| Background and Objective:Colorectal cancer has become the most common malignancy of the digestive system,causing a huge burden of disease worldwide.Although some progress has been made in the diagnosis and treatment of colorectal cancer,the disease is increasing and growing younger,which poses a more serious challenge to the prevention and treatment of the disease.Therefore,the search for more effective diagnostic markers and therapeutic targets is of great significance for the early diagnosis and treatment of colorectal cancer.Alternative splicing maintains normal biological functions by regulating the formation of different transcript variants and the expression of protein-coding genes,yet dysregulation of alternative splicing is considered to be a new landmark event in tumors.Differential expression of splicingassociated factor genes is a direct mechanism of action leading to tumorigenesis.Through bioinformatic analysis,we identified CLK2 as a hub splicing-associated factor gene in colorectal cancer.In this study,we further detected the expression level of CLK2 in colorectal cancer and analyzed its relationship with clinicopathological parameters and prognosis of patients,and investigated the effect of CLK2 on the biological function of colorectal cancer cells and explored the molecular mechanisms involved,aiming to provide new targets for intervention in the clinical diagnosis and treatment of colorectal cancer.Methods:1、By merging the expression profile data and the list of splicing-associated factor genes from colorectal cancer samples in the TCGA database,the samples were categorized using NMF clustering analysis and confirmed using the GSE17536dataset;the hub splicing-associated factor genes in colorectal cancer were identified by combining three machine learning algorithms.2、The expression levels of CLK2 in 64 pairs of fresh colorectal cancer tissues and paracancerous tissues were detected by q RT-PCR and Western blotting assay,respectively;the expression of CLK2 protein in 120 colorectal cancer tissues was also detected by immunohistochemistry,and the correlation between CLK2 protein expression levels and patients’ clinical data was analyzed based on the staining score results.3、The expression level of CLK2 in six colorectal cancer cell lines and normal intestinal epithelial cells was detected by q RT-PCR and Western blotting;two highly expressed cells were selected to construct stable expression lines by transfection of CLK2-RNA interference lentiviral vector,and two less expressed cells were selected to construct the CLK2-overexpressing cell line with stable gene expression;The effects of CLK2 on the proliferation,invasion and angiogenesis of colorectal cancer cells in vitro were examined by CCK8 assay,Ed U assay,plate clone formation assay,wound healing assay,transwell migration and invasion assay,EMT-related marker assay and tube formation assay,respectively.4、The effect of CLK2 on colorectal cancer cell tumor formation and metastasis in animals was assessed by constructing a subcutaneous tumor formation and liver metastasis model in nude mice.5、The gene set enrichment analysis was performed on CLK2 high and low expression groups,and Western blotting was used to detect the protein expression of signaling pathways involved in CLK2 regulation.6 、 The reciprocal proteins of CLK2 were identified and analyzed by constructing overexpression tag plasmids and using protein immunoprecipitation combined with mass spectrometry,and the interactions between the proteins were further verified using immunoprecipitation and cellular immunofluorescence assays.Results:1、The NMF clustering analysis algorithm was able to classify colorectal cancer samples in the TCGA database into C1 and C2 subtypes based on splicing-associated factor genes,and the typing results were also validated in the validation set GSE17536;the difference analysis results showed significant survival differences between C1 and C2 subtypes.Six hub colorectal cancer splicing-associated factor genes were further identified by combining three machine learning algorithms,with CLK2 being the most significantly different gene.2、The results of q RT-PCR and Western blotting experiments showed that the expression level of CLK2 in colorectal cancer tissues was significantly higher than that in paracancerous tissues,and the difference was statistically significant(p<0.05);the immunohistochemical results revealed that the increased expression of CLK2 protein correlated with the pathological stage,depth of tumor infiltration and lymph node metastasis of colorectal cancer patients(p<0.05),and was strongly correlated with poor prognosis of colorectal cancer patients(p<0.005).3 、 The expression level of CLK2 was increased in colorectal cancer cells.SW480 and SW620 with higher expression levels were used to construct the CLK2-interfering cell line with stable gene expression,while HCT8 and HCT116 with lower expression levels were used to construct the CLK2-overexpressing cell line with stable gene expression.The results of CCK8 assay,Ed U assay and plate clone formation assay showed that overexpression of CLK2 significantly promoted cell proliferation compared to the control group(p<0.05);next,the results of wound healing assay,transwell migration and invasion assay and EMT-related markers showed that overexpression of CLK2 also promoted cell migration and invasion compared to the control group(p<0.05).Further,the results of the tube formation assay indicated that overexpression of CLK2 also significantly promoted the angiogenesis compared to the control group(p<0.05);In contrast,interference with CLK2 expression inhibited the effect.4 、 In vivo experiments showed that colorectal cancer cells overexpressing CLK2 significantly enhanced the tumorigenic ability of the cells compared with the control group(p<0.05),and the immunohistochemical results showed higher Ki-67 expression in the tumor;at the same time,colorectal cancer cells overexpressing CLK2 also had stronger metastatic ability;while interference with CLK2 expression inhibited the tumorigenic and metastatic ability of colorectal cancer cells.5、The results of the gene set enrichment analysis showed that CLK2 may regulate the progression of colorectal cancer through the Wnt/β-catenin signaling pathway,and the results of Western blotting experiments indicated that overexpression of CLK2 could promote the increase of total β-catenin protein and the accumulation of β-catenin in the nucleus.6、SRSF7 was found to be a significantly different protein molecule by protein immunoprecipitation combined with mass spectrometry,and may be a reciprocal molecule of CLK2.The existence of protein interactions between CLK2 and SRSF7 was confirmed by endogenous and exogenous immunoprecipitation experiments,and cellular immunofluorescence verified a high degree of co-localization in the spatial structure with a correlation coefficient greater than 0.85.Conclusions:The increased expression of CLK2 in colorectal cancer tissues is positively correlated with clinicopathological parameters such as pathological stage and depth of tumor infiltration in colorectal cancer patients,and patients with colorectal cancer with high CLK2 expression had a poor prognosis,which is expected to be used as a prognostic indicator for colorectal cancer.In vitro and in vivo experiments have demonstrated that CLK2 can promote various malignant biological behaviours such as proliferation,invasion and metastasis of colorectal cancer cells,indicating that CLK2 act as an oncogene in colorectal cancer.In addition,CLK2 can mediate the progression of colorectal cancer by regulating the Wnt/β-catenin signaling pathway,and the protein interactions between CLK2 and SRSF7 may be a potential target for colorectal cancer therapy. |
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