The Efficacy And Mechanism Of Niltotinib As A Novel ILK Inhibitor In Esophageal Squamous Cell Carcinoma Based On Molecular Docking Technology

Objective: Based on the effect of ILK in esophageal squamous cell carcinoma,this study was designed and implemented to screen ILK inhibitors from clinical drugs approved by FDA and explore whether the inhibitors have anti-tumor effects on esophageal squamous cell carcinoma,so as to provide experimental basis for molecular therapy of esophageal squamous cell carcinoma.Methods: 1)The expression and prognostic significance of ILK in pan-carcinoma were comprehensively analyzed using multiple databases such as c Bio Portal,Timer2 and GEPIA,and the expression of ILK in esophageal squamous cell carcinoma was detected by q RT-PCR and Western blotting(WB).Linked Omics and UALCAN were used to focus on exploring the potential mechanism of ILK in esophageal squamous cell carcinoma.2)Molecular docking technology was used to screen the drugs with the strongest binding ability to ILK protein structure from FDA clinical drug library,SPR technology was used to verify the affinity between screened Nilotinib and Teniposide and ILK,and CCK8 was used to detect the IC50 concentration of inhibitors.The antitumor effects of Nilotinib and Teniposide on ESCC cells were investigated by clonal formation assay and flow cytometry.3)In vitro experiments,CCK8,LDH,clonogenesis assay,EDU,flow cytometry,Transwell and WB were used to verify the targeting relationship between Nilotinib and ILK as well as the effect of Nilotinib on ESCC cell function.The effect of Nilotinib stimulation on cell structure was observed by electron microscopy.The effects of Nilotinib on mitochondrial function were detected by flow cytometry using a variety of fluorescent probes.The effects of Nilotinib on radiotherapy sensitivity of ESCC cells were detected by cloning assay and flow cytometry.The effect of Nilotinib treatment on tumor size and quality was observed in nude mouse transplantation tumor model.The damage of tumor tissue after Nilotinib treatment was observed in HE,and the effect of Nilotinib treatment on Ki67 and cleave-caspase3 expression in tissue was detected by IHC.4)RNA sequencing was performed on Nilotinib-treated TE-1 cells to find downstream regulatory molecules,and q RT-PCR was used to detect the difference in expression of downstream AIRE between ESCC and normal esophageal epithelial cells.Cloning formation assay,EDU kit,flow cytometry,Transwell invasion,scratch assay,WB to verify the role of downstream molecule AIRE on ESCC malignant biology.WB was used to detect the influence of Nilotinib treatment and silencing ILK on AIRE expression and the influence of Nilotinib treatment on the stability of AIRE protein.Results: Part I: Analysis of the biogenesis of ILK in pan-carcinoma: ILK was highly expressed in most tumors,and was negatively correlated with OS and DFS;The high expression of ILK was positively correlated with multiple immune checkpoint genes,and ILK may be a potential immunotherapeutic target.The main type of ILK gene alteration is mutation,and its mutation level is positively correlated with good prognosis in some mutated tumors,such as UCEC.ILK was highly expressed in ESCC cells.ILK ESCC patients with high expression had poor prognosis with the same pathological grade.Among the 10 co-expressed core genes screened,high expression of FN1 and BGN was significantly correlated with poor prognosis of ESCC,which may mediate the involvement of ILK in the development of ESCC.The second part:From more than 1600 clinical drugs approved by FDA,the two drugs with the highest binding force to ILK--Nilotinib and Teniposide were selected by molecular docking technology.The affinity between the two drug candidates and ILK protein was verified by surface plasmon resonance experiment.The 24 h IC50 value of Nilotinib in TE-1 cells and KYSE150 cells was 49.52μM and 55.2μM respectively,respectively.The 48 h IC50 value of TE-1 and KYSE150 cells was 36.07μM and 38.17μM respectively.The 24-h IC50 value of Teniposide was 1059 n M in TE-1 cells and 313.6n M in KYSE150 cells,respectively.The 48 h IC50 value of TE-1 and KYSE150 cells was 396.2n M and 162.7n M respectively.The results of cloning and flow cytometry showed that Nilotinib and Teniposide could significantly inhibit the proliferation and promote apoptosis of ESCC cells.Part III: EDU,clonal formation,flow cytometry and WB results showed that overexpression of ILK could reverse the inhibitory effect of Nilotinib on proliferation and apoptosis of ESCC cells.Nilotinib significantly inhibited the survival rate,proliferation,invasion and migration ability of TE-1 and KYSE150 cells of esophageal squamous cell carcinoma,and promoted apoptosis.The results of electron microscopy and flow cytometry showed that Nilotinib affected cell mitochondrial function,and the above effects were enhanced with the increase of drug concentration.The results of clonal formation showed that Nilotinib could increase the sensitivity of esophageal squamous cell to radiation(SER=1.23).Nilotinib could increase the apoptosis rate and G2/M phase arrest of ESCC cells after irradiation.In vivo transplanted tumor model of nude mice showed that the tumor volume and mass of Nilotinib treatment group were smaller than that of the control group.HE staining showed large necrotic area of tumor tissue and severe necrotic degree of tumor cells in the treatment group.The expression of Ki67 decreased and the expression of cleave-caspase3 increased significantly in the treatment group as detected by IHC.Part IV: High-throughput sequencing analysis of cells treated with different concentrations of Nilotinib.There were 142 differentially changed differentially genes in the three groups,among which 63 differentially up-regulated genes were co-expressed,such as ABCG2,ANGPTL4,APOL6,etc.There were 72down-regulated differential genes,such as AIRE,ALPI,ART1,etc.Comparing two selected three group were significant differences in gene expression downgrade AIRE molecules for subsequent research object,q RT-PCR detection AIRE,KYSE150 cells in esophageal squamous carcinoma TE-1 m RNA level higher than that of normal esophageal epithelium in HET-1A cell.AIRE silenced lentivirus significantly inhibited the proliferation,invasion,migration and DNA replication of esophageal squamous cell carcinoma cells,and promoted the apoptosis of tumor cells.Overexpression of AIRE could promote the ability of cell cloning,but the EDU results showed that overexpression of AIRE inhibited the ability of DNA replication of cells and promoted cell apoptosis,which was unclear and needed further verification.WB results showed that Nilotinib treatment and ILK-silencing lentivirus infection both caused downregulation of AIRE expression.The detection of AIRE expression after Nilotinib treatment at different time points showed that the drug promoted the degradation of AIRE protein.Conclusion: ILK is a gene with research value as a potential tumor therapeutic target,and it is also relevant to immunotherapy.Nilotinib and Teniposide were selected as novel ILK inhibitors with potential anti-ESCC effects.In vitro and in vivo experiments,Nilotinib proved that targeting ILK affects the stability of AIRE protein and plays an anti-esophageal squamous cell carcinoma role,providing research value for targeting ILK in the treatment of esophageal squamous cell carcinoma,and laying a foundation for developing new targets and new application scope of Nilotinib before clinical trials.