Expression And Functional Study Of EMT Related MicroRNA In Colorectal Cancer

[Background]Colorectal cancer (CRC) is one of the most common malignant tumor, which90%of cancer patients die of tumor metastasis in clinical. Its process is difficult to be effectively reversed by conventional surgery, chemotherapy or radiation therapy, effective early detection and target point treatment of tumor invasion and metastasis, in order to find a new tumor marker for early diagnosis and individualized treatment is the key to reduce the malignant behavior of cancer,it could be also Contribute to prevention and treatment of colorectal cancer. Epithelial-mesenchymal transition (EMT) refers to the phenomenon of transformation of epithelial cells to mesenchymal cells in a specific physiological and pathological circumstances. The study found that of EMT phenomenon is closely related to tumor invasion and metastasis, EMT plays an important role in many types of cancer in situ infiltration and distant metastasis. miRNA (miRNA) are endogenous non-coding small RNAs (usually length in18-25nt) general. miRNA inhibition of gene transcription after the translation, the main target gene mRNA3'untranslated region (3'untranslational region,3'UTR) combined biological effect of inhibiting protein translation. Recent discovery of miRNA on EMT has a strong regulatory role. With a In-depth study in recent years, the relationship between the miRNA and the tumor EMT research is gradually increasing, However, the study of EMT-related miRNA in colorectal cancer is still the initial stage.[Research Methods and Properties]EGF and TGF-1were applied to induce EMT in human colon cancer cell line SW480. Our preliminary study had analyzed different time points miRNAs expression during the course of this EMT model. miRNA expression profile were analyzed and3miRNAs including has-miR-138, has-miR-139-5p, has-miR-1180were most differentially expressed. Real-time PCR validated the result. Our project are aimed to further confirm these miRNA's expression in cellular and clinical tissue biopsy and try to explore its cellular and molecular biological characteristics. Bioinformatics prediction and target validation were also applied to elucidate the regulation mechanism of the EMT process in CRC.[The Test Results]1) Expression of EMT-related miRNA in colorectal cancer cells and clinical tissue samples:(1) Real-time PCR at multiple time points to verify the detection of miR-138, miR-139-5p, miR-1180expression.(2) Real-time PCR detection of miR-138expression in24pairs of cancer tissue and normal control group, the results are shown that the expression of miR-138in2pairs were elevated, while22pairs were significantly reduced in CRC cancer tissues, Scatter plot prompted the CA group of miR-138expression was significantly lower expression than normal control group, the two group have significant difference, Control group is2.66times higher than the CA group; Real-time PCR detection of miR-139-5p expression in18cancer tissues and normal control group, the results show no significant difference in expression between the two groups.(3) In situ hybridization detection of MiR-138in colorectal cancer, adjacent noncancerous mucosa and lymph nodes express, results show that the expression of miR-138in colorectal cancer tissues was significantly lower than the adjacent normal tissue and cancer distal normal mucosa,the two group have significant difference; no significant difference between normal mucosa adjacent noncancerous and normal mucosa of cancer remote; Colorectal primary tumor expression rate was significantly lower than the positive expression in lymph node metastases, There were significant differences between the two groups.(4)Different clinical stages of the prognosis of CRC clinical and pathological data analysis:statistics in situ hybridization-tissue microarray results showed that the MiR-138-positive expression with age, degree of differentiation and lymph node metastases, and no correlation with other factors.2) Biological functions of EMT-related miRNA in colorectal cancer cells:Transwell invasion assay results show that miR-138can weaken the ability of migration and invasion in SW480cell,miR-139-5p can enhance the ability of migration and invasion in SW480cell. flow cytometry experimental results indicate that of miR-138overexpression could significantly promote cell apoptosis,and slow down cell cycle progression. But miR-139-5p is not obvious on the cell cycle and apoptosis, MTT and the scratch test showed that colon cancer cells up-regulated the expression of miR-138not only inhibits the proliferation of HT-29cells, but also can inhibit the migration ability of SW480cell movement.3) The identification of miR-138, miR-139-5p target gene:(1) Real-time PCR detection both of SW480cells transiently transfected with transfection of miR-138, miR-139-5p expression and inhibit the expression of the target gene expression vector and TGF-β treatment of SW480cells48h in order to occured EMT, were transiently transfected with miR-138, miR-139-5p expression vector and inhibit the expression vector, The results prompted that EZR is the miR-138target gene, CDH1and ZEB2are miR-139-5p target gene.(2) Luciferase experiments: Built-PMIR vector containing EZRIN and miR-138binding sites and its deletion mutant, Built-PMIR vector containing CDH1and ZEB2miR-139-5p binding sites and its deletion mutant, the results suggest that EZR is the miR-138target gene, CDH1and ZEB2are miR-139-5p target gene.(3) Western-blot further validation were transfected miR-138mimics, and miR-139-5p mimics, And its target gene EZRIN of CDH1protein expression down-regulated, These results suggest that:EZRIN is the target gene of miR-138, CDH1is the target genes of miR-139-5p.[Conclusion]1. miR-138downregulated in colorectal cancer tissues and TGF-β-induced EMT transformation of colon cancer cell line SW480.2. Cell biology function tests suggest that miR-138inhibit HT-29cell growth and proliferation, inhibition of SW480cells of the migration and invasion, significantly reduced apoptosis, slowing cell cycle progression, miR-138may play a role in inhibition of colorectal cancer in EMT by target genes EZRIN.3. MiR-139-5p were upregulated in colorectal cancer tissues and TGF-β-induced colon cancer cells SW480EMT, it can enhance the migration and invasion of SW480cells, may play a role in promoting CRC EMT by directly targeting on the CDH1gene.