MiR-429Inhibit TGF-β1Induced Epithelial-Mesenchymal Transition By Onecut2in Colorectal Cancer

[Background]Colorectal cancer is a worldwide common gastrointestinal cancer. It is an important pathological features about multi-stage and multi-genes characteritics in the incidence and development of colorectal cancer. With the improvement of genomics and transcriptomics technology, the dynamic expression pattern of coding RNA and non-coding RNA may be studied from the overall level in the different clinical stages of colorectal cancer. In our previous researches, we has constructed colorectal cancer dynamic gene and miRNA expression profiles in different clinical stages by using the latest genome-wide chip and micro-RNA chip, and then constructed the microRNA-GO network, and found that miR-429was located in the core of the microRNA-GO network,which was a vital microRNA in the developing process of colorectal cancer. Therefore, on the basis of the preliminary work, we choose the miR-429to confirm its dynamic expression pattern in the clinical stage developmental process of colorectal cancer, and its molecular biological function and mechanism of action in the colorectal cancer.[Method]Firstly, real-time PCR was used to detect the expression of miR-429in colorectal cancer cells and tissues (colorectal carcinoma and the paired remote normal tissue specimens of colorectal cancer were18cases,respectively). Secondly, the dynamic expression of miR-429also has been confirmed in the different clinical stages of CRC by Real-time PCR with85samples (TNM stage Ⅰ:11cases, stage Ⅱ,18cases, stage Ⅲ,20cases, stage iV,12cases and normal colonic mucosa,24cases). In situ hybridization were used to further detect the dynamic expression model in the CRC tissue array with103cases colorectal carcinoma. Kaplan-Meier survival analysis was used to analyze the effect of miR-429expression on the prognosis of CRC patients. Finally, the biological phenotype-related experiments were to study the effect of miR-429on EMT(epithelial-mesenchymal transition), and node subcutaneous transplanted model has been established. On the this basis, we further study the effect and mechanism of miR-429on TGF-betal-induced EMT by targeting Onecut2in colorectal carcinoam by morphology and gene expression analysis.[Results]1.miR-429has shown the dynamic expression pattens during the initiation and development process of colorectal cancer, and it expression is closely related with the prognosis of CRC patients.By the methods of Real-time PCR and situ hybridization, we detected the expression of miR-429in different clinical progressive stages of colorectal cancer and found that the low expression of miR-429has been lowed obviously in most of the colorectal cancers.The dynamic expression of miR-429showed in different clinical progressive stages of colorectal cancer is same as the dynamic differential expression patterns of colorectal cancer which was acquired by miRNA array during our preliminary project.By means of hybridization in situ analyzing the expression of miR-429in the103colorectal cancer cases and the correlations with clinical and pathological features of the patients,we found the lower expression of miR-429in colorectal cancer is close related with the age of the patients and the degree of tumor differentiation.The lower expression is close related with the development of stage Ⅱ and stage Ⅲ of colorectal cancer’s TNM staging, while is unrelated with the stage Ⅰ and stage Ⅳ. In addition,the expression of miR-429closely related with survival,and the survival rate of the patients who are with lower expression of miR-429is lower than the patients with higher expression and so is the poor prognosis, according to Kaplan-Meier survival analysis.2.miR-429has potential to suppress the epithelial-mesenchymal transition of colorectal cancer cells by targeting Onecut2.Firstly, we detected the colorectal cancer cell proliferation inhibition ability by expression of miR-429in vitro transfected, according to the results of MTT,the most obvious inhibitory effect is that when miR-429is in2.5pmol/L concentration of48-72hours.Meanwhile,we also have done a test of colorectal cancer cells transplanted into nude mice,applying multi-point injecting miR-429mimics within the tumor,observing the growth inhibitory effect of the colorectal cancer cells transplanted into nude mice by miR-429,which turned out that miR-429could obviously inhibit the growth of colorectal cancer subcutaneous tumor which was transplanted into nude mice.The result shows that miR-429has taken part in the development of colorectal cancer,and it would be a very good small molecule target of action in curing colorectal cancer.Secondly in order to detect the possible molecular mechanism of miR-429working as small molecule therapeutic target, and with overall consideration of the works our project had done before,we selected and verified that Onecut2is the target gene of miR-429which could lateral control the expression of Onecut2in or after transcription.Our research also proved that there is expression negative correlation between miR-429and Onecut2in colorectal cancer tissue.Finally we had done a further research on the impact of miR-429to EMT Related biological behaviour.The results we have studied shows that miR-429has inhibit the Migration and invasion of colorectal cancer cell SW620.Meanwhile,the Onecut2with interfere endogenous could also inhibit the migration of cell SW620.Over-expression Onecut2can recover the invasion inhibition of cell SW620mediated by miR-429.These indicate that miR-429could inhibit migration and invasion of colorectal cancer cell SW620by the expression of targeting Onecut2.miR-429can inhibit the epithelial-mesenchymal transition of TGF-betal-induced.3.miR-429can reverse the expression changes of transcription factors and genes which is related to EMT induced by TGF-betal in colorectal cancer cells.In addition to the obvious change of cell morphology characteristics, there were also a series of characteristic changes of the biomolecule during the EMT formation. Our study suggested that the EMT-related transcription factors and gene expression changes of colorectal cancer SW620cells induced by TGF-β1could be reversed by the over-expression of miR-429or the expression of interfering endogenous Onecut2. Under the premise of TGF-β1inducting, the over-expression of miR-429or the expression of interfering endogenous Onecut2increased the expression of the catonated molecules (E-cadherin,CTNNA1andCTNNB1) and fibronectin (FN),and decreased the expression of heterologous adhesion molecules CD44and matrix metalloproteinase expression of MMP2. Our study also showed that TGF-β1induced the expression of Onecut2,ZEB1,ZEB2,Snail andSLUG of colorectal cancer SW620cells,miR-429inhibited the expression of Onecut2, ZEB1,ZEB2,SNAIL and SLUG,and interfering with endogenous Onecut2had the same effect as the over-expression of miR-429.In addition,we also confirmed the above EMT-related expression changes of transcription factors and genes in the experiment of transplanting colorectal cancer cells into nude mice by immunohistochemistry.The above results showed The EMT-related transcription factors and gene expression of colorectal cancer cells induced by TGF-β1could be reversed by targeting Onecut2of miR-429. At the same time, Onecut2also could also be used as EMT-related transcription factor,directly involved in the colorectal cancer cell EMT activation. [Conclusion]1.It was affirmed the miR-429showing dynamic expression pattens in the development of colorectal cancer, and is closely related with the prognosis of patients.2.It was affirmed the miR-429can inhibit the epithelial-mesenchymal transition of TGF-betal-induced by targeting Onecut2.3.It was affirmed miR-429can reverse the expression changes of transcription factors and genes expression which is related to EMT induced by TGF-betal in the colorectal cancer cells.22Figures,6tables and70references.