Interferon Regulatory Factor-1 Reverses Chemotherapy Resistance By Downregulating The Expression Of RAD51 In Gastric Cancer

To study the mechanism of interferon regulatory factor-1(IRF-1)in reversing chemotherapy resistance in gastric cancer.To reveal the impact of IRF-1 on DNA damage repair.To investigate the correlation between the expression of IRF-1 and RAD51 in clinical specimens of gastric cancer,and to provide the important theoretical basis for reversing chemotherapy resistance in gastric cancer.Methods1.Two Multi-drug resistant(MDR)gastric cancer(GC)cell lines were cultured.Transcript sequencing was performed on chemotherapy-resistant cell lines and parental cell lines to analyze differential expression.Western blot(WB)and PCR were used to detect the expression of IRF-1 and RAD51 in gastric cancer resistant cell lines and parental cell lines.2.By regulating the expression of IRF-1,the sensitivity of gastric cancer resistant cell lines to chemotherapeutic drugs was detected,and at the same time,RAD51 was overexpressed on the basis of overexpression of IRF-1,and it was tested whether it can rescue the resistance effect of IRF-1 reverse transformation therapy.Then,the effects of IRF-1 expression on DNA damage repair ability were investigated by immunofluorescence and comet experiments.3.The regulation of RAD51 were analysed by regulating the expression of IRF-1 in chemotherapy-resistant gastric cancer cell lines.Furthermore,we clarified the regulatory mechanism of IRF-1 on RAD51 expression through luciferase experiments and chromatin immunoprecipitation experiments.4.A gastric cancer resistant cell line(SGC7901 / IRF-1)with doxycycline-induced stable expression of IRF-1 was injected subcutaneously into nude mice at 4 weeks of age to establish an animal model.Nude mice were divided into control group,IRF-1 overexpression group and IRF-1 + RAD51 co-overexpression group.The three groups were injected intraperitoneally with the same dose of chemotherapy drugs(doxorubicin,cisplatin,and 5-fluorouracil).The tumor growth(weight and volume)was then analyzed.Immunohistochemistry(IHC)was used to detect the expression of IRF-1 and RAD51 in tumors.5.Collecting clinical gastric cancer tissue specimens and their clinical data,and analyzing the expression of IRF-1 and RAD51 by immunohistochemistry to explore the correlation between the expression of the two.Above results and with the TCGA database gastric cancer gene expression profile data to explore the relationship between RAD51 expression and gastric cancer prognosis.Results1.Compared with parental cell lines,DNA damage repair related genes were highly expressed in gastric cancer resistant cell lines.RAD51 was highly expressed in gastric cancer resistant cell lines and clinical samples of gastric cancer,and IRF-1 expression was low in gastric cancer resistant cell lines and clinical samples of gastric cancer.2.IRF-1 can increase the sensitivity of gastric cancer resistant cell lines to chemotherapy drugs.After IRF-1 overexpression,gastric cancer resistant cells were treated with doxorubicin,cisplatin,and 5-fluorouracil at different concentrations,which increase apoptosis and inhibited proliferation.After IRF-1 knockdown,apoptosis and proliferation were reversely expressed.Over-expression of RAD51 on the basis of over-expression of IRF-1 can partially rescue the reversal of drug resistance of IRF-1.It is further verified that RAD51 protein plays a key role in IRF-1 expression reversing chemotherapy resistance of gastric cancer.3.In gastric cancer cells,IRF-1 can inhibit DNA damage repair.After IRF-1 was overexpressed,gastric cancer cells were treated with doxorubicin at an appropriate concentration,and DNA damage was increased.In contrast,knockdown of IRF-1 expression reduced DNA damage in gastric cancer cells.4.In gastric cancer cells,IRF-1 regulates RAD51 expression.When IRF-1 is overexpressed,RAD51 expression decreases.In contrast,RAD51 expression increase after IRF-1 knockdown.5.IRF-1 inhibits RAD51 promoter activity.Chromatin immunoprecipitation was used to verify that IRF-1 can bind to the transcriptional binding site of the RAD51 promoter.The luciferase experiment further prove that the expression of IRF-1 has an inhibitory effect on the promoter activity of the RAD51 protein gene.6.IRF-1 overexpression reverses gastric cancer chemotherapy resistance in animals.After constructing the animal model via intraperitoneal injection of chemotherapy drugs(doxorubicin,cisplatin,and 5-fluorouracil),the weight and volume of xenograft tumors in the IRF-1 overexpression group were significantly reduced after treatment,while the IRF-1 and RAD51 overexpression groups recive partial recovery in weight and volume of xenograft tumors.WB,PCR,and immunohistochemical methods confirmed that the expression of RAD51 protein was significantly lower in the IRF-1 expression group than in the control group.7.Statistical analysis of gastric cancer patient specimens and TCGA database data showed that IRF-1 expression was significantly correlated with RAD51 expression.High expression of RAD51 and low expression of IRF-1 are associated with poor prognosis in patients with gastric cancer.Conclusion1.IRF-1 reverses the MDR of GC in vivo and in vitro.2.IRF-1 regulates DNA damage repair.3.IRF-1 downregulates RAD51 expression in gastric cancer.4.IRF-1 suppresses RAD51 promoter activity at the transcriptional level.5.IRF-1 and RAD51 expression levels are correlated in gastric cancer and are independently predictive of poor prognosis.