Inhibition Effect And Mechanism Of URI On Potassium Dichromate-induced Oxidative Stress And DNA Damage In SGC-7901 Cells

Background: Chromium(Cr) is a natural element and it is widely found in the atmosphere, soil, rocks, microbes, plants and animals. It exists mainly in two valence state, i.e., trivalent chromium [(III) Cr] and hexavalent chromium [Cr(VI)]. Cr(VI) is a known carcinogen. Cr(VI) from industrial production process is an important cause of workers’ occupational lung cancer. Long-term exposure to Cr(VI) contaminated environment will have a serious health hazard, such as liver and kidney organ damage, contact dermatitis and skin cancer. Cr(VI) –induced oxidative stress and DNA damage are main reasons for tumor formation. In addition to lung cancer, Cr(VI) can also lead to gastrointestinal tumors, but the exact mechanism remains to be studied. URI(unconventional prefoldin RPB5 interactor, URI) is a member of the prefoldin family of molecular chaperones. It can interact with RPB5, mainly involving the regulation of gene transcription. Studies found that URI plays an important role in maintain genomic stability in C. elegans and Drosophila. URI silence can lead to the accumulation of DNA damage resulting in cell apoptosis. URI is considered as an oncoprotein, which can promote proliferation of tumor cells and resistance to apoptosis. URI is also a radiation-sensitive protein. Potassium dichromate, a common Cr(VI) salt, has been confirmed as a chemical carcinogen. However, the mechanism of Cr(VI)-induced tumor formation is unclear so far.Objective: Potassium dichromate has cytotoxic and tumorigenicity for normal cells. But what will be the reaction of tumor cells after treated with potassium dichromate is unclear. Facing cytotoxicity, strengthening DNA damage repair and escaping death are characteristics of tumor cells. URI is an oncoprotein, whether URI has protective effects on potassium dichromate induced oxidative stress and DNA damage in tumor cells are largely unknown. In this study, we will explore the effect of URI on oxidative stress, DNA damage repair and cell death in SGC-7901 cells induced by potassium dichromate through URI knockdown. This study will further enrich our understanding of URI functions, and provide new ideas of the relationship between URI and potassium dichromate-induced tumor formation.Materials and Methods:(1) si RNA silencing method was used to knockdown URI gene in human gastric cancer SGC-7901 cells,and set si RNA-A group, Scrambled group and Blank group.(2) The specific fluorescent dye DCFH-DA was used to detect the intracellular ROS level.(3) OTM value were estimated by comet assay in potassium dichromate-treated cells, which can reflect the extent of cell DNA damage and repair efficiency, and set the concentration of potassium dichromate(1μM,10μM).(4) Annexin V / PI staining kit was used to measure cell death through flow cytometry after treated with potassium dichromate.(5) The expressions of corresponding protein, including anti-apoptotic protein Bcl-2, Mcl-1 and pro-apoptotic protein Bax and apoptosis-inducing factor AIF, etc in cells of each group were detected by western blot and immunofluorescence methods after potassium dichromate treatment.(6) NAD + level were determined in potassium dichromate-treated cells using NAD + assay kit.Results:(1) Potassium dichromate-induced ROS was increased significantly in si RNA-A group.(2) In si RNA-A group, potassium dichromate-induced DNA damage was significantly increased, and after treated with a relatively high concentration of potassium dichromate, DNA damage repair ability was dramatically suppressed.(3) SGC-7901 cell death induced by relatively low concentrations of potassium dichromate was dependent largely on mitochondrial apoptosis pathway, and by the relatively high concentration of potassium dichromate was mainly dependent on PARP1 necrosis pathway. Moreover,URI has a protective effect in these two kinds of cell death pathways.Conclusions:(1) URI can inhibit ROS generation, indicted that URI is involved in the regulation of cellular oxidative stress induced by potassium dichromate.(2) URI is able to reduce excessive DNA damage induced by potassium dichromate. URI interference significantly inhibited cell repair efficiency after 10μM concentration of potassium dichromate treatment. These suggested that URI could influence the potassium dichromate-induced DNA damage and its repair.(3) URI has a protective effect on potassium dichromate-induced cell death, indicted that URI is essential for cell survival under the effect of potassium dichromate.