BRCA1/2Gene And Drug Sensitivity Of Eμ-Myc P19^Arf-/- Cells And Its Expression In Breast Cancer Clinical Singnificance

DNA damage response （DDR）, which is essential for maintaining genome integrity, isthe main defense system to the exogenous injury for cells. Breast cancer susceptibilitygene1（breast cancer susceptibility gene-1, BRCA1） and breast cancer susceptibilitygene2（breast cancer susceptibility gene-2, BRCA2） play an important role inhomologous recombination repair of DNA double-strand breaks. Mutations of BRCA1orBRCA2gene would cause DNA double-strand break repair defects. The accumulation ofsomatic mutations may cause many kinds of cancers such as breast cancer and ovariancancer. Considering the important role in DNA damage repair that BRCA1, BRCA2gene played, we designed this study to investigate the possible consequence of abnormalBRCA1or BRCA2gene expression in tumor cells and breast cancer patients.Part1Effect of BRCA1/2knockdown on drugs sensitivity inEμ-Myc p19^Arf-/- cellObjective: Construction of BRCA1/2knockdown Eμ-Myc p19^Arf-/- cell through RNAi.Investigate the change of drug sensitivity after BRCA1/2knockdown in Eμ-Mycp19^Arf-/- cell.Methods: We used the retroviruses expressing BRCA1and BRCA2shRNA to transfectEμ-Myc p19^Arf-/- cell. Then use more than30drugs to test the change of sensitivity ofeach drug.Results: The BRCA1/2were successfully knocked down in Eμ-Myc p19^Arf-/- cell line.The sensitivities of seven drugs, DDP, CPT, SAHA, AZD2281, Ara-C, clofarabine and5-FU significant enhanced after BRCA1gene knockdown. And sensitivities of DDP, CPT,17-AAG, AZD2281, TFT also significant enhanced after BRCA2gene knockdown.Conclusions: BRCA1knockdown made Eμ-Myc p19^Arf-/- cell more sensitive to DDP,CPT, SAHA, AZD2281, Ara-C, clofarabine and5-FU. And BRCA2knockdown madeEμ-Myc p19^Arf-/- cell more sensitive to DDP、CPT、17-AAG、AZD2281、TFT. That maybecome a new therapy selection for BRCA1/2related cancer patients.Part2Effect of cytarabine in BRCA1/2-deficient Eμ-Myc p19^Arf-/- cell line on DNA damage,cell cycle and apoptosisObjective：Test the change of the expression of γH2AX, cell cycle and apoptosis inBRCA1/2-deficient Eμ-Myc p19^Arf-/- cell line after treatment of cytarabine.Methods: Transfected the Eμ-Myc p19^Arf-/- cell with BRCA1and BRCA2-shRNA byretrovirus-mediated transfection. Treat the cell with appropriate concentration ofcytarabine. Test the expression of γH2AX through western blot, and test the change ofcell cycle and apoptosis in the cell after treatment of cytarabine through flow cytometer.-4- Results: After treatment of cytarabine, the expression ofγH2AX was significantincreased, and the expression ofγH2AX was higher in BRCA1-deficient Eμ-Mycp19^Arf-/- cells. But the expression ofγH2AX was equal in BRCA2-proficient andBRCA2-deficient Eμ-Myc p19^Arf-/- cells after treatment of cytarabine. Both of theBRCA1/2-deficient and the BRCA1/2-proficient Eμ-Myc p19^Arf-/- cells showedsignificant G1phase cycle arrest and apoptosis. And the BRCA1-deficient Eμ-Mycp19^Arf-/- cells showed an increased early and late apoptosis rate compared toBRCA1-proficient cell. But the BRCA2-deficient Eμ-Myc p19^Arf-/- cell fail the showedany difference in apoptosis as BRCA2-proficient one.Conclusions: The treatment of olaparib would lead to accumulation of DSB in Eμ-Mycp19^Arf-/- cells. After treatment of cytarabine, BRCA1/2-deficient Eμ-Myc p19^Arf-/- cellsshowed cell cycle arrest in G1followed by apoptosis.Part3Expression of BRCA1/2in breast cancer and its clinicalsignificanceObjective: To detect expression of BRCA1/2in breast cancer by immunohistochemistry（IHC） and explore their correlation with clinicopathological factors. And discuss thepotential of these two molecules being prognostic and predictive factor for breast cancerpatients.Methods:434cases of breast cancer tissue and135cases of adjacent noncanceroustissue were collected to construct tissue microarrays （TMAs）. Use IHC to detectBRCA1/2expression. Collect the relevant clinical information to perform correlationanalysis.Results: The expression of BRCA1and BRCA2were significant higher in adjacentnoncancerous tissue than in breast cancer tissue. BRCA1expression was significanthigher in axillary lymph node metastasis negative group, PR positive group andnon-triple negative breast cancer group. BRCA2expression was significant higher in PRnegative group and triple negative breast cancer group. In multivariate analysis, PRdemonstrated significant association with BRCA1. No significant difference was foundin overall survival of the patients with or without BRCA1/2expression. Clinical stagesand PR expression were independent prognostic factors for breast cancer patients.Conclusions：BRCA1and BRCA2expression were significant lower in breast cancertissue. Lymph node metastasis status, PR stasus and whether or not triple negative breastcancer was significant associated with BRCA1expression. PR status and whether or nottriple negative breast cancer was significant associated with BRCA2expression. Clinicalstages and PR expression were independent prognostic factors for breast cancer patients.