| Background:Colorectal cancer(CRC)is the third most common cancer in the world,and the fifth leading cause of cancer-related mortality.It is also the leading cause of cancer-related death and the major cause of cancer-related death.For decades,surgical technique,chemotherapy and radiotherapy for the treatment of colorectal cancer,surgery is the main treatment,early detection,early diagnosis and early treatment is essential,and simple surgical treatment can not cure CRC,even if combined with chemotherapy and radiotherapy still has high recurrence and metastasis.In recent years,biological targeted therapy has been gradually applied to the diagnosis and treatment of CRC,and has achieved some results.The focus of the current research is focused on the search and screening of tumor markers that can detect CRC early.In the diagnosis,electronic colonoscopy examination is still the most important means in the screening of CRC,it is found that the main method of early CRC,but pathology remains the gold standard for diagnosis of CRC,however,colonoscopy and pathological examination as an expensive and low compliance of invasive examination still,with great limitations.Therefore,it is necessary to find a non-invasive diagnostic marker for CRC detection.In recent years,scientists have been searching for biological markers for the diagnosis and prognosis of CRC.Recent studies have shown that long noncoding RNA plays an important role in the development and progression of CRC.Aberrant expression of microRNA(miRNA),as a tumor suppressor or oncogene,plays an important role in the development and invasion of colorectal cancer.In addition,miRNA can target hundreds of messenger RNA(mRNA)and interfere with cell signaling pathways.Especially circulating miRNAs is widely regarded as non-invasive detection of early colorectal cancer biological marker,which is helpful to understand the etiology and biological characteristics of colorectal cancer so as to improve the diagnosis of CRC and abundant treatments of CRC may be provided.In recent years,more and more studies have shown that microRNA(miRNA)abnormalities can be used as a potential biomarker for cancer diagnosis.A high-throughput transcriptome technology research from the transcriptome changes or differences in CRC,and elucidate the molecular mechanism of disease progression of CRC,has important significance to find the molecular marker of early intervention and prognosis of tumor.In addition,changes in DNA methylation can also play a role in tumor gene expression during tumor formation and progression.The Cancer Genome Atlas(TCGA)data portal contains information,clinical data and genomic characterization of colorectal adenocarcinoma(case group)and normal colorectal mucosa(control group)sequence analysis of high level,so it can provide the opportunity hitherto unknown for revealing the molecular mechanism of CRC.In recent years,There is increasing evidence that the pathology of miRNAs in colorectal cancer occurrence,development and transfer of science research is an important regulatory factor,some miRNAs is considered to be the regulation of colorectal cancer and cancer gene in the development process or tumor suppressor gene.Our previous studies showed that play an important role in the occurrence and development process of miR-335 in CRC,but its at the molecular level of the specific mechanism is still not clear.In this study,we studied the colorectal cancer associated miRNAs,and identified the differentially expressed genes of CRC,and constructed and regulated the regulatory network of colorectal cancer related transcription factors.This paper consists of two parts.In the first part,we analyzed the expression characteristics of microRNA/mRNA in colorectal cancer,and explored the function of miR-335 in colorectal cancer.The second part focuses on the study of miR-335 in CRC,The Twistl is involved in miR-335 mediated tumor cell migration,invasion and epithelial mesenchymal transition(EMT)research,to explore and reveal the miR-335 inhibit the development of CRC,the molecular mechanism of invasion and transfer processPart I:a comprehensive analysis of the expression characteristics of microRNA/mRNA in colorectal cancer.Objective:(1)To identify the characteristics of miRNA and mRNA in colon adenocarcinoma.(2)To investigate the function of miR-335 in CRC.Methods:(1)by January 5,2016,the clinical data of 459 patients were collected from the genomic Genome Atlas(TCGA)of the cancer genome for the analysis of gene mapping data for colorectal adenocarcinoma.(2)The differential expression of miRNA and mRNA in colorectal cancer and adjacent normal tissues was calculated by R-bioconductor software deseq2.Benjamini and Hochberg methods were used for multiple comparisons to obtain the false discovery rate.The absolute values of log2 multiples were determined by the volcano clustering map of miRNAs and mRNA,so as to find the differentially expressed miRNA and mRNA.(3)the expression of miRNA learning algorithm to predict the differences in the use of biological information,via a variety of algorithm selected miRNA-mRNA target gene,using visualization software(http://www.cytoscape.org/)and finally construct the target gene regulation network of miRNA,power can be enriched by gene pathway annotation and function using Gene Codis online software,finally explore the potential targets for rectal carcinoma miRNA.(4)Using COHCAP(https://sourceforge.net/projects/cohcap/)to determine the differentially expressed methylation genes between colorectal adenocarcinoma and adjacent normal tissue adjacent to the tumor.DNA methylation was applied to the methylation 450 platform of methyljhu USC,and the differentially expressed genes were negatively correlated with the level of DNA methylation by using COHCAP online software package.(5)From five cases of resection of tumors obtained normal colorectal tissue colon carcinoma tissues and para carcinoma specimens,respectively PCR and RT-PCR quantitative experiment for the analysis of gene expression using 2-??CT method using human 18SrRNA and U6 as the analysis of mRNA and miRNA expression in the control(reference).From.gene expression library(GEO)to obtain a colorectal cancer miRNA expression data set(GSE83924)used to verify the expression of miRNAs,to evaluate the diagnostic efficacy of the differential expression of miRNA as a biomarker for colorectal cancer.A R-bioconductor software package was used to deal with the differential expression of miRNA between CRC patients and healthy controls,and the receiver operating characteristic curve(ROC)was obtained.Results:(1)Through the comparative study,we found that the expression of 55 differentially expressed miRNA and 1291 different mRNA in colorectal adenocarcinoma and miRNA showed an overall upward trend,the software algorithm can not only predict the 58 of the miRNA-mRNA gene,but also found the negative correlation among has-mir-141,has-mir-19a,closely related to the expression of has-mir-20a and the increase of has-mir-335 and the carcinogenesis of colorectal cancer.(2)Functional enrichment analysis showed that the expression of miRNA gene in pancreatic juice,saliva,gastric juice and bile secretion increased significantly.This study identified a robust identifica-tion marker comprising 58 tiny RNA genes and identified 11 genes negatively associated with DNA methylation levels.Among them,the MSX1 and KRT7 genes in colorectal cancer tissues were down regulated and showed obvious DNA methylation.(3)The results of this study showed that up regulation of miR-141 expression and downregulation of RSP02 in colorectal cancer can modulate most of the targets,suggesting that miR-141 is a potential biomarker for the early diagnosis of colorectal adenocarcinoma.(4)the expression of miR-20a in colorectal cancer is up regulated significantly.The ROC curve of miR-20a suggests its value in the diagnosis of colorectal cancer.It can be used as a potential biological marker.(5)the expression of mir-590 in colorectal cancer is up regulated,and mir-590 may be a key regulator of the carcino-genesis of colorectal cancer.(6)the expression level of miR-335 in colorectal cancer is up-regulated,which may play an important role in the development and progression of colorectal cancer.Conclusion:(1)This study identified eight differentially expressed miRNA in colorectal cancer and normal colorectal tissues,all of which are up-regulated miRNAs,which can be used as biomarkers for the early diagnosis of colorectal cancer.(2)The study of colorectal cancer tissues compared to adjacent to the 11 adjacent normal colorectal tissue expression and methylation of genes were identified,high methylation can inhibit the expression of these 11 genes,the methylation detection of MSX1 and KRT7 for the diagnosis of colorectal cancer is more significant.(3)This study shows that up regulation of miR-141 expression and downregulation of RSP02 in colorectal cancer can modulate most of the targets,suggesting that miR-141 is a potential early biomarker for the diagnosis of colorectal adenocarcinoma.(4)The results of this study showed that miR-20a expression was significantly up-regulated in colorectal cancer,The ROC curve of miR-20a suggests its value in the diagnosis of colorectal cancer.It can be used as a potential biological marker.(5)The results of this study show that the expression of mir-590 in colorectal cancer is significantly up-regulated,and is a key regulator in the carcinogenesis and development of colorectal cancer.(6)The results of this study show that the expression level of miR-335 in colorectal cancer is up-regulated and plays an important role in the development and progression of CRC.Part II:MicroRNA-335 targeting Twistl inhibits the growth and epithelial mesenchymal transition of colorectal cancer HCT116 cells.Objective:(1)To investigate the specific role of miR-335 in the develop-ment and progression of colorectal cancer.(2)Through the study of Twistl involved in miR-335 mediated tumor cell migration,invasion and epithelial mesenchymal transition(EMT)research,to explore and reveal the miR-335 inhibit the development of colorectal cancer,the molecular mechanism of invasion and transfer process.Methods:(1)Using human colorectal cancer HCT116 cells,the expression of miR-335 in colorectal cancer cells was altered by transfection of miR-335 analogues and inhibitors.(2)The application of CCK-8 method,flow cytometry,Transwell assay and Western blot assay were used to detect tumor cell survival rate,cell apoptosis,cell migration,invasion and epithelial mesenchymal transition(EMT)related proteins,further evaluation of miR-335 function in colorectal cancer cells.(3)The expression of Twistl protein was detected by double luciferase assay.(4)The growth and metastasis of cells were assessed by cell co transfection of miR-335 inhibitors and silencing of Twist1 protein by siRNA.Results:(1)The results of this study show that the expression level of miR-335 in colorectal cancer is up-regulated,and may play an important role in the development and progression of CRC.(2)The results of this study showed that overexpression of miR-335 inhibited cell survival,migration and invasion,and promoted the rate of apoptosis.Overexpression of miR-335 can up regulate E-calcium binding proteins and down regulate N-calcium binding proteins,vimentin and snail proteins.(3)The results of this study show that Twistl is a direct target of miR-335,Twistl gene silencing can promote tumor cell apoptosis,inhibit tumor cell viability,reduce the abnormal expression of EMT related protein;inhibition of miR-335 can inhibit the apoptosis of tumor cells,increase cell viability,increase the abnormal expression of EMT related proteins.(4)The results of this study show that silent Twist1 can terminate the activation of miR-335 induced by the inhibition of p65 and I?B?,and terminate the inhibition of miR-335 induced by Wnt3a,Wnt5a,and?-Catenin upregulation.Conclusion:(1)The results of this study show that miR-335 can inhibit the growth,migration,invasion and EMT of HCT116 cells,and play an inhibitory role in the development and progression of colorectal cancer.(2)Twistl is a direct target of miR-335,and miR-335 can inhibit the invasion and metastasis of colorectal cancer cells by inhibiting Twistl.(3)the NF-?B signaling pathway and the Wnt/?-Catenin signaling pathway may be involved in the whole process of miR-335 targeting Twistl regulation of CRC growth,migration and invasion.(4)Silencing of Twistl could terminate the activation of p65 and I?B? induced miR-335 inhibition,and terminate the inhibition of miR-335 induced by upregulation of Wnt3a,Wnt5a,and ?-Catenin.In summary:microRNA is potentially valuable in the early diagnosis of colorectal adenocarcinoma,and a specific miRNA signature may be an emerging tumor marker of digestive tract cancer.Based on the analysis of the degree of miR-335 and Twistl expression in colorectal cancer tissue samples,combined with the evidence offered in this study,we believe that miR-335 play a role in tumor suppression in colorectal cancer development process,the process of miR-335 can inhibit the growth of colon cancer cells HCT116,migration,invasion and ETM.Twistl is a direct target of miR-335,and miR-335 may inhibit the invasion and metastasis of colorectal cancer cells by inhibiting Twistl.The NF-?B signaling pathway and the Wnt/?-Catenin signaling pathway may be involved in the whole process of miR-335 targeting Twistl regulation in colorectal carcinogenesis and progression.MiR-335 could be a novel target for metastatic CRC,antisense technology and application of miR-335 to the growth and invasion,effectively inhibit CRC,has a certain application prospect,may become a new means of adjuvant therapy of CRC,further research on its mechanism will be helpful to understand the nature of colorectal cancer invasion and metastasis. |
PDF Full Text Request