Wumei Wan Attenuates Colorectal Tumorigenesis By Inhibiting IRAK1

Objective:To elucidate the mechanism of IRAK1 in the colorectal tumorigenesis and explore the prevention of Wumei Wan.Method:1.The expression of irakl in normal colon tissues,adenoma tissues,and tumor tissues was explored by searching the GEO database.Clinical samples were used to verify the expression of IRAK1 and its phosphorylated protein in normal intestinal tissue,adenoma tissue and tumor a tissue.Western blot was used to detect the expression level of IRAK1 protein in colorectal cancer cell lines.HCT116 and RKO cells lines were selected according as tool cells for further research.Stable cell lines with IRAK 1 overexpression and knockdown were constructed in HCT116 cell line,and IRAK1 knockdown were constructed in RKO cell line by lentiviral transfection.The protein expression levels of IRAK1 in HCT 116 and RKO stable cell lines were verified by Western blot.CCK-8 assay was utilized to investigate the effect of IRAK1 on the cell viability of human colorectal cancer cells in vitro.Colony formation assay was utilized to investigate the effect of IRAK1 on the proliferative capacity of human colorectal cancer cells in vitro.CCK-8 and colony formation assays were also performed using 15409 to study the effect of pharmacological inhibition of IRAK1 on cell proliferation.Flow cytometry was used to investigate the effect of IRAK on apoptosis and cell cycle of human colorectal cancer cells in vitro.Western blot was used to detect the expression of cell cycle related proteins.The effect of pharmacological inhibition of IRAK1 on apoptosis and cell cycle of colorectal cancer cells was studied by 15409.2.GSEA was utilized to mine the potential function of IRAK1 in adenoma tissues.The possible mechanism by which IRAK1 promotes colorectal tumorigenesis was screened out as regulation of MYC expression.Co-IP,qPCR,Western blot as well as IHC were utilized to validate the relationship of IRAK1 and MYC.Using the IRAK1 inhibitor i5409 and the c-Myc inhibitor 10058-F4 in HCT116 and RKO cell lines,it was clarified by CCK-8 as well as colony formation assays whether IRAK1 affects cell proliferation by regulating c-Myc.Western blot was used to screen the related changes of factors in the pathway downstream of IRAK1,and the possible pathway of c-Myc regulation by IRAK1 was mined.The p38 inhibitor SB203580 was utilized to verify the alteration of c-Myc after overexpression of IRAK1 by inhibition of p38 in the rescue experiments.Using 15409 and SB203580 in HCT116 and RKO cell lines,we verified whether the regulation of c-Myc by IRAK1 was p38 dependent by Western blot and qPCR.Using SB203580 and 10058-F4 to determine whether p38 affects cell proliferation by regulating c-Myc using CCK-8 as well as colony formation.The effect of c-Myc on IRAK1 activation was examined by Western blot using 10058-F4.3.The representative components of Wumei Wan were obtained by HPLC.Through molecular docking operations,mining for components of which could potentially act on IRAK1.Azoxymethane(AOM)with dextran sulfate sodium(DSS)induced colon tumor model in mice was established to explore the alleviative effect of Wumei Wan as well as IRAK1 inhibitor 15409 on it.The survival status,the number of colon tumorigenesis and tumor size of mice in each group were calculated.The colon pathological changes were verified by HE staining.The expression of IRAK1/p38/c-Myc in colon tissues of mice in each group was detected by Western blot.DSS induced mice colitis model was established and the alleviating effects of Wumei Wan as well as 15409 on enteritis were evaluated by colon length and HE staining.The expression of IRAK1/p38/c-Myc in colon tissues of each group was detected by Western blot.The GEO database was utilized to validate the correlation between IRAK1 and MYC expression in colon tissues of IBD patients.Result:1.GEO databases(GSE117606,GSE37364,GSE41258 and GSE71187)confirmed that IRAK1 expression was gradually elevated in normal tissues,adenomas and tumors(P<0.05).The expression of IRAK1 protein and its phosphorylation in normal tissues,adenomas and tumors of patients with adenoma and adenocarcinoma increased gradually(P<0.05).Kaplan Meier curve analysis of TCGA colon cancer data showed that patients with high IRAK1 level had a short overall survival(Log rank P<0.05).IRAK1 expression was significantly higher in cell lines with HCT116 overexpressing cell line than in the vector control.And IRAK1 expression was significantly lower in HCT116 and RKO cell lines in which IRAK1 was knocked down than in the vector control cells.IRAK1 overexpression increased cell viability in HCT116,whereas different IRAK1 interfering fragments suppressed cell viability in both HCT116 and RKO cells(P<0.05).IRAK1 overexpression increased the colony formation rate of HCT116,whereas different IRAK1 interfering fragments suppressed colony formation rate in both HCT116 and RKO cells(P<0.05).The IRAK1 inhibitor 15409 inhibited the cell viability of HCT116 and RKO cells(P<0.05).15409 inhibited cell colony formation in HCT116 and RKO cells(P<0.05).Knockdown of IRAK1 did not affect the apoptosis rate of HCT116 and RKO cells compared with vehicle control.The proportion of HCT116 cells in the G2 phase decreased after IRAK1 overexpression,whereas HCT116 and RKO cells arrested in the G2 phase after IRAK1 interference(P<0.05).The expression of cyclinB1,CDK1 was increased and that of P21 was decreased in IRAK1 overexpressing HCT116 cells.The expression of cyclinB1 and CDK1 was decreased and P21 was increased in HCT116 and RKO cells after IRAK1 interference.15409 did not affect the apoptosis rate of HCT116 and RKO cells.15409 inhibited cell cycle arrest in G2 phase of HCT116 and RKO cells(P<0.05),decreased the expression of cyclinB1 and CDK1,and increased the expression of P21.2.GSEA analysis showed that IRAK1 expression in adenoma tissues was significantly enriched in MYC target related functional genes.EHC detection of clinical samples confirmed the highly positive correlation between the expression of IRAK1 and c-M yc in colorectal adenoma tissues(P<0.05).Co-IP confirmed the existence of an interaction relationship between IRAK1 and c-Myc protein in HCT116 and RKO cells.The expression of MYC in HCT116 cells with irak1 overexpression was increased,while the expression of MYC in HCT116 and RKO cells with irakl knockdown was decreased(P<0.05).In HCT116 and RKO cells,c-Myc protein expression was reduced by 15409,which was reversed by MG132.Both 15409 and 10058-F4 reduced HCT116 and RKO cell viability(P<0.05),15409 combination was unable to further increase the effect of 10058-F4.Both 15409 and 10058-F4 reduced HCT116 and RKO colony formation(P<0.05),15409 combination also could not further increase the effect of 10058-F4.MAPK p38 was activated in HCT116 with IRAK1 overexpression,but NF-KB-p65 was not significantly changed,while ERK1/2 was inhibited.MAPK p38 and NF-?B-p65 in IRAK1 knockdown HCT116 were inhibited,but ERK1/2 activity had no significant change.The activities of p38,NF-?B-p65 and ERK1/2 were inhibited in IRAK1 knockdown RKO cell line.SB203580 was able to reverse the increase in c-Myc caused by IRAK1 overexpression.15409 or SB203580 both reduced the mRNA and protein expression of MYC in HCT116 and RKO.SB203580 and 10058-F4 both decreased HCT116 and RKO cell viability(P<0.05);SB203580 combination with 10058-F4 was unable to increase the effect of 10058-F4.Both SB203580 and 10058-F4 reduced HCT116 and RKO colony formation(P<0.05);SB203580 in combination was unable to increase the effect of 10058-F4 inhibition.IRAK1 expression was decreased and p38 activation was inhibited in 10058-F4 treated HCT116 and RKO cells.3.The 11 chemical components of Wumei Wan were identified by HPLC,including citric acid,berberine,berberine,sinomenine,coptisine,epiberberine,palmatine,6-gingerol,hydroxyl-?-Zanthonin,asaronin,ferulic acid.Citric acid and berberine were higher than other components.Molecular docking results suggest that berberine and phellodendron can bind to the phosphorylation site T387 of IRAK1.The model of intestinal tumor induced by AOM/DSS was successfully constructed.The survival rate of the model animals was significantly improved by taking Wumei Wan 6g/kg.The number of intestinal tumors was reduced(P<0.05),and the volume of the tumor was also decreased(P<0.05).Pharmacological inhibition of IRAK1 did not reduce tumorigenesis but significantly reduced the tumor volume(P<0.05).Oral Wumei Wan with 6g/kg effectively alleviated the destruction of colon mucosa.Western blot showed that IRAK1 was activated at Thr387 in model mice,accompanied by the activation of c-Myc and p38.Wumei Wan and 15409 could reduce the activation of IRAK1,p38 and c-Myc.The inhibition of Wumei Wan and 15409 in the colitis induced by DSS can improve the colon length(P<0.05).The pathology suggested that pharmacological intervention could alleviate the situation of mucosal damage.IRAK1 was activated at Thr387,and p38 was activated in colitis.The expression of c-Myc in colon tissues of the colitis mice was decreased.The active IRAK1 and p38 were decreased by Wumei Wan and 15409.The expression of IRAK1 and MYC in IBD patients were highly positive correlation in GSE3629 and GSE75214.Conclusion:IRAK1 upregulates MYC expression by activating p38 to promote tumor proliferation during tumorigenesis.Wumei Wan may act as a chemopreventive drug by inhibiting IRAK1.