The Expression Of MiR-92a In Colon Carcinoma And Its Influences On HT-29 Cells’ Chemosensitivity

Micro RNAs are a class of endogenously expressed non-coding small RNAs of approximately 17~25 nucleotides. These micro RNAs can decrease translational efficiency of target genes’ m RNA via pairing with m RNAs of 3’ protein-encoding areas. They contributes to individual development, cell apoptosis, cell proliferation, degration and any other life activities. They are closely related with tumors’ orign, metastasis, chemoresistance or other pathology process. It had been confirmed that the expression of mi RNA in tissue or cells were significantly correlated with tumorigenesis, had tissue specificity and stability. In colon carcinoma cells specific suppression or upregulation of mi RNA is correlated with colon carcinoma development, evolution, translation or chemoresistance. As an important member of mi R-17~92 clusters mi R-92 a express abnormally in a variety of tumors and is correlated with proliferation or apoptosis of cancer cells. We use fluorescent quantitative PCR method to detect expression of mi R-92 a in colon cancer tissue and evaluated its clinical significance. Then we selected the cell lines HT-29 which mi R-92 a expression was upregulated. This cell lines were found to have chemoresistance. We use liposome and antisense oligonucleotides transfecte HT-29 to inhibite the expression of mi R-92 a. Then we examed the change of its chemoresistance.Objective: To test expression of mi R-92 a in colon carcinoma tissues and evaluate its clinical significante. Research the influence that inhibite mi R-92 a to HT-29 s’ chemoresistance.Methods: 1. Use q RT-PCR to detect the relative expression levels of mi R-92 a in 75 cases of colon carcinoma. Follow-up the 75 patients with colon carcinoma and regard disease-free survival(DFS) and overall survival(OS) as evaluation index. Make Kaplan Meier survival curve analysis the relationship between mi R-92 a expression and DFS, OS in patients postoperatively with colon carcinoma. Detect the survival by the Log-rank test.2. Subculture HT-29, SW-480, HCT-8 colon cancer cells, extract total RNA in logarithmic phase cells, to detect the level of mi R-92 a expression by fluorescentquantitative PCR method. Screening the cell line which express the highest level of mi R-92 a, then transfect mi R-92 a inhibitor. Detect the mi R-92 a again. If the level of mi R-92 a is downregulated meas the transfection is effective. Choose this cell line to do the subsequent research.3. Transfecting mi R-92 a inhibitor into HT-29 cell line mediated by riboFECT. Liposomes group only transfected liposomes,negative control group transfected negative control oligonucleotides while blank group didn’t add anything. After transfection every group add oxalipolatin effect for a period of time. Then measured the proliferation of colon cell by CCK-8 assay; the apoptosis rate were assayed by TUNEL assay.Results: 1. q RT-PCR detection found that the relative expression level of mi R-92 a in colon carcinoma tissues from tissue adjacent to carcinoma increased obviously, its relative expression level is 3.57±1.41. The mi R-92 a expression was no significant with anatomic sites, invasion depth, lymph node metastasis, vascular tumor emboliand tissue differentiation(P=0.508, 0.163, 0.808, 0.334, 0.556, 0.808). Low level expression of mi R-92 a is better than that of high expression in DFS and OS( all P<0.0001). Low level expression of mi R-92 a median DFS is 17 month while the high level expression of mi R-92 a is 31 month.The median OS of these two group is 20, 34 month.2. Use qRT-PCR method detected four colon carcinoma cell lines. The expression level of HT-29 is the highest upregulated 4.15±0.13. After transfection mi R-92 a inhibitor the mi R-92 a expression downregulated to 0.05±0.10.3. The proliferation ability were obviously inhibited than the lipsomes group, blank group or negative control group when the concentration of mi R-92 a inhibitor was used by CCK-8 test in HT-29 cell line, P<0.05; Through TUNEL experiments tumor cell apoptosis rate at 72 h by mi R-92 a inhibitor was higher compared with other three groups, P<0.05.Conclusion: mi R-92 a expression in colon carcinoma tissue is higher than tissue adjacent to carcinoma significantly. The level of mi R-92 a expression was interrelatedwith disease-free survival and overall survival. The expression level in different colon carcinoma cell lines were different and it may influence the cell chemoresistance. Inhibit its expression can supress the cell proliferation after chemotherapy, inhance the cell apoptosis rate. mi R-92 a may be the new target of patients who had chemoresistance.