Screening And Function Studies Of MicroRNA Targeting CREPT In Colorectal Cancer

Micro RNAs(mi RNA) are a group of endogenous small non-coding RNAs with20-22 nucleotides, they can regulate target genes at post-transcription level through causing target gene m RNA degradation or translation inhibition of translation resulting in target gene. mi RNAs may have close relationship with the growth, proliferation,metastasis and apoptosis in tumor tissues.They may have causal roles in numerous normal cellular and tumor processes, such as development, differentiation, proliferation and apoptosis. mi RNAs target in tumor related genes, and further can inhibit the growth of tumor the growth of tumor cells, cells, and promote its ageing their senescence and inhibiting the tumor cell proliferation of tumor cells and so on. CREPT is an oncogene that newly was found a cancer gene in malignant tumor recently, and is expected to It has been found up-regulated in many tumor tissues, and was becoming a new potential therapeutic targets. Now, however, these previous studies only focused on the function of CREPT in the tumorigenesis and the crystal structure of CREPT, and protein in the process of tumor occurrence and development of the downstream gene regulation mechanism research and the structure of the protein crystal revealed that focus on the downstream CREPT protein biological function, and the molecular mechanism about the reason CREPT high expressed in tumor tissue was not elucidated yet.Gene expression level itself in tumor tissues increased molecular mechanism is unclear.In our experiment, CREPT m RNA was regarded as a therapeutic target, and screened the mi RNAs targeting CREPT m RNA through prediction software and related articles retrieval. After a series of verifying experiment, we screened out that mi R-194 was the most potential mi RNA targeting CREPT m RNA. Furthermore,we determined the precise binding sites of mi Rn and CREPT m RNA 3’-UTR by luciferase assay. After that, we studied the effect of mi R-194 on the CREPT expression in colorectal cell lines by western blotting. By cell counting, MTT assay, colony formation assay, we systematically studied the effects of mi R-194 on the growth, proliferation, colony formation of HCT 116β/W cell line. By flow cytometry, the effect of mi R-194 on cell cycle was further explored. Theseresults showed that after targeting CREPT, mi R-194 suppressed HCT 116β/w growth and proliferation, regulated cell cycle of cells, and promote cell cycle arrest in S phase. At last,we determined the effect of mi R-194 on the protein expression of cell cycle-related proteins and cell apoptosis related. Our results showed that after targeting CREPT,mi R-194 inhibit the Wnt signaling pathway to a certain extent. Meanwhile,after targeting CREPT, mi R-194 down-regulated the expression of tumor-cycle-related proteins CDK2,CDK4, Cyclin D1, Cyclin E and C-myc protein,which were targeted genes of CREPT.And up-regulated the apoptosis related protein NF-κB p65, speculated that mi R-194 can regulate the NF-κB signaling pathway.In this thesis, we screened out the mi RNA targeting CREPT m RNA by software prediction and verifying experiment, namely mi R-194.On the basis of it, we further explored the changes of its downstream genes and the effect of cell cycle after mi R-194 targeting CREPT. Our study will provide a theoretical basis to the mechanism for high expression of CREPT in tumor tissues.