miR-34a in vitro and in vivo and suggested that the256-579nt fragment in miR-34a promoter and the AT rich sequences within it as critical for SATB1binding and regulation. Finally, we show that stimulation of Hacat cells with UV results in increased binding activity of SATB1to the miR-34a promoter, suggests a feedback regulation of SATB1and miR34a for the protection against UV stimulation.In summary, our investigation first reveals that SATB1and miR-34a participate in the skin basal cancer induced by UV and forms regulatory feedback loop with each other.