| Background:Colorectal cancer is one of the most common malignant tumors of the digestive tract in China and the world with high morbidity and mortality.Although the research on colorectal cancer continues to deepen,the techniques for prevention,diagnosis and treatment of colorectal cancer continue to develop,but some patients still suffer from late pathological stage when be diagnosed,early invasive metastasis,and tolerance to radiotherapy and chemotherapy during treatment.These situations have greatly threatened the health of people.In recent years,the second generation sequencing technology has developed rapidly and been applied in biomedicine.With its advantages of high throughput and low cost,it has rapidly become one of the most important means to study human diseases.Through the second generation sequencing technology,oncology researchers can explore the genome and transcriptome changes of malignant tumors,which provides a possibility for the overall control mechanism of malignant tumors.According to the current understanding of colorectal cancer,more than 80% of colorectal cancers have undergone normal-adenomas-carcinogenesis process,and a considerable number of patients have lymph node metastasis in the early stage of tumorigenesis.Therefore,the evolutionary mechanism and molecular characteristics of adenoma carcinogenesis and early invasion of colorectal cancer are a very meaningful study for the prevention and treatment of colorectal cancer.However,there are some problems in the study of early evolution mechanism and molecular characteristics of colorectal cancer by the second generation sequencing technology,such as the large bias of samples not taken from the same individual,the small number of sequencing samples with limited funds,and the inability to repeat them.In addition,at the transcriptome level,long non-coding RNA has become a hot topic for colorectal cancer researchers.Long non-coding RNA can achieve fine regulation of tumors through competitive endogenous RNA networks.With the second generation sequencing technology and bioinformatics,the relationship oflnc RNA-mi RNA-m RNA is constructed,and further experimental verification of cells has become a common technique used by cancer researchers.This study intends to use the sequencing data of this study and the sequencing data of public databases to explore the early evolutionary mechanism and molecular characteristics of colorectal cancer.Then construct a competitive endogenous RNA network of colorectal cancer for further screening of target lnc RNA and explore related mechanisms.Part I Overview of whole-exome and transcriptomesequencing resultsObjective The aim of this study was to explore the key GO function and KEGG pathway and driver genes in adenoma carcinogenesis and early invasion of colorectal cancer.Methods 1.Fresh specimens coexisting with colorectal adenoma and colorectal cancer were collected.Normal tissues,paracancerous adenoma tissues and cancer tissues were collected.2.We performed high-throughput sequencing of transcriptome and whole exomes on quality-qualified specimens,and strictly controlled the sequencing results.3.GO function tree and KEGG signal pathway interaction network enrichment analysis were performed at the transcriptome level for differentially expressed genes at the whole level(tumors VS normal,adenomas VS normal)and subgroup level(T1-2N0M0 tumors VS normal,T1-2N1-2M0 tumors VS normal,T3-4N1-2M0-1 tumors VS normal).The results were used to analyze the GO function and KEGG pathway associated with adenoma carcinogenesis,early lymph node metastasis of colorectal cancer.4.At the level of whole exome sequencing,SNV mutation genes were analyzed in paired samples of each patient and in three subgroups of T1-2N0M0,T1-2N1-2M0,and T3-4N1-2M0-1.Then,by analyzing the common genes of mutations,the potential driver genes are explored.Results 1.Both of sequencing samples and results are qualified.2.At the transcriptome level,adenomas have fewer differentially expressed genes than tumors,and enriched GO function and KEGG pathway are less than tumors.The differentially expressed genes of adenomas have been significantlyenriched in some functions and pathways such as cell proliferation,invasion,and migration,but less than tumors.The T1-2N1-2M0 subgroup has unique enrichment in cell differentiation,cell maturation,extracellular matrix formation,phospholipase C activation of G protein-coupled receptor signaling pathways,and other signaling pathways.3.At the level of the whole exome sequencing,paired samples did not find the common mutation genes of adenoma-tumor,only some local common mutation genes,such as APC(adenoma and cancer tissues of patients No.8,11 and 14),KRAS(adenoma and cancer tissues of patients No.4,7 and 8),TTN(adenoma and cancer tissues of patients No.11 and 12),etc.Conclusions 1.In terms of GO function and KEGG pathway,differentially expressed genes in adenoma tissues have been significantly enriched in cell proliferation,invasion,migration-related functions and signaling pathways,but relatively less than in tumor tissues,which conforms to a certain law of molecular evolution.2.The differentially expressed genes in the T1-2N1-2M0 subgroup have uniquely enriched GO function and KEGG pathway,which may promote their early invasion events.3.Due to the heterogeneity of the tumor,no common mutant gene was found,but some local common mutant genes were found.Part II Deep analysis of bioinformatics of earlyevolutionary mechanism and molecular features ofcolorectal cancer based on sequencing sample data of thisstudy and TCGA databaseObjective The purpose of this part is to explore the unique genome and expression profiles of T1-2N1-2M0 subgroup in genome and transcriptome and reveal the evolutionary mechanism of the T1-2N1-2M0 subgroup that is different from the T1-2N0M0 subgroup and the T3-4N1-2M0-1 subgroup.The results were used to explore the early evolutionary events and molecular characteristics of colorectal cancer.Methods 1.The mutation types of colorectal cancer samples sequenced in this study were summarized and classified.Non-negative matrix factorization algorithmwas used to obtain the mutation signatures of the samples.We clustered the mutation signatures and the 30 mutation signatures published in the COSMIC database to obtain the mutation signatures which were similar to COSMIC database.The samples were clustered according to the mutation signatures,and the regularity between the sample groups was explored.2.We found that the mutation signature annotations were related to APOBECs,and then we analyzed the existence of APOBECs of differentially expressed genes in different groups and validated them in TCGA database.3.We explored the SNVs of APOBECs in different groups of large sample data from the TCGA database and analyzed them with clinical pathology data.4.We combined the expression profile data to analyze the relationship between SNVs and m RNA expression of APOBECs.5.The CNVs of APOBECs were analyzed,and the CNVs were analyzed combinded with expression profiles.6.We analyzed the combined expression profiles of significant mutation genes in different groups.Results 1.The mutations enriched in colorectal adenoma tissue and tumor tissue are mainly derived from AID/APOBECs-driven mutations.There are significant differences in the mutation signatures and mutation spectrum among the T1-2N0M0 group,the T1-2N1-2M0 group,the T3-4N1-2M0-1 group.2.The 11 genes of APOBECs were different quantities’ differentially expressed in colorectal adenomas and cancer tissues in T1-2N0M0 group and T3-4N1-2M0-1 group,but no significant difference was found in T1-2N1-2M0 group.3.The SNVs of APOBECs occurred in the TNM stage I and stage II were significantly higher than that of stage III and stage IV.The SNVs of APOBECs were not found in T1-2N1-2M0 group,but found in both the T1-2N0M0 group and T3-4N1-2M0-1group.However,due to the small sample size,there is no statistically significant difference among them.The data from TCGA database showed that the expression level of APOBEC1 mutation types were lower than that of wild types(P=0.027),and the expression level of APOBEC4 mutation types were lower than that of wild types(P=0.012).4.There were significant differences in m RNA expression among three or four groups of copy number of APOBEC1,APOBEC2,APOBEC3 B,APOBEC3C,APOBEC3 D,APOBEC3F,APOBEC3 G and APOBEC3H(P < 0.05),and we foundthat the m RNA expression level increased gradually with the increase of the copy number,with a few exceptions(APOBEC3D,APOBEC3G).5.The expression trend of significantly mutated genes in T1-2N1-2M0 group is different from that in T1-2N0M0 group and T3-4N1-2M0-1 group,which reveals the particularity of genome and transcriptome in T1-2N1-2M0 group.Conclusion Most of the evolutionary process of colorectal cancer is accompanied by abnormal expression of APOBECs.The mutation types are driven by APOBECs.However,colorectal cancer with lymphatic metastasis in early T stage may have different evolutionary mechanisms and expression profiles than those driven by APOBECs.Part III Study on tumor immunocyte infiltration incolorectal cancer tissue samples based on TCGA databaseObjective The aim of this study was to explore 22 types of immunocyte infiltration patterns in different grouped tumor samples in TCGA database and the prognostic-associated immunoinfiltrating cells.Methods 1.The TCGA database samples’ expression profile data were normalized and the unqualified samples were removed.2.In the R3.5.0,the CIBERSORT code was loaded to calculate the composition of the 22 immunocytes of the samples,then subgroups analysis were performed.3.We used univariate COX regression to analyze prognostic immune cells in combination with survival data.Results 1.There were no statistically significant immunoinfiltrating cells in the T1-2N1-2M0 group,and there were different numbers of similarly statistically significant immunoinfiltrating cells in the T1-2N0M0 group and the T3-4N1-2M0-1 group.2.The infiltration levels of dendritic cells resting in different TNM stages were significantly different and showed a certain trend.The infiltration level was significantly correlated with prognosis.Conclusion The T1-2N1-2M0 group has different immune infiltration patterns than the T1-2N0M0 group and T3-4N1-2M0-1 group,which may be related to their evolutionary mechanisms.Dendritic cells resting are significantly associated with prognosis in patients with colorectal cancer.Part IV Screening and Validation of lnc RNA in ColorectalCancerObjective The aim of this study was to construct a ce RNA regulatory network for colorectal cancer,and the results were used to screen and validate the candidate lnc RNA.Methods 1.Target mi RNAs that regulate key genes in the early stages of colorectal cancer evolution are explored based on target gene prediction softwares.2.The ce RNA network of colorectal cancer was constructed based on differentially expressed m RNAs,lnc RNAs and target gene prediction softwares.3.The key lnc RNA-mi RNA-m RNA relationship was screened to identify and validate the candidate lnc RNA.Results 1.Target gene prediction softwares predict that mi R-137 is a target mi RNA for a large part of APOBECs,significant mutation genes.2.The ce RNA network of colorectal cancer has been successfully constructed,and a large proportion of lnc RNAs in the network have been reported in the literature.3.Lnc RNA-TINCR was significantly down-regulated in the sequencing samples of this study and was validated in the TCGA database to determine the target lnc RNA.Conclusion Mi R-137 plays an important role in the early evolution mechanism of colorectal cancer.The ce RNA network of colorectal cancer provides a reference for understanding the regulation system of colorectal cancer.The target lnc RNA-mi RNA-m RNA relationship was determined by the ce RNA network.Part V Study on the relationship between long non-codingRNA-TINCR and radioresistance of colorectal cancer cellsObjective The aim of this study was to explore the mechanism of radioresistance of colorectal cancer associated with lnc RNA-TINCR.Methods 1.The proliferation and migration ability of colorectal cancer cells after overexpression of TINCR were detected by CCK-8,scratch healing and transwell assay.2.CCK-8 assay was used to detect the proliferation of SW620 R and SW620 cells.Tumor sphere formation assay was used to detect the spherical formation ability of SW620 R and SW620 cells.Clonogenic survival assay was used to detect the cellsurvival rate of SW620 R and SW620 cells under different doses of X-ray irradiation(0,2,4,6 and 8 Gy).3.We used q PCR to detect the expression level of TINCR in SW620 R and SW620 cells.Changes in proliferation and migration ability after overexpression of TINCR were detected by CCK-8,scratch healing and transwell assay.TINCR of SW620 R cells was knocked out by si RNA of TINCR.The effect of TINCR on radiation resistance of SW620 R cells was confirmed by clonogenic survival assay.4.QPCR and Western blot were used to detect the RNA and protein levels of TCF4 in SW620 R cells after TINCR was knocked out.Tumor sphere formation assay was used to test the spherical formation ability,and q PCR was used to detect the expression level of stemness related genes.5.We used a dual-luciferase assay to detect whether TINCR is a direct target of mi R-137.The expression level of mi R-137 was detected by q PCR after TINCR was knocked out.Results 1.Compared with the control group,the proliferation and migration ability of SW620 and HTC116 cells in TINCR overexpression group were decreased.2.CCK-8 assay showed that the proliferation ability of SW620 R cells was higher than that of SW620 cells.Tumor sphere formation assay show that SW620 R cells have higher spherical formation ability than SW620 cells.Clonogenic survival assay showed that SW620 R cells had higher radioresistance than SW620 cells under different doses(0,2,4,6 and 8 Gy)of X-ray irradiation.3.CCK-8 assay,scratch healing assay and transwell assay showed that overexpression of TINCR still inhibited the proliferation and migration of SW620 R cells compared with the control group.When TINCR was knocked out,the resistance of SW620 R cells to radiation was reduced.4.After TINCR was knocked out,TCF4’s expression was decreased at the RNA level and protein level.When TINCR was knocked out,the spherical formation ability of SW620 R cells decreased.The expression levels of OCT4 and SOX2 in TINCR knockout group were lower than those in control group.The expression of OCT4,SOX2 and NANOG in SW620 cells was lower than that in SW620 R cells.5.The dual-luciferase assays showed that TINCR was the direct target of micro RNA-137.After TINCR was knocked out,the expression of mi R-137 was significantly increased.Compared with the control group,the difference was statistically significant(P<0.05).Conclusion TINCR can inhibit the proliferation and migration of colorectal cancer cells,but promote their radioresistance.Radioresistance of colorectal cancer cells is closely related to the stemness of cancer cells that was regulated by mi R-137.Summary1.Our results show that most of the evolutionary process of colorectal cancer is accompanied by abnormal expression of APOBECs,and the mutation types are driven by APOBECs.However,colorectal cancer with lymphatic metastasis in early T stage has different early evolutionary molecular characteristics from those driven by APOBECs.2.The uniqueness of colorectal cancer with lymphatic metastasis in early T stage is mainly manifested in the uniqueness of the enrichment of GO functions and KEGG pathways,the uniqueness of APOBECs’ expression,the uniqueness of APOBECs’ SNVs,the uniqueness of the number and expression pattern of significant mutation genes,and the uniqueness of immune microenvironment.3.The unique molecular characteristics of colorectal cancer with lymphatic metastasis in early T stage suggest that the treatment of this kind of patients should be different from the traditional TNM staging.The results indicate the research direction for clinical precise therapy.4.TINCR plays an important role in the regulation of stemness in CRC cancer cells,which directly affects the radioresistance of CRC cancer cells and has potential as a diagnostic marker or therapeutic target of radioresistance. |
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