| Purpose:1.To elucidate the roles and molecular mechanisms of interferon regulatory factor-1(IRF-1)in reversing chemotherapy resistance of gastric cancer2.To determine the expression of IRF-1 and P-glycoprotein in clinical gastric cancer samples,and to explore the application value of IRF-1 in gastric cancer chemotherapyMethods1.Two chemotherapy-resistant gastric cancer cell lines were cultured,and the expression of IRF-1 in chemotherapy-resistant cell lines and parental cell lines were detected,and its correlation with chemotherapy resistance of gastric cancer was analyzed.2.The effect of IRF-1 on chemoresistance of gastric cancer and the regulation of P-glycoprotein were analyzed via regulating the expression of IRF-1.Furthermore,chromatin immunoprecipitation and luciferase reporter assay were used to elucidate the regulation mechanisms of IRF-1 on P-glycoprotein expression3.The expression of IRF-1 in gastric cancer cells was induced by IFN-γ,and its effect on P-glycoprotein expression was explored4.Doxycycline-induced IRF-1 expression system was established in gastric cancer cell line(MKN45/IRF-1),and 6-week BALB/c nude mice were used to establish an animal model of gastric cancer,which was divided into IRF-1 expression group(Doxycycline-induced expression)and control group,the above two groups were intraperitoneal injected with chemotherapeutic drugs(Cisplatin,5-fluorouracil and Doxorubicin)to analyze the tumor growth and the expression of IRF-1.In vivo studies of IRF-1 reversal of chemoresi stance in gastric cancer were conducted5.Clinical gastric cancer samples and the related clinical pathological data were collected and combined with TCGA database gastric cancer gene expression profile data to analyze the clinical correlation between IRF-1 expression and gastric cancer chemotherapy resistance.Results:1.IRF-1 is relatively low expression in both chemotherapy-resistant gastric cancer tissues and chemotherapy-resistant gastric cancer cells.2.IRF-1 regulates chemotherapy resistance in gastric cancer cells:after IRF-1 overexpression,gastric cancer cells are treated with different concentrations of chemotherapeutic drugs(Cisplatin,5-Fluorouracil,Doxorubicin),cell proliferation was inhibited,and apoptosis was increased.After IRF-1 knockdown,increased proliferation be observed.Moreover,the role of IRF-1 in regulating chemotherapy resistance of gastric cancer is related to the change of intracellular chemotherapy drug concentration.3.IRF-1 regulates P-glycoprotein expression in gastric cancer cells:P-glycoprotein expression is inhibited after IRF-1 overexpression;After IRF-1 knockdown,P-glycoprotein expression was increased.Further rescue experiments confirmed that P-glycoprotein plays a pivotal role in reversing the chemoresistance of gastric cancer by IRF-1 expression.4.IFN-y inhibits P-glycoprotein expression in gastric cancer cells:IFN-y increases IRF-1 expression,and P-glycoprotein is inhibited in a dose-dependent manner.After IFN-y treatment,the inhibition rate of chemotherapeutic drugs on cells was significantly increased,which was related to the decrease of P-glycoprotein expression and the increase of intracellular drug concentration on the cell membrane.5.IRF-1 inhibits P-glycoprotein promoter activity:chromatin immunoprecipitation confirms the transcriptional binding site of IRF-1 on the P-glycoprotein gene promoter,and luciferase assay technology further demonstrates IRF-1 expression has an inhibitory effect on the promoter activity of the P-glycoprotein gene.6.Increased expression of IRF-1 reverses chemotherapy resistance of gastric cancer in vivo:after intraperitoneal injection of chemotherapeutic drugs(Cisplatin,5-Fluorouracil and Doxorubicin),the volume and weight of xenograft tumors were significantly reduced after treatment.Immunohi stochemi stry,Western-blotting and RT-RCR assays confirmed that the expression of P-glycoprotein in the IRF-1 expression group was significantly lower than that in the control group.7.Statistical analysis of clinical pathological data and TCGA databases showed that low expression of IRF-1 was associated with elevated P-glycoprotein and poor prognosis of gastric cancer patients,and that IRF-1 expression was an independent prognostic factor for total survival of patients with gastric cancerConclusions:1.IRF-1 reverses chemoresistance of gastric cancer in vivo and in vitro2.IRF-1 reverses chemoresi stance by down-regulating the expression of P-gp in gastric cancer cells3.IRF-1 mediates the inhibition of P-gly coprotein expression by IFN-γ4.IRF-1 suppresses the promoter activity of P-gp gene and the expression of P-gp at the transcriptional level5.The expression of IRF-1 is negatively related to P-gp expression and can be used as an independent prognostic biomarker for gastric cancer patients. |
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