Expression And Bioinformatics Analysis Of CKMT2 In Colorectal Cancer Based On Two-dimensional Liquid Chromatography-mass Spectrometry

Objective:In this study,bioinformatics analysis,two-dimensional liquid chromatographymass spectrometry,western blot and immunohistochemistry are used to detect the difference in CKMT2 expression in Colorectal cancer(CRC)tissues and adjacent tissues.The expression level of CKMT2 in serum of colorectal cancer patients and healthy subjects was analyzed by ELISA and the relationship between the expression level of CKMT2 and clinicopathological data of colorectal cancer patients was analyzed,and the value of CKMT2 in clinical diagnosis and prognosis of colorectal cancer was preliminarily discussed.Method:1.Use public databases to conduct bioinformatics analysis on CKMT2,collate data,and explore its relationship with colorectal cancer.2.Differential protein expression profiles of colorectal cancer were constructed by two-dimensional liquid chromatography-mass spectrometry,indicating that CKMT2 is up-regulated in colorectal cancer tissues.3.Western blot was used to verify the expression of CKMT2 in colorectal cancer tissues and adjacent tissues.4.Immunohistochemistry was used to verify the expression of CKMT2 in colorectal cancer tissues and adjacent tissues.5.Enzyme Linked immunosorbent assay(ELISA)was used to detect the expression level of CKMT2 in serum of colorectal cancer patients and healthy subjects,and to analyze the relationship between the expression level of CKMT2 and clinicopathological data of colorectal cancer patients.Result:1.Analysis results of Oncomine database and Timer2.0 database showed that CKMT2 m RNA was up-regulated in colorectal cancer tissues compared with normal tissues.TISIDB database analysis showed that the expression of CKMT2 gene was significantly different in various molecular subtypes of colon cancer and rectal cancer.The Progno Scan database analysis showed that CKMT2 expression was not significantly associated with overall survival,disease-specific survival,and complete remission disease-free survival.Results from the C-BIO Portal database showed that the CKMT2 gene had 12 mutation sites(11 missense mutations and 1 in-box mutation)between amino acids 0 and 419.CKMT2 gene was co-expressed in colorectal cancer,and the top 10 genes with the strongest association were SLC6A4,PLA2G12 B,ACSL6,LY6G6 D,SLC30A2,CEL,OIT3,CELP,SLC22A11 and SLC22A11.Among them,SLC6A4 gene was the most co-expressed gene with CKMT2,and its m RNA level was positively correlated with CKMT2(Spearman's Correlation=0.60,Pearson=0.34).According to STRING database,GAMT,MB,CKM,ALDOA,LDHAL6 A,LDHAL6B,RAB28,LDHC,LDHB and other proteins are closely related to CKMT2 protein,and it is found that the main molecular functions of CKMT2 protein are Llactate dehydrogenase activity,creatine kinase activity,catalytic activity,etc.The main biological processes involved include creatine phosphate biosynthesis,creatine metabolism,muscle contraction,pyruvate metabolism,cell-modified amino acid biosynthesis and so on.2.Results of 2d liquid chromatography-mass spectrometry :(1)Venn diagram showed that there were 381 common differential proteins in stage ? colorectal cancer and adjacent tissues,and stage ? colorectal cancer and adjacent tissues.(2)The IPA software analyzes the classic Pathway of different proteins.The results showed that in stage ? colorectal cancer,compared with para-cancer tissues,protein synthesis,oxidative phosphorylation and TCA cycle were significantly activated,while cytoskeleton and Rho GDI pathways were significantly inhibited.In stage ? colorectal cancer,protein synthesis,PI3 K,AKT,PKA and other pathways were significantly activated in colorectal cancer tissues compared with adjacent tissues.(3)CKMT2 is one of the up-regulated proteins.3.Western blot results: the protein expression of CKMT2 in colorectal cancer tissues was significantly higher than that in adjacent tissues.4.Immunohistochemical results: the protein expression of CKMT2 in colorectal cancer tissues was significantly higher than that in adjacent tissues.5.ELISA results: Serum CKMT2 protein concentration in colorectal cancer patients was significantly higher than that in healthy subjects,P<0.05.There was no significant correlation between the expression level of CKMT2 protein and gender,age,lymph node metastasis,tumor location,tumor differentiation level and tumor size.Conclusion:1.CKMT2 is highly expressed in colorectal cancer tissues.2.CKMT2 is highly expressed in the serum of patients with colorectal cancer.3.CKMT2 is expected to be a valuable biomarker for colorectal cancer.