8-chloro-adenosine Inhibits The Proliferation,migration And Invasion Of Breast Cancer Cells By Down-regulating RNA Editing Enzyme ADAR1

Objective:By studying the effect of 8-Chlor-Adenosine on the proliferation,migration and invasion of breast cancer cells and the transcriptional regulation mechanism of ADAR1 promoter in breast cancer,search for the mechanism of 8-Cl-Ado in anti-breast cancer and the corresponding new target of targeted therapy,and lay a reliable scientific foundation for 8-Cl-Ado treatment of breast cancer.And the new scientific basis and research basis of targeted gene therapy.Methods:Different breast cancer cell lines MCF-7 and MDA-MB-231 were treated with 10μmol/L 8-Chloro-Adenosine for different times.Cell proliferation was detected by CCK-8 and plate cloning.Cell migration and invasion were detected by Transwell technology.The cycle and apoptosis were detected by flow cytometry.CCK-8 was used to detect cell proliferation and Transwell to detect cell migration and invasion after ADAR1 overexpression.After MDA-MB-231 and MCF-7 were treated with 10 μmol/L8-Cl-Ado for different times,ADAR1 m RNA expression level was detected by RT-PCR,and ADAR1 protein expression level was detected by Western blotting.The PE-GFP-N1 fluorescence vector of Ad AR1 promoter and the corresponding double luciferase reporter vector were constructed.After 48 h treatment with 10 μmol/L 8-Cl-Ado,the corresponding GFP protein expression level was detected by Western blotting,and the fluorescence intensity of the cells was measured by inverted microscope.The transcription factor fragments were found and bioinformatics analysis was performed to determine the transcription factor;Double Luciferase Report Assay and Chromatin Immunoprecipitation were used to verify transcriptional activity.Transcription factor knockdown is used to detect cell migration and invasion.Results:1.10 μmol/L 8-Cl-Ado inhibited the proliferation,migration and invasion of breast cancer cell lines MCF-7 and MDA-MB-231;2.In breast cancer cell lines MCF-7 and MDA-MB-231,the overexpression of ADAR1 can enhance their proliferation,migration and invasion ability;3.The expression levels of ADAR1 protein and m RNA in breast cancer cells decreased gradually with the increase of 8-Cl-Ado treatment time;4.After 8-Cl-Ado treatment of different fragments of ADAR1 promoter fluorescence vectors,the fluorescence intensity was changed,and the promoter fragments containing related regulatory transcription factors could be found;5.The double luciferase reporting experiment confirmed that the transcription factor was PU.1;6.Chromatin immunoco-precipitation verified the transcription factor PU.1;7.PU.1 knockdown affects the proliferation,migration and invasion of breast cancer cells;Conclusions:1.8-Cl-Ado could inhibit the proliferation,migration and invasion of breast cancer cell lines MCF-7 and MDA-MB-231,and the effect was better at 48 h and 72h;2.ADAR1 can regulate the proliferation,migration and invasion of breast cancer cell lines MCF-7 and MDA-MB-231;3.8-Cl-Ado can affect the proliferation,migration and invasion of breast cancer cell lines MCF-7 and MDA-MB-231 by affecting the expression of ADAR1;4.8-Cl-Ado regulates proliferation,migration and invasion of breast cancer cells MCF-7 and MDA-MB-231 by affecting transcription factors on ADAR1 promoter to down-regulate ADAR1;5.PU.1 regulates the proliferation,migration and invasion of breast cancer cells MCF-7 and MDA-MB-231;6.8-Cl-Ado regulates the proliferation,migration and invasion of breast cancer cells through PU.1 and ADAR1.