| BackgroundColon cancer is the third most common tumor in the world,caused hundreds of thousands of deaths each year.In China,the number of new cases of colon cancer ranks the second among all tumors,which seriously threatens the life safety of our Chinese people.As per histological classification,colon cancer can be mainly divided into three types:adenocarcinoma,adenosquamous cell carcinoma and undifferentiated carcinoma,among which the colon adenocarcinoma(COAD)is the main type.Due to the insidious onset,most patients present in the middle or advanced stages of progression.At present,even if surgery and chemotherapy are used for combined therapy for colon cancer of intermediate and advanced stages,the patients can only achieve limit efficacy because of relatively high mutation rate of colon cancer.However,the common targeted drugs cetuximab and bevacizumab can only benefit a limit range of patients due to the receptor mutability and side effects,respectively.So trying to find new therapeutic targets for colon cancer and to perform in-depth study towards their occurrence and development mechanism are of far-reaching significance for improving the prognosis of colon cancer patients.Cold Shock Protein(CSPs),a kind of relatively conserved protein in the process of biological evolution,which plays a critical role of some adverse conditions,such as low temperature,poisoning and so on,produced to adapt to the environmental changes,can change tumor-cell senescence process.Moreover,its structure is of a high-degree homology,and the current research found that it is widely expressed in diverse tumors.Because of its non-mutant characteristics,it becomes a good anti-tumor target.YBOX3,which is encoded by YBX3 gene,is also the new discovered member of the Y-box protein family,which plays an important role in regulating transcription,m RNA alternative splicing,DNA repair,cell proliferation and regeneration.The expression of YBX3 in normal tissues is relatively weak,whereas the expression level in tumor tissues is significantly increased.However,the specific effect and mechanism of YBX3 upregulation in colon cancer and its role in vivo still need to be further investigated.Objective1.To explore the expression of YBX3 in colon cancer and its influence on the clinical significance of colon cancer;2.To explore the influence of YBX3 on the biological function of colon cancer;3.To explore the mechanism of YBX3 upregulation and the specific molecular mechanism of its role in colon cancer.Methods1.To study the expression of YBX3 in COAD and its effect on the clinical significance of COADThe data acquired by RNA-seq of 521 COAD cases and some paracancerous patients in the TCGA-COAD database were used to explore the expression patterns of YBX3 in COAD and its corresponding paracancerous tissues,and further,the internal cohort clinical samples were also used to verify the difference between the expression of YBX3 in cancer and paracancerous groups.Finally,the prognostic data were used to analyze the impact of YBX3 on the prognosis of colon cancer cohort in our hospital.2.Exploration of the biological function of YBX3 in colon cancer2.1 The corresponding stable YBX3-knockdown cell lines HCT116-YBX3-sh1/2 and SW620-YBX3-sh1/2,the stable YBX3 over-expression cell lines HCT116-oe YBX3 and SW620-oe YBX3 were all constructed based on the colon cancer cell lines HCT116 and SW620.2.2 HCT116-YBX3-sh NC and SW620-YBX3-sh NC were used as their respective negative controls.CCK8,Transwell,scratch assay and clone formation assay were performed using the above cell lines,and the effects of YBX3 expression on biological functions such as proliferation,invasion and migration in colon cancer cell lines were detected in vitro by relevant experiments which were listed above.3.The upregulation mechanism of YBX3 in colon cancer and the exploration of its specific mechanism of promoting the progression of colon cancer3.1 Florescent real-time quantitative PCR(q PCR)was used to detect differential RNA methylases in normal colonic epithelium cell lines(FHCs)and colon cancer cell lines,and the statistically significant RNA methylases were analyzed for western blotting(WB).Specific RNA methylases were selected for RNA methylation co-immunoprecipitation(Me RIP)analysis,the effect of RNA methylation on YBX3 expression in colon cancer cell lines was explored.Moreover,the corresponding small interference RNA(si RNA)of methylase was used to further validate the role of RNA methylase on YBX3 expression in colon cancer cell lines.3.2 The RNA sequencing data of colon cancer samples obtained from TCGA-COAD database were divided into 2 groups according to YBX3 expression level.YBX3 expression lower than the first quartile interval(0-25%)was named as the low expression group,and the level of YBX3 expression in the samples higher than the last quartile interval(75-100%)was named as the high expression group.Finally,YBX3 single gene differential analysis were also performed to find the genes that YBX3 may regulate in colon cancer.3.3 Given that the RNA-binding properties of YBX3,using transcriptome sequencing(RNA-seq)to search for differentially regulating downstream target m RNAs in SW620-YBX3-sh1/2 vs.SW620-YBX3-sh NC and HCT116-YBX3-sh1/2 vs.HCT116-YBX3-sh NC.At the same time,RNA Immunoprecipitation Sequencing(RIP-seq)was used to detect the target m RNA that binds to YBX3 in SW620-oe YBX3 vs.SW620-YBX3-oe NC.Finally,intersected with the above results to explore the downstream target m RNA of YBX3-bound and YBX3-regulated in colon cancer cell lines.The top m RNAs were comprehensively screened according to log FC and P values for further discussion.Results1.The expression of YBX3 was upregulated among multiple tumors across TCGA database.Among the multiple immune subtypes of colon cancer,the expression level of YBX3 was highest in IFN-γ-dominated type.Among the many molecular subtypes,YBX3 expression is highest in the HM-indel subtype.In the cohort of gastrointestinal tumors(esophageal adenocarcinoma,ESAD;stomach adenocarcinoma,STAD),patients with low expression of YBX3 have better disease-free survival.YBX3 expression has high diagnostic value in colon cancer,and analysis towards different subgroup of colon cancer cohorts shows that YBX3 expression promotes the malignant phenotype of different clinical subtypes.Meanwhile,the upregulation of YBX3 expression predicts poor clinical outcomes among patients with colon cancer of advanced stages,with lymph node metastasis,and with upregulated level of tumor biomarkers.Single gene differential analysis of YBX3 revealed that the main Hub gene network pointed to nucleic acid binding,corneous vesicular formation,and regulation of tumor cell necrosis,etc.Immune infiltration analysis showed that YBX3 was positively correlated with various immune cell infiltration in COAD immune microenvironment.Single-cell analysis showed that YBX3 in colon cancer was significantly enriched in the epithelial cell subset,and in the TCGA colon cancer chemotherapy cohort,YBX3 expression was higher in those with high chemotherapy responsiveness.Based on the results of YBX3 immunohistochemical staining of tissue chip derived from internal colon cancer cohort,the extent of YBX3 staining gradually increased with the increase of pathological stage of colon cancer,and the expression level of YBX3 was higher in the colon cancer cohort of advanced stages(the prognosis was also worse).2.The upregulation of YBX3 effectively promotes the malignant phenotype of colon cancer cell lines.The results of four experimental results,CCK8,scratch,Transwell and clone formation assay based on YBX3 steady over-expression/knockdown cell lines which had already been constructed in above parts,reveals that colon cancer cell lines with high YBX3 expression showing higher proliferation,invasion and migration capabilities.However,the ability of colon cancer cells with low YBX3 expression to proliferation,invasion and migration decreased significantly.3.The upregulation of YBX3 expression in colon cancer is due to its positive-feedback regulation of m RNA m6A-methylation modification.Methylation level detection experiment showed that compared with the normal colonic epithelial cell line FHC,the level of YBX3 RNA m6A-methylation modification in colon cancer cell lines,SW620 and LOVO,was significantly increased.The test of polymethylased enzymes showed that compared with normal colon epithelium,m6 A writer enzyme—methyltransferase-like 3(METTL3),m6 A Reader protein YTHDC1(YTH domain-containing protein 1)in colon cancer cell lines were significantly increased.After the transfection of the corresponding small interfering RNA targeting methylase,the expression of YBX3 in colon cancer significantly decreased.At the same time,RIP-PCR showed that YBX3 can bind and inhibit the m RNA level of ALKBH5,thereby reversing demethylation,consequently maintaining its high hypermethylation state.4.YBX3 enhances the stability of the m RNAs of MYC zinc finger protein,MAZ,and MYC-binding protein,MYCBP2,finally promoting the c-Myc expression and the malignant phenotype.Combined with RIP-seq data acquired from SW620-oe YBX3 vs.SW620-YBX3-oe NC,RNA-seq based on SW620-YBX3-sh1/2 vs.SW620-YBX3-sh NC and HCT116-YBX3-sh1/2 vs.HCT116-YBX3-sh NC,the results showed that YBX3 can bind and regulate various m RNAs.And the combined analysis showed that there were 244 upregulating m RNAs,and 138 m RNAs were bound and downregulated,among which,MAZ,MYCBP2,RUNX3 and UBE2 H were selected as potential target m RNAs after screening.RIP-q PCR also showed that YBX3 bound with MAZ and MYCBP2.If the level of YBX3 expression decreased,the expression of MAZ and MYCBP2 were downregulated accordingly,thereby,the expression level of c-Myc was also decreased.ConclusionCold shock protein YBX3 is regulated by methylase METTL3 and m6 A binding protein——YTHDC1 in colon cancer,and it inhibits the expression of demethylase ALKBH5 to maintain its own high level of m6A-methylation modification.Moreover,it upregulates the expression of c-Myc via binding with the m RNAs of MYBP2 and MAZ to promote the malignant progression of colon cancer. |
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