ZFX Facilitates Resistance To 5-Fluorouracil Through Inducing Autophagy Via ERK-MAPK Pathway In Colorectal Cancer

OBJECTIVE 1.To explore the biological role and related molecular mechanism of Zinc-finger protein X-linked(ZFX)in the initiation and chemoresistance of colorectal cancer(CRC).2.To investigate the prognostic significance of ZFX in II/III stage colorectal cancer patients with postoperative chemotherapy.MATERIALS AND METHODS 1.Real-time PC R was used to detect the expression of ZFX in different colorectal cancer cel s.2.Lentivirus-mediated ZFX knockdown in CRC cells were constructed.The MTT assays,clonogenic assay,and flow cytometry was applied to investigate the biological role of cancer cells.The western-blot was applied to detect the expression of authphagy biomarker LC3-II/LC3-I.The effect of ZFX knockdown on drug sensitivity was analyzed by measuring the cytotoxicity of a chemotherapeutic drug,5-Fu,with the MTT method,which was used to calculate their IC50 values(?M).3.The effect of ZFX on in vivo tumorigenicity was investigated using a xenograft model in which nude mice were subcutaneously injected with SW620 and SW480 cells transfected with NC or ZFX sh RNA.4.Microarray analysis was performed using SW620 cells transfected with ZFX and NC sh RNA to explore the potential molecular mechanism of the ZFX contribution to the malignant characteristics of C RC cells.According to GO enrichment analysis and pathway analysis,genes that might be linked with the regulation of MAPK signaling pathway were selected.Western blot analysis was performed to further validate the results.5.Small interfering RN A(si RNA)was performed to downregulate DUSP5 expression in colorectal cancer cells with transfected ZFX sh RNA.MTT assays was used to determine whether DUSP5 downregulation could rescue the growth suppressive effects of ZFX knockdown.6.An interaction network was then established using the Reactome database by automatically adding key pathway molecules(MAPK1,MAPK3 and MAPK11)to the downstream of DUSP5.Western blot analysis was performed to further validate these pathway molecules.7.Immunohistochemistry was performed to detect expression of ZFX in 290 primary CRC tissues and their matched normal tissues,and its clinical significance was evaluated by statistical methods.RESULT 1.RT-PCR shows ZFX m RNA expression was higher in SW620 and SW480 than other colorectal cancer lines.2.After downregulating ZFX expression,cell proliferation,apoptosis resistance,authphagy and chemoresistance were dramatically inhibited.3.In vivo experiments showed that ZFX knockdown suppressed the tumorigenic ability of SW480 and SW620.4.Microarray analysis was performed using SW620 cells transfected with ZFX and NC sh RNA.Based on the inclusion criteria,a total of 290 up-regulated genes and 196 down-regulated genes were identified following ZFX knockdown.GO enrichment analysis,pathway analysis and bioinformatics analysis,fi Ve genes related to MAPK signaling [RAP1A,Dual specifiity phosphatase 5(DUSP5),Ets variant 1(ETV1),FOS-like antigen 1(FOSL1),and Tumor necrosis factor superfamily member 10(TNFSF10)] were selected from the microarray data.Wes tern blot analysis and Reactome and O ncomine database proposed that DUSP5 is the dominant target of ZFX in SW620 and SW480 cells.5.Si RNA was applied to ablate DUSP5 in ZFX-KD SW620 and SW480 cells.MTT assays showed that DUSP5 downregulation rescued the ZFX-mediated suppression in cell proliferation.6.An interaction network was then established using the Reactome database by automatically adding key pathway molecules(MAPK1,MAPK3 and MAPK11)to the downstream of DUSP5.To further validate these pathway molecules,western blot analysis was performed and the results suggested that the phosphorylation of ERK was inhibited,whereas the phosphorylation of MAPK3 and MAPK11 and the expression of ERK,MAPK3 and MAPK11 had no effect in in ZFX-KD SW620 cells.7.ZFX expression was statistically higher than that in normal tissues(P<0.001).ZFX expression correlated with tumor differentiation(p= 0.005)and TNM stage(p= 0.003),while no correlation was observed with other clinical parameters,including gender(p =1.000),age(p= 0.900),tumor location(p= 0.706)and tumor size(p= 0.799).multivariate analysis confirmed that only ZFX expression and TNM stage were independent prognostic factors for OS in stage II and III patients.A complete cohort with higher ZFX expression was observed to have statistically lower OS and DFS rates than a cohort with lower ZFX expression.Further analysis of the patients,stratified based on postoperative chemotherapy,also revealed a positive correlation between ZFX expression and low OS and DFS rates.This was not observed exclusively in stage II patients,but also occurred in stage III patients.However,for stage II patients treated without postoperative chemotherapy,there was no significant correlation between ZFX expression and OS.CONCLUSION ZFX palys an important role in inducing autophagy to faciliate the 5-FU resistance of colorectal cancer cells.We revealed ZFX may mediate autophagy to acquire chemoresistance by activating ERK-MAPK signaling pathways in colorectal cancer cell.Survival analysis indicated that II/III stage colorectal cancer patients with postoperative chemotherapy with high ZFX expression had poorer overall and disease-free survival.Finally,we will comprehensively explore the function characterization of ZFX in vivo and in vitro from clinical samples,cell,and animal levels.This will provide a novel molecular target and dynamic and warning marker for therapy and monitoring of II/III colorectal cancer patients with postoperative chemotherapy and improve their prognosis.