Study Of The Protective Effect Of RAD51 Against Oxidative Stress In Human Ovarian Cancer Cells

[Background]Ovarian malignant tumors, also known as ovarian cancer, is a common tumor of female reproductive organs. Ovarian cancer is the most common cause of deaths caused by female reproductive system tumors. Generally ovarian cancer at early stages has no clinical symptoms,70% of the patients are diagnosed at late stage. So far there have been no effective treatments for ovarian cancer. With the commonly used combination surgery and chemotherapy, the 5-year survival rate is only 30%. Therefore, to understand the malignant behavior of ovarian cancer becomes especially important.The integrity and stability of the DNA ensure cell survival and proper function. However, metabolites and many extrinsic factors like chemotherapy drugs, ionizing radiation can cause various types of DNA damage. DNA double-strand breaks (DSBs) and DNA interstrand crosslinks (ICLs) are the most serious form of DNA damage,which can lead to cell death or senescence and also is the main mechanism of radiation therapy and some chemotherapy drugs. The major repair ways for DSBs include homologous recombination repair (HRR) and non-homologous end joining repair (NHEJ). By homologous recombination repair, the damaged chromosome can use its intact sister chromatid to repair itself. RAD51 protein functions as a recombinase in eukaryotes. So high expression of RAD51 can inevitably increase the ability of cells to repair DNA damage, so as to protect the cell viability and maintain the normal life activities.Many studies have shown that RAD51 is highly expressed and confers resistance to chemotherapy in a variety of tumors.Free radicals are indispensable for many cellular processes. Under normal circumstances, a set of intracellular antioxidant system can maintain metabolic balance of free radicals. But when reactive oxygen species (reactive oxygen species, ROS) and reactive nitrogen free radicals (reactive nitrogen species, RNS) are produced too much under some stressful conditions or when the antioxidant system is compromised, oxidative stress occurs, which may cause damage to various macromolecules including DNA.RAD51 is highly expressed and and positively correlated with the degree of tumor malignancy. In addition to participating in classical HR, RAD51 is also known to play importance roles in restart of stalled replication forks and in repair of cross-links. Whether or not RAD51 possesses other functions remains to be studied. This study explores the role of RAD51 in cellular defense against oxidative stress.We employed A2780 ovarian cancer cells that overexpress RAD51 and their control cells. We found that RAD51 overexpression can effectively repair DNA damage and reduce the formation of DNA double-strand breaks compared to the control, and it also can effectively reduce ROS levels, confer increased resistance to H2O2. We also studied the effect of RAD51 downergulation by RNA interfering and found that RAD51 depletion resulted in increased ROS, produced more DNA damage and inhibited cell proliferation. However, the defects associated with RAD51 depletion can be rescued by antioxidant. We also that NRF2 and NRF2 target genes were downregulated by RAD51 depletion.[Methods]1. Clonogenic assay was employed to assess proliferation of A2780-mRAD51 and A2780-pcDNA3.1 by treating with H2O2.2. Flow cytometry method was used to determine the ROS level in A2780-mRAD51 and A2780-pcDNA3.1 by treating with H2O2.3. Immunofluorescence method was used to detect the marker of DNA double strands break gamma-H2AX foci formation in A2780-mRAD51 and A2780-pcDNA3.1 after treating with H2O2/TH287.4. Using flow cytometry and immunofluorescence method to detect ROS levels in RAD51 depleted cells.5. Immunofluorescence method was used to detect 8-OHdG formation in RAD51 depleted cells.6. Clonogenic assay and EdU cooperation was employed to assess proliferation of ovarican cancer cells treated with siRAD51 and NAC.7. Real-time PCR was used to determine the mRNA expression level of redox genes NRF2 and the target genes NQO1 and HO-1 in RAD51 depleted cells.[Results]1. A2780-pcDNA3.1 exhibit a distinct growth inhibition compared with A2780-mRad51 after treating with H2O2, it indicate Rad51 overexpression help cell’s to tolerant the oxidative stress2. The ROS level have a significantly increase in A2780-pcDNA3.1 when treated by H2O2,while having a unconspicuous increase in A2780-mRad51.3. A2780-pcDNA3.1 have a significantly increase of DNA damage when treated by H2O2/TH287,while in A2780-mRad51 have a low level.4. Immunofluorescence method and flow cytometry showed that the ROS level have a significantly increase after RAD51 depletion.5. Plate cloning experiments found that the depletion of RAD51 can cause a high production of 8-OHdG,it suggest intracellular oxidative stress caused DNA base damage.6. Antioxidants NAC can significantly rescuue cell proliferation disorder in ovarian cancer siRAD51 cells, which means the cells proliferation inhibition caused by depletion of RAD51 was paritlymediated by ROS.7. The mRNA expression of NRF2 and its target genes NQO1、HO-1 have a decrease in RAD51 depleted cancers.[Conclusions]:In addition to performing the classic HR function, RAD51 have a role in cellular defense against oxidative stress by reducing the intracellular ROS level.