The Identification Of Sternness-related Genes In Colon Cancer And The Functional Validation Of CCDC34

Background:Colon cancer is the fourth most common malignancy in the world,and it ranks fifth in the primary cause of death in neoplastic diseases.In China,colon cancer is the top-ranked cancer in newly diagnosed and the major cause for cancer-related mortality.Only in2020,over 560,000 colon cancer were diagnosed,which covered twelve percent of total newly diagnosed.Although there have been several important advances in surgery,chemotherapy and targeted agents over the past few decades,the overall survival of colon cancer patients is still unsatisfactory.Hence,it is critical to explore the cancer biomarker in this complicated malignancy.By precisely exploring the molecules mechanism in cancer,it may help us to improve patient’s clinical outcome.Based on tumor heterogeneity theory,cancer stem cells（CSCs）,having the principal properties of self-renewal,clonal tumor initiation capacity and clonal long-term repopulation potential,play an important role in cell proliferation,migration,invasion,and chemotherapy resistance.Therefore,screening and identification of genes that modulated cancer stemness may help us to understand the potential molecular mechanisms,search for novel biomarkers on target therapy and improve patients’prognosis.Methods:Key genes involved in colon cancer stemness regulation were identified according to the one-class logistic regression（OCLR）,weighted gene co-expression network analysis（WGCNA）and differential expression analysis method.The RNA-seq matrix and corresponding clinicopathologic information of The Cancer Genome Atlas（TCGA-COAD）were retrieved from the Genomic Data Commons Data Portal.The expression of candidate gene（CCDC34）was confirmed in data from Gene Expression Omnibus（GEO）and paired tissues from our hospital.Target gene stable knockdown cell lines were constructed to explore its impact on cancer stemness,growth,migration,and invasion,also in vivo mouse model was next applied.Then generation sequencing was performed searching the downstream pathways and the key proteins were confirmed by Western blottingResults:1.The level of m RNAsi was significantly up-regulated in colon cancer tissue comparing to the normal（p<0.001）,and patient with higher m RNAsi suffered worse prognosis（p=0.011）.There were 3765differential expression genes（DEGs）between normal and cancer tissues,including 2208 upregulated genes and 1557 downregulated genes,with the criterion that|log₂（Fold change）|>1&FDR<0.05.2.There were 13 modules generated from WGCNA,and blue module showed most closely correlated with the m RNAsi,（cor=0.82 and p<0.001）.The blue module contained 595 genes,and there were 10 hub genes that had potential in regulating colon cancer stemness,including CHEK1,TTK,NUP107,SGO1,CIP2A,KIF18A,CCDC34,PLK4,SGO and BUB1.3.The expression level of CCDC34 was remarkably upregulated in colon cancer tissues,comparing to the normal.Colon cancer patients with higher CCDC34 expression level demonstrated low grade differentiation tumor tissue（p=0.048）and more lymphatic metastasis（p=0.034）.And The expression of CCDC34 notably increased in colon cancer cell lines both in m RNA and protein level.4.We successfully constructed stable CCDC34 knockdown HT29and SW480 cell line with lentiviral vectors,with more than 80%knockdown efficiency.Knockdown of CCDC34 inhibits the stemness,proliferation,migration,and invasion of colon cancer in vitro（All p<0.05）.Knockdown CCDC34 impaired the growth and metastasis of xenografts in vivo（All p<0.05）.And overexpression of CCDC34enhanced the ability of stemness,proliferation,migration,and invasion of colon cancer（All p<0.05）.5.There were 1035 DEGs after CCDC34 overexpression in HCT116cell line detected by next generation sequencing.The functional enrichment result showed overexpression of CCDC34 activated the HEDGEHO and EPITHELIAL MESENCHYMAL TRANSITION pathways which were classical cancer stemness related pathways.Conclusions:1.m RNAsi is upregulated in colon cancer tissues compared to normal tissues.And COAD patients with a higher m RNAsi suffer a worse prognosis.2.CCDC34 is significantly upregulated in colon cancer,and it is strongly related to m RNAsi,the degree of tumor differentiation and the number of metastatic lymph node.3.Knockdown of CCDC34 inhibited colon cancer stemness,cell proliferation,migration,and invasion.4.Overexpression of CCDC34 activated HEDGEHO、EPITHELIAL MESENCHYMAL TRANSITION pathways.5.CCDC34 can act as a potential therapeutic target of colon cancer.