The Role Of Kinesin Binding Protein KTN1 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC),characterized with dormant onset,difficult early diagnosis and poor prognosis,is one of the main factors threatening human health.The incidence and mortality of HCC are increasing year by year in China and even in the world.Although some earlier diagnoses and therapies of HCC have been made,it is still a huge challenge to further explore the pathogenesis and find new therapeutic targets of HCC.KTN1 is an endoplasmic reticulum membrane protein,which is involved in many biological processes such as maintenance of endoplasmic reticulum morphology,cell axonal transport and cell mitosis.In addition,KTN1 protein binds to translation elongation factor δ(EF-δ)to participate in the assembly of EF-1 complex and regulate the expression of other proteins.Current studies have confirmed that the expression of KTN1 is closely related to the occurrence of various tumors.It was observed that KTN1 was significantly upregulated in cutaneous squamous cell carcinoma(CSCC)and involved in the MALAT1-KTN1-EGFR axis to promote the development of CSCC cells.The expression of KTN1 in giant cell tumor of bone is significantly increased,which promotes the migration of giant cell tumor of bone.Our research group previously found a high positive rate of anti-KTN1 antibody in the sera of HCC patients,and a high positive rate in the sera of AFP negative patients,which is expected to be a marker for early diagnosis of HCC.However,the expression of KTN1 in HCC tissues and its mechanism in the occurrence and development of HCC are still unclear.Objective: 1.To investigate the expression of KTN1 mRNA in liver cancer tissues and its clinical significance.2.Explore the biological function of KTN1 in liver cancer cells.3.Explore the changes in the expression profile of liver cancer cell lines after KTN1 knockout.4.Explore the protein interacting with KTN1.Method: 1.73 HCC datasets were collected from Gene Expression Omnibus(GEO),Oncomine,Arlay Express,and The Cancer Genome Atlas(TCGA)databases,including 3772 HCC tissue samples and 2998 non-HCC liver tissues.Basing on these 73 datasets,Meta-analysis results which was performed by software Stata;The ROC curve and SROC curve were drawn by combining the data of each platform;Four data sets were used to draw the survival analysis K-M curve;After the Barcelona Clinic Liver Cancer(BCLC)staging and TNM staging respectively,by comparing the expression level of KTN1 gene;QRT-PCR was used to detect the expression level of KTN1 in the clinical tissues.2.The CRISPR/Cas9 gene editing technique was used in this study to knock out KTN1 gene in HCC cell line Huh7 in this study;The expression of KTN1 protein in the cells before and after knockout was detected by q RT-PCR and Western blot;Cell proliferation was detected by CCK-8 assay;Cell cycle was analyzed by flow cytometry;Cell migration and invasion ability were detected by transwell chamber assay.3.The RNA-seq technology was used to detect the changes in the genome-wide expression profile of the HCC cell line Huh7 after knocking out the KTN1 gene,and the bioinformatics database GO and Pathway were used to enrich the differential genes and screen out cancer-related genes.4.Co-IP technology was used to pull down the KTN1 protein-bound protein in HCC cell line Huh7,hepatoblastoma cell line Hep G2 and normal hepatocyte line WRL68,and Mass Spectrometry technology was used to identify the protiens;The bioinformatics database GO and Pathway were used to analyze the enriched proteins.Results: 1.The forest plot shows that the combined SMD value is 0.2(P<0.001);The areas under the curve(AUC)were higher than 0.5,the SROC curve AUC is 0.7,sensitivity is 0.44,specificity is 0.81,positive likelihood ratio is 2.36,negative likelihood ratio is 0.69.The results suggest that KTN1 can distinguish HCC and non-HCC tissues well;The overall survival time and disease-free survival time of the KTN1 high expression group were shorter than the KTN1 low expression group;The expression of KTN1 in tumor tissues of the three stages of I,II,and IV was significantly higher than that of the control group,and the expression in stages I,II,and IV showed a gradual upward trend;the expression level of KTN1 mRNA in liver cancer tissues was higher than that of matched cancer The adjacent tissues are high,and the difference is statistically significant(P<0.05).This result is consistent with the big data analysis of the significant increase in mRNA expression of KTN1 in liver cancer tissues.2.After KTN1 gene knockout,the proliferation ability of Huh7 cells was inhibited,and the number of early and late apoptotic cells were significantly increased,and the cell migration and invasion capabilities were also significantly reduced.3.Comparing the KTN1 knockout KO group with the untreated WT group,there are a total of 1070 differentially expressed genes,of which 793 were up-regulated in the KO group and 277 down-regulated;54cancer-related genes were screened out to be mainly enriched in chemical carcinogenesis,Pathways such as PPAR signaling pathway,retinol metabolism and cell-adhesion molecules;Cox regression to construct a risk model showed that SELL,ITGB4,ALDH3B1,THBS4,and ADH4 may be protective factors for HCC,while UGT2B17,NAT2,FABP3,CDH2,ACSL5 and ADH6 It may be a risk factor for HCC.The combination of KTN1 gene and these genes can predict the prognosis of HCC patients.4.A total of 30 KTN1 binding proteins were identified by Mass Spectrometry.Among them,PRL,CASPE,ACTS,and PRDX1 were only detected in samples of normal liver cell line WRL68.Compared with normal liver cell line WEL68,VIME was found in liver cancer cells.Both strains Huh7 and Hep G2 were elevated,while ACTN4,H2A1 J,H12,RS25,HBB,and DCD were all reduced in hepatocarcinoma cell lines Huh7 and Hep G2.We performed GO function annotation analysis on 30 proteins which bind to KTN1.These 30 proteins can be enriched in 18 biological processes,among which the cellular process,metabolic process,biological regulation,response to stimulus,multicellular organismal process,developmental process and other enriched proteins are more abundant.These proteins are also enriched in 12 cell components,of which cell part,organelle,extracellular region part,and organelle part are more enriched in protein.For molecular functions,these proteins are enriched in a total of 8 molecular functions,the most of which is binding,and a total of 27 proteins are enriched.Pathway annotates the pathways involved in the KTN1 binding proteins.These genes are mainly enriched in the Alcoholism and Systemic lupus erythematosus pathways.Conclusion: 1.The mRNA expression of KTN1 in HCC tissues is higher than that in non-HCC liver tissues.KTN1 mRNA expression can better diagnose HCC and is related to prognosis and grade.2.Knockout of KTN1 gene will inhibit the proliferation,migration and invasion of Huh7 cells,and promote cell apoptosis.3.Knockout of KTN1 will cause a large number of gene expression changes,which will affect a variety of biological functions.4.Identified the KTN1 interacting proteins and involved pathways.In conclusion,this study preliminarily verified that KTN1 was expected to be a molecular marker for the diagnosis and prognosis of HCC.RNA-seq preliminarily revealed the changes in the expression profile of HCC cells after the knockout of KTN1 gene,and the involvement of different genes in multiple cancer-related pathways.The results of Co-IP combined with mass spectrometry technology showed the interaction of the differential expression proteins and their tumor-related pathways in knocking out the KTN1 gene normal cells and HCC cell lines,laying a foundation for further research on the mechanism of KTN1 protein involvement in the occurrence and development of HCC.