| Objective:1.To explore IQGAP1(IQ motif containing GTPase activating protein)expression in ESCC(esophageal squamous cell carcinoma)tissues and its relationship with prognosis.2.To explore the effect of IQGAP1 expression on paclitaxel resistance.3.To explore the effect of IQGAP1 expression on ferroptosis.4.To study the mechanism of IQGAP1 affecting paclitaxel resistance via regulating ferroptosis in ESCC cells.5.To study the mechanism of IQGAP1 modulating ferroptosis via targeting YAP1(Yes-associated protein 1)to affect paclitaxel resistance.Methods:1.Analysis of IQGAP1 expression and prognosis by bioinformatics and clinical samples(1)The expression of IQGAP1 in pan-cancer was analyzed using the TIMER(tumor immune estimation resource)database.The data of ESCA(esophageal carcinoma)tissues and non-tumor tissues were downloaded through the TCGA(the cancer genome atlas)database to analyze the differential expression of IQGAP1 in ESCA tissues and adjacent normal tissues,and ROC(receiver operating characteristic)curve was drawn to evaluate the ability of IQGAP1 to distinguish ESCA from adjacent normal tissues.(2)100 cases of ESCC patients and 69 cases of adjacent normal tissues and their clinicopathological characteristics were collected,the expression differences of IQGAP1 in ESCC and adjacent normal tissues were analyzed by immunohistochemistry analysis.ROC curve was drawn to evaluate the ability of IQGAP1 to distinguish ESCC from adjacent normal tissues,and the relationship between IQGAP1 expression levels and clinicopathological characteristics was analyzed.K-M(Kaplan-Meier)survival curve and forest plot were further drawn based on the survival data,and the relationship between IQGAP1 expression and prognosis was analyzed by COX regression.2.The effect of IQGAP1 on the survival and death of paclitaxel-treated ESCC cells(1)IQGAP1 overexpression cell lines were established by stably transfecting GFP-IQGAP1 plasmid into EC9706 cells,and IQGAP1 sh RNA(short hairpin RNA)plasmid was stably transfected into KYSE150 cells to construct IQGAP1 knockdown cell lines,and IQGAP1 expression was identified by Western blot.(2)Different concentrations of paclitaxel were used to treat IQGAP1 overexpression and control cells and IQGAP1 knockdown cells and control cells,respectively.MTT assay was detected the viability of ESCC cells,clone formation and PI(propidium iodide)staining assays were used to detect proliferation and death of ESCC cells,respectively.3.The effect of IQGAP1 on ferroptosis in ESCC cells Using GSEA(gene set enrichment analysis)software to analyze the enrichment pathway of IQGAP1 expression in ESCA.DHE(dihydroethidium)staining detected the effect of IQGAP1 overexpression or knockdown on the ROS(reactive oxygen species)levels of ESCC cells by fluorescence microscopy and flow cytometry.The effects of IQGAP1 overexpression or knockdown on MDA and GSH levels in ESCC cells were detected by MDA(malondialdehyde)and GSH(glutathione)kits,respectively.The effect of IQGAP1 overexpression or knockdown on the expression levels of ferroptosis-related proteins GPX4(glutathione peroxidase 4)and SLC7A11(recombinant solute carrier family 7,member 11)in ESCC cells was detected by Western blot.4.IQGAP1 affects paclitaxel resistance by regulating ferroptosis in ESCC cells The ferroptosis inducer(RSL3)or the ferroptosis inhibitor(ferrostain-1,Fer-1)was used to deal with paclitaxel-treated IQGAP1 overexpression or knockdown cell lines,and the proliferation and death of ESCC were detected by colony formation and PI staining assays,respectively.The levels of ROS,MDA,GSH and the expression levels of GPX4 and SLC7A11 proteins were detected by DHE staining,MDA,GSH kits and Western blot.5.IQGAP1 affects paclitaxel resistance by YAP1-regulated ferroptosis The effect of IQGAP1 overexpression or knockdown on the expression of YAP1 in ESCC cells was detected by Western blot,and the TIMER database analyzed the correlation between IQGAP1 and YAP1 in ESCA.Subsequently,Western blot was used to dectect the effect of the YAP inhibitor VP(Verteporfin)on YAP1 expression in paclitaxel-treated IQGAP1 overexpression cell lines.The proliferation and death of ESCC cells were detected by clonal formation and PI staining assays,respectively.DHE staining,MDA,GSH kits and Western blot assays were used to detect ROS,MDA,GSH levels and ferroptosis-related protein expression levels.Results:1.Bioinformatics and clinical sample analysis of the relationship between IQGAP1 expression and prognosis(1)The expression of IQGAP1 levels were significantly higher in tumor tissues than that of normal tissues analyzed by the TIMER database,including CHOL(cholangiocarcinoma),COAD(colon adenocarcinoma),ESCA,HNSC(head and neck squamous cell carcinoma),STAD(stomach adenocarcinoma),and THCA(thyroid carcinoma).TCGA data analysis further verified that the expression of IQGAP1 in ESCA was significantly higher than adjacent normal tissues,and ROC curve showed that IQGAP1 can be used as a biomarker to distinguish ESCA tissues from adjacent normal tissues.(2)Immunohistochemistry results showed that the expression of IQGAP1 in ESCC tissues was significantly higher than that in adjacent normal tissues,and ROC curve showed that IQGAP1 can be used as a biomarker to distinguish ESCC tissues from adjacent normal tissues,and clinical data analysis showed a positive correlation between IQGAP1 expression in ESCC tissues and N stage,and IQGAP1 expression with five-year overall survival(OS),IQGAP1 was a poor prognostic factor of OS in ESCC patients.2.The effect of IQGAP1 on the survival and death of paclitaxel-treated ESCC cells(1)The results showed that the expression of GFP-IQGAP1 fusion protein was found in IQGAP1 overexpression cell lines but not in the control cells,and the IQGAP1 expression was significantly lower in knockdown cell lines than the control cells.These data suggested the successful establishment of IQGAP1 overexpression and knockdown cell lines.(2)After treatment with different concentrations of paclitaxel,it was found that the survival rate and proliferation ability of IQGAP1 overexpression cells were significantly higher than that of control cells,and the number of cell death was significantly lowerthan that of control cells,while the survival rate and proliferation ability of IQGAP1 knockdown cells were significantly lower than that of control cells,and the number of cell death was significantly higher than that of control cells.3.The effect of IQGAP1 on ferroptosis in ESCC cells GSEA enrichment analysis showed that the IQGAP1 low expression group was enriched in the ETC(electron transport chain)and oxidative phosphorylation in mitochondria,and the IQGAP1 high expression group was enriched in glycolysis and hypoxia.ROS and MDA levels in IQGAP1 overexpression cells were significantly lower than those of control cells.GSH levels,GPX4 and SLC7A11 expression were significantly higher in IQGAP1 overexpression cells than control cells.ROS and MDA levels in IQGAP1 knockdown cells were significantly higher than those of control cells.GSH levels,GPX4 and SLC7A11 expression levels were significantly lower in IQGAP1 knockdown cells than those of control cells.4.The mechanism of IQGAP1 affecting paclitaxel resistance by regulating ferroptosis in ESCC cells The results showed that RSL3(ferroptosis inducer)could reverse the effects of IQGAP1 overexpression on paclitaxel-treated proliferation,death,ROS,MDA,GSH,and GPX4,SLC7A11 expression.Fer-1(ferroptosis inhibitor)could reverse the effects of IQGAP1 knockdown on paclitaxel-treated proliferation,death,ROS,MDA,GSH,and GPX4 and SLC7A11 expression levels.5.IQGAP1 affects paclitaxel resistance by YAP1-regulated ferroptosis Western blot showed the expression levels of YAP1 elevated in IQGAP1 overexpression cells,YAP1 decreased in IQGAP1 knockdown cells.TIMER database analysis showed a positive correlation between IQGAP1 and YAP1 expression in ESCA.Using the combination of VP and paclitaxel in IQGAP1 overexpression cell lines,and Western blot showed that YAP1 expression was downregulated compared with the control group.VP could reverse the effects of IQGAP1 overexpression on the proliferation,death,ROS,MDA,GSH levels,and GPX4,SLC7A11 expression levels in paclitaxel-treated IQGAP1 overexpression cells.Conclusion:1.IQGAP1 is highly expressed in ESCC tissues and associated with a poor prognosis in ESCC patients,and IQGAP1 is a poor prognostic molecule for ESCC patients.2.IQGAP1 overexpression can reduce the sensitivity of ESCC cells to paclitaxel,and IQGAP1 knockdown can enhance the sensitivity of ESCC cells to paclitaxel.3.IQGAP1 overexpression can inhibit ferroptosis in ESCC cells,and IQGAP1 knockdown can promote ferroptosis in ESCC cells.4.IQGAP1 overexpression leads to paclitaxel resistance in ESCC cells by inhibiting ferroptosis,and IQGAP1 knockdown can reverse paclitaxel resistance in ESCC cells by promoting ferroptosis.5.IQGAP1 affects paclitaxel resistance by YAP1–regulated ferroptosis in ESCC cells. |
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