Studies Of URI Regulating DNA Damage And Repair In Gastric Cancer Cells Induced By Cisplatin Via ATM/CHK2 Pathway

Background Gastric cancer is one of the most common gastrointestinal malignancies,and the mortality rate of which is second only to that of liver and lung cancer.Clinically,the diagnostic rate of gastric cancer at early stage is low,while surgical resection combined with chemotherapy or chemotherapy alone become the major treatment option for patients with gastric cancer at middle or late stage.Chemo-resistance is not uncommon in gastric cancer,which severely affects the prognosis.Cisplatin is one of the chemotherapy drugs and the resistance of gastric cancer cells to cisplatin limited its clinical application.Previously,multiple studies have demonstrated that the unconventional prefoldin RPB5 interactor URI is an oncoprotein participating in regulation of gene transcription by interacting with RPB5.In addition,our previous studies have shown that URI enhances the resistance of multiple cancer cells to common chemotherapeutic drugs,mitigates potassium dichromate-induced DNA damage as well as promotes repair after the damage.It has been reported that URI plays an important role in maintaining the genomic stability in the studies of C.elegans and Drosopliha.It is noteworthy that the increase of DNA damage repair is considered to be one of the mechanisms of cisplatin resistance.In this study,we will investigate the mechanism of cisplatin resistance of gastric cancer cells based on its role of URI in DNA damage repair in gastric cancer cells.Objectives In this study,we will explore the effect of URI on drug resistance and DNA damage repair in cisplatin-treated human gastric cancer cell lines MGC-803 with low differentiation and SGC-7901 which came from lymphonodus metastatic lesions derived from stomach cancer with poor differentiation,as well as investigate whether URI promotes DNA damage repair through ATM/CHK2 pathway in gastric cancer cells via URI knockdown.The study aims to further explore the potential mechanism of drug resistance in gastric cancer cells through URI knockdown.Materials and Methods(1)URI specific si RNA-A was transfected into human gastric cancer cells(MGC-803 and SGC-7901)with chemical transfection reagents Hiperfect to establish URI knockdown cell lines,while Scrambled groups and Untransfected groups was used as control groups.The expression of URI in all groups was detected by q RT-PCR and Western blot in order to verify the result of URI knockdown.(2)Comet assay was taken to measure the DNA damage in gastric cancer cells treated with different concentrations of cisplatin.The expression of γH2AX in gastric cancer cells with or without cisplatin treatment was detected by Western blot.(3)The viability of gastric cancer cells treated with different concentrations of cisplatin was estimated via CCK-8 assay,and the IC50 of cisplatin was calculated according to the vitality.The EDU detection kit was taken to detect the proliferation of gastric cancer cells treated with cisplatin.(4)The apoptotic cells in URI knockdown groups and control groups with cisplatin treatment were detected via flow cytometry.(5)The proteins relating to DNA damage repair pathways in all groups with or without cisplatin treatment were measured by Western blot after repairing for 0,6,12 hours.(6)The flow cytometry was used to detect the cell cycle distribution in cisplatin-treated cells after repairing for 12 hours.Results(1)URI expression in two gastric cancer cell lines after transfection with si RNA-A.The results of q RT-PCR and Western blot showed that the m RNA and protein levels of URI in two gastric cancer cell lines(MGC-803 and SGC-7901)after transfection with URI specific si RNA-A were obviously lower than the control groups.(2)Cisplatin results in DNA damage in gastric cancer cells and the damage was significantly increased in URI knockdown cells.In the test of comet assay,the OTM values were increased in two gastric cancer cell lines with cisplatin treatment and were increased while the cisplatin concentration was increased.The results indicated that the degree of DNA damage was increased with increasing concentration of cisplatin.The western blot results showed that the protein expression of γH2AX was increased after cisplatin treatment,and the expression of γH2AX in URI knockdown groups was obviously higher than the control groups.This indicated that URI can attenuate the DNA damage induced by cisplatin.(3)URI knockdown reduces the viability and proliferation of gastric cancer cells.The results of CCK-8 cell viability assay showed that the cell viability and the mean IC50 of URI knockdown groups was lower than control groups,which indicated that URI promotes resistance to cisplatin in gastric cancer cells.The EDU results showed that the proliferation of URI knockdown groups was lower than the control groups under cisplatin treatment.(4)URI reduces the cisplatin-induced apoptosis in gastric cancer cells.The early cisplatin-induced apoptosis in MGC-803 cells and the late apoptosis in SGC-7901 cells as well as the total apoptosis in the two gastric cancer cell lines with URI knockdown were increased.(5)URI influences the expression of relative protein in DNA damage repair pathway under cisplatin treatment.The Western blot results showed that the levels of P-ATM and P-CHK2 in DNA damage pathways significantly increased after treated with cisplatin.There was little difference between URI knockdown groups and control groups on the expression of P-ATM and P-CHK2 after repairing for 0 hour or 6 hours,but the P-ATM and P-CHK2 expression in URI knockdown groups was obviously lower than control groups when the cells repaired for 12 hours after cisplatin treatment,suggesting that URI knockdown reduced activity of ATM/CHK2 repair pathways.(6)URI promotes the DNA damage repair induced by cisplatin in gastric cancer cells.URI knockdown did not affect the cell cycle of gastric cancer cells without cisplatin treatment,but the G1 phase retardation obviously appeared in cisplatin-treated cells after repairing for 12 hours,suggesting that URI could reduce the DNA damage and promote the ability of damage repair in gastric cancer cells,as well as promote the cells to go through the G1 phase and enhance the drug resistance of gastric cancer cells as a result.Conclusions(1)URI reduces DNA damage and promotes DNA repair efficiency in gastric cancer cells under cisplatin treatment.(2)URI promotes DNA damage repair via ATM/CHK2 pathway,and enhances resistance of gastric cancer cells to cisplatin.