MicroRNA-133b Inhibits CTGF And Regulating EMT In Colorectal Cancer

Backgroud:Colorectal cancer is one of the high incidence and mortality of digestive tract malignant tumor in the world. There is an upward trend incidence of colorectal cancer in China, which causes serious effect to people’s health and the quality of life. Tumor metastasis is the major cause of death in colorectal cancer, and epithelial-mesenchymal transition (EMT) of tumor cell is the key to tumor metastasis. EMT is refers to the phenomenon that the epithelial cells transformed to mesenchymal cells in specific physiological and pathological conditions. The study found that EMT phenomenon is closely related to the tumor invasion and metastasis, which plays an important role in a variety of in situ invasion and distant metastasis of cancer. MicroRNA is a kind of endogenous non-coding RNA, which is composed of about22nucleotides, complementary base pairing with target mRNA3’UTR for inhibition of mRNA translation or directly making its degradation. The expression of target genes is restrained in the transcription level, thus regulate gene expression. The current research of the biological functions of miRNAs are still unclear, but what is certain is that miRNAs play a very important role in the progression of the organism, growth, differentiation, apoptosis and tumor formation, especially in some malignant tumors, miRNAs target different genes which is similar to the original oncogenes or tumor suppressor genes. Recent research results confirmed that the abnormal expression of microRNA-133b(miR-133b) is an important part of the tumor progression, has promoting effect to the tumorigenesis, differentially expressed of miR-133b have been reported in colon cancer, but its mechanism of tumor metastasis is not fully clear in colorectal cancer.Methods:Previous studies in this study group have confirmed that miR-133b is a fresh discovered miRNAs with tumor suppression effect in colorectal cancer, but there are still many key problems to be further studied. On the basis of previous work, This study is proposed to carry out the research work as follows:1) Systems analysis and in-depth research, via target validation and cell function verification on target genes of miR-133b, is expected to clarify the role of miR-133b in colorectal cancer.2) Analyse miR-133b with various clinical pathological parameters in colorectal cancer via the detection of miR-133b and the target gene expression in colorectal cancer specimens.3) Analyse the possible reasons for colorectal cancer metastasis through the molecular regulation mechanism of EMT process in colorectal cancer cells.Results:1. Target validation between miR-133b and CTGF in colorectal cancerThe result of bioinformatics website prediction and gene chip suggested that the potential target genes of miR-133b were involved in TGF-(3, Pathway in cancer, colorectal cancer Pathway. Then we selected CTGF gene for further validation. Real-time PCR detected the expression of target genes in SW620, HT29cells which were transfection with over expression and inhibiting expression miR-133b vector. Preliminary results suggested that CTGF was the target genes of miR-133b. Dual luciferase report gene system:built its mutant pYr-miR vector including the CTGF and miR-133b, results suggested:CTGF was regulatory target genes of miR-133b. Western blot experiment confirmed that CTGF protein expression was changed after transfection miR-133b, show that CTGF was post transcriptional regulation by miR-133b.2. Correlation analysis between miR-133b and CTGF expression in colorectal cancer tissue samples with clinical pathological characteristicsReal-time PCR detected the expression of miR-133b in71human CRC tissues and its paired normal tissues. Results show that in71paired samples, miR-133b expression were decreased in66samples, only5cases increased of miR-133b expression in cancerous tissue, it has significant difference. The immunohistochemical results of CTGF in colorectal cancer showed that CTGF expression in colorectal cancer tissues were significantly higher than the carcinoma adjacent normal tissues, there are significant differences. The Correlation analysis of CRC clinical pathological features:on the basis of qRT-PCR and immunohistochemical results data, we determined the low and high expression groups by a median value of miR-133b and CTGF in71CRC specimens. The incidence of miR-133b showed low expression in specimens with poor cell differentiation, lymph node metastasis and advanced clinical stages. However, no significant relationship was found between the level of miR-133b expression and the age, sex, tumor site, extramural vascular invasion status. In addition, the incidence of CTGF showed high expression in specimens with advanced clinical stages (stage Ⅲ, Ⅳ vs. Ⅰ, Ⅱ) and lymph node metastasis. There is no statistically significant difference of CTGF expression in clinicopathologic data, such as age, sex, tumor site, tumor differentiation and extramural vascular invasion status.3. Molecular regulation mechanism analysis of miR-133b mediated the process of EMT in colorectal cancer cellsAfter transfection pre-miR-133b plasmid, cells change from spindle shape to round, immune fluorescence intensity of E-cadherin increased, immune fluorescence intensity of vimentin decreased. RT-PCR and Western blot confirmed on behalf of the epithelial cell phenotype of E-cadherin expression level raised, on behalf of stromal cell phenotype of vimentin expression level decreased. Transwell experiments confirm that invasion and migration ability decreased in colorectal cancer cell after transfection pre-miR-133b plasmid. After combination transfection pre-miR-133b plasmid and CTGF expression plasmid, immune fluorescence intensity of vimentin increased, immune fluorescence intensity of E-cadherin decreased. RT-PCR and Western blot confirmed on behalf of the epithelial cell phenotype of E-cadherin expression level decreased, on behalf of stromal cell phenotype of vimentin expression level increased. Transwell experiments confirm that invasion and migration ability increased in colorectal cancer cell after combination transfection pre-miR-133b plasmid and CTGF expression plasmid. Compared with normal colorectal tissues, E-cadherin expression level decreased, CTGF and vimentin expression level increased obviously in colorectal cancer tissue. CTGF expression level, negatively correlated with E-cadherin, and positively correlated with vimentin, were closely related patients with lymph node metastasis and clinical progression.Conclusions:1. miR-133b is a tumor suppressor gene in colorectal cancer, can inhibit the target gene CTGF mediate effect of suppressing tumor invasion and metastasis.2. CTGF played a key role that miR-133b regulated the process of EMT in colorectal cancer, miR-133b participate in the regulation of colorectal cancer EMT.