RAD51C Gene And Drug Sensitivity Of Eμ-Myc P19^Arf-/- Cells And Its Expression In Breast Cancer Clinical Significance

Homologous recombination （HR） is a fundamental cellular process conserved in allorganisms.It maintains genome integrity by repairing endogenous and exogenous DNAdouble-strand breaks （DSBs）. Cells rely on two major pathways to repair DSBs: HR, theerror free mechanism of DNA repair, and non-homologous end joining, which canintroduce changes in the DNA sequence at the repair site. HR is necessary for most of thecells.RAD51C plays an important role in DNA damage response （DDR）. It acts as a transducerof the damage signal to ensure engagement of the HR pathway of repair. RAD51Clocalizes to the sites of DNA damage few minutes after the damage appeared, indicatingthat RAD51C has a role in the early stage of HR. Besides, RAD51C is required forphosphorylation of CHEK2by ATM, which is required for checkpoint activation. Thatindicates RAD51C is required for effcient checkpoint signalling to delay cell cycleprogression in response to DNA damage. And the RAD51C foci persisted long afterRAD51disappeared. So it is possible that RAD51C plays a role both early and late in theHR reaction. Many other studies have proved that RAD51C involves in the late stage ofHR. They found that RAD51C regulates resolution of Holliday Junction （HJ） at the laterstages of HR. And the RAD51C-deficient hamster cells and MEFs have reduced HJresolution activity.Because of the important role that HR pathway plays in DDR, the mutations of HR relatedgene may cause the accumulation of unrepaired DNA damage. And at last can lead to thehappening of carcinogenesis. Studies showed that mutations of RAD51C increased the riskof breast cancer and ovarian cancer. Besides, research shows that mutation of RAD51Cleaded to FA-like disorder.Considering the important role of RAD51C in DNA repair, the study was designed toinvestigate the possible correlation between RAD51C expression and drug sensitivity incancer cell and explored its relationship with clinicopathological factors of breast cancer.The whole study was divided into three parts. In the first part, we used more than30drugsto test the survival of RAD51C-deficient Eμ-Myc p19^Arf-/-cell. Then to find out that thesensitivity of which drug would be changed after the RAD51C gene knockdown. In thesecond part, the experiment was designed to find out what kind of change in cell cycle andapoptosis would appear in RAD51C-deficient Eμ-Myc p19^Arf-/-cell after olaparibtreatment. In the third part, the study was performed to detect expression of RAD51C in breast cancer by immunohistochemistry （IHC） and explore their correlation withclinicopathological factors.Part1Effect of knockdown of RAD51C in Eμ-Myc p19^Arf-/-cell lineon anti-cancer drugs sensitivityObjective: To investigate the change of sensitivity of anti-cancer drugs inRAD51C-deficient Eμ-Myc p19^Arf-/-cell.Methods: Eμ-Myc p19^Arf-/-cell was stably transfected with retroviruses coexpressingRAD51C-shRNA and green fluorescent protein （GFP）. More than30drugs were chosed inthis study. We used a single-cell flow cytometry-based GFP competition assay to test thechange of sensitivity of each drug. GFP-negative cells in the same population serve as aninternal control.Results: Transfection of RAD51C-shRNA could effectively reduce the RAD51C proteinexpression in Eμ-Myc p19^Arf-/-cell line. The sensitivities of four drugs, CPT, DDP,17-AAG and olaparib, significant increased after RAD51C gene knockdown.Conclusions: The sensitivities of four drugs, CPT, DDP,17-AAG and olaparib, weresignificant increased after suppression of RAD51C expression in Eμ-Myc p19^Arf-/-cell.Part2Effect of olaparib in RAD51C-deficient Eμ-Myc p19^Arf-/-cellline on DNA damage, cell cycle and apoptosisObjective: To test the Effect of olaparib in RAD51C-deficient Eμ-Myc p19^Arf-/-cell lineon the expression of γ-H2AX, cell cycle and apoptosis.Methods: The Eμ-Myc p19^Arf-/-cells were stably transfected with RAD51C-shRNA usingretrovirus-mediated transfection. Then we test the expression of γ-H2AX through westernblot, and test the cell cycle and apoptosis of the cell through FCM after olaparib treatment.Results: After treatment of olaparib, the expression ofγ-H2AX was significant increased,and the expression ofγ-H2AX was higher in RAD51C-deficient Eμ-Myc p19^Arf-/-cells.Both of the RAD51C-deficient and RAD51C-proficient Eμ-Myc p19^Arf-/-cells showedsignificant S phase cycle arrest and apoptosis after treatment of olaparib. And theRAD51C-deficient Eμ-Myc p19^Arf-/-cells showed an increased early and late apoptosisrate compared to RAD51C-proficient cells.Conclusions: The treatment of olaparib would lead to accumulation of DSB in Eμ-Myc p19^Arf-/-cells. And the treatment of olaparib on RAD51C-deficient Eμ-Myc p19^Arf-/-cellswould lead to an S phase cycle arrest followed by apoptosis.Part3Expression of RAD51C in breast cancer and its clinicalsignificanceObjective: To detect expression of RAD51C in breast cancer by immunohistochemistry（IHC） and explore their correlation with clinicopathological factors.Methods:213cases of breast cancer tissue and99cases of adjacent noncancerous tissuewere collected to construct tissue microarrays （TMAs）. IHC was used to detect proteinexpression of RAD51C. Relevant clinical information was collected to perform correlationanalysis.Results: There was no significant difference between cancer and adjacent noncanceroustissues in the expression of RAD51C protein. RAD51C expression was significantly higherin HER-2overexpression group. The overall survival of the patients withRAD51C-negative expression was longer than positive ones. RAD51C expression was anindependent prognostic factor for breast cancer patients.Conclusions: The expression of RAD51C was positively correlated with the expression ofHER-2. The overall survival of the patients with RAD51C-negative expression wassignificant longer. And RAD51C expression was an independent prognostic factor forbreast cancer patients. All of these indicated that the test of RAD51C expression by IHCmay have prognostic value.