Experimental Study Of AKAP12 Regulating Breast Cancer Cell Proliferation And Radiation Sensitivity

Objective To explore the influence of AKAP12 on the proliferation and radio-sensitivity of human breast cancer cells and its relevant mechanism.Methods Bioinformatics network was used to analyze AKAP12 expression in different molecular subtypes of breast cancer.AKAP12 protein expression in different molecular subtypes of breast cancer were examined by western blot.Tumor cell were infected with lentiviral vectors,after that AKAP12 protein was interfered or overexpressed.Western blot was used to detect AKAP12 protein expression in tumor cells after interference or exogenous overexpression of AKAP12.The influence of interference and overexpression of AKAP12 on cells proliferation was measured by EdU cell proliferation assay,CCK-8 cell proliferation assay and cell plate clone formation assay,respectively.Western blot was used to detect the expression of the proteins participated in cell cycle and apoptosis mechanism related to proliferate after interference or exogenous overexpression of AKAP12 in breast cancer.After Irradiated cells with X-ray in vitro,DNA double strand breaks(DSBs)labeled protein ?-H2AX in tumor cells was examined at different time points by immunofluorescence.Interference with AKAP12 expression and corresponding interference with empty vector of tumor cells were Irradiated with X-ray.Then western blot was used to detect cell cycle regulation related proteins and double strand break marker proteins.Results According to the bioinformatics analysis of breast cancer,the expression level of AKAP12 in different molecular subtypes of breast cancer cell lines was significant statistical different,which include that the expression of AKAP12 in luminal breast cancer cell lines(such as MCF-7)was higher than that in triple negative breast cancer cell lines(such as MDA-MB-231).The expression of AKAP12 protein in different molecular type breast cancer cell lines cultured in vitro is consistent with that outcome derive from bioinformatics analysis.The expression of AKAP12 in breast cancer cell lines is related to molecular subtype.Western blot confirmed that cells were successfully transfected.Estrogen receptor positive MCF-7 cell line was relatively high expression.Interfering with the AKAP12 protein in MCF-7 cells could accelerate the cell proliferation rate.Western blot showed that the expression of CyclinD1 was upregulated by downregulating the expression of p21,p27 and CyclinDl.Then accelerate G1/S phase transformation of tumor cells to promote the proliferation of MCF-7 cells.Low expression of AKAP12 protein in MDA-MB-231 of TNBC cells,AKAP12 accelerates the proliferation of MDA-MB-231 cells after Exogenous overexpression.Western blot analysis showed that exogenous overexpression of AKAP12 could promote the proliferation of MDA-MB-231 cells by downregulating the expression of p21,p27,CyclinDl and Bcl-2/Bax.Simultaneously,exogenous overexpression of AKAP12 could accelerate the G1/S phase transformation of tumor cells and increase the resistance to apoptosis.After cell treated by irradiated at room temperature,the repair time of DNA double strand break(DSB)in MCF-7 cells interfering with AKAP12 expression was longer than vector group.Western blot showed that interfering with AKAP12 expression could bring MCF-7 cells about escaping from G1/S cycle checkpoint by destroying G1/S cycle checkpoint of MCF-7 cellsConclusion AKAP12 affects the proliferation of breast cancer cells by regulating Gl/S cycle checkpoint.Interfering with the expression of AKAP12 promotes the proliferation of MCF-7 cells(ER+,wild-type p53)by accelerating G1/S cycle transformation.Exogenous overexpression of AKAP12 promotes the proliferation of MDA-MB-231 cells(ER-,PR-,HER-2,mutant p53)by accelerating G1/S cycle transition and increasing apoptosis resistance.Otherwise,interfering with the expression of AKAP12 prompted MCF-7 cells escape from the G1/S cycle checkpoint,thus increasing their radio-sensitivityFigure 15 table 12 reference 58...