Regulation And Function Of Micrornas Deregulated In Cancer Development

Cancer remains to be a huge threat to human life. Clinical managments such as operation, radiotherapy and chemotherapy had limited efficacy largely due to the unknown nature of human carcinogenesis. Studies on molecular mechanisms of tumor initiation and development will stimulate the development of new treatments with batter clinical efficiency, microRNA(miRNA), a micro-molecule with less than30nucleotides has been another hot spot in medical science field because of its various impacts on cell biological behavior. Although the close link between miRNA and tumor has been recognized, the intimate molecular mechanisms are not thoroughly explored. We expect to investigate the regulation of expression and metabolism between miRNA and cancer on the base of existing research results, and help to provide new targets for cancer therapy.Part one:The regulation of miRNA expression by Ras signaling pathwayObjective:To explore the regulation of miRNA expression by Ras signaling pathway.Methods:We firstly screened differentially expressed miRNAs between normal NIH3T3cells and RAS protein transformed NIH3T3cells (RAS/NIH3T3) by microarray. The results were further validated by quantitative real-time PCR. Six obviously changed miRNAs were selected to determine the expression level of different structures (primary, precursor, mature) by conventional PCR and real-time PCR. The results were further analyzed to explore the cause of deregulation. Recombinant of miRNA gene promoter containing pGL2vector was used to assess the promoter activity of miRNAs by luciferase activity assay.Results:①A large number of differentially expressed miRNAs were screened by microarray;②real-time PCR further verified the microarray results and six obviously changed miRNAs were selected for further study;③Analysis of the expression level of different structures indicated that six miRNAs were subjected to transcriptional regulation;④The luciferase activity assay of promoter region containing luciferase reporter vector showed active fragments of the promoter region.Conclusion:Different regulation mechanism of miRNAs in RAS/NIH3T3cell contributed to its distinctive expression pattern.Part two:microRNA and glutamine metabolism regulation in tumor cellsObjective:To explore roles and expression regulation mechanism of glutaminase (GLS) in colorectal cancer. To exploit intervention strategy against tumor cells abnormal metabolism by sruding its epigenetic basics.Methods:①We determined the expression level of GLS protein by western blot;②The effect of GLS knockdown by siRNA in colorectal cancer cell lines was estimated by MTS assay;③Apoptosis was measured by flow cytometry after GLS knockdown with or without OAA/NAC administration;④Cell energy variation after suppression of GLS protein was evaluated by ATP kit;⑤he expression level of miR-137in colorectal cancer tissue and cells was detected by real-time PCR;⑥The effect of GLS knockdown by miR-137mimic in colorectal cancer cell lines was estimated by MTS assay;⑦The relationship of miR-137and GLS was analyzed by application of a cell model in which endognous miR-137was induced by enhanced APC protein;⑧To directly demonstrate interaction of miR-137and GLS by wild type and mutant pMIR-GLS-3'-UTR luciferase reporter assay.Results:①Western Blot showed GLS was up-regulated in colorectal cancer cell lines;②Knockdown of GLS by siRNA apparently suppressed cell growth and proliferation;③Depletion of GLS facilitated cell apoptosis and could be partially rescued by OAA/NAC;④Diminished GLS was accompanied with unexpected elevated ATP;⑤miR-137was down-regulated in colorectal cancer tissue and cells;⑥miR-137mimic suppressed growth and proliferation of colorectal cancer cells;⑦Endognous miR-137inhibited GLS protein;⑧Luciferase activity assay showed the interaction of miR-137and GLS.Conclusion:①GLS protein was overexpressed in colorectal cancer cells, which was much possibly related to silence of miR-137by CpG island hypermethylation;②Inhibition of GLS suppressed cell proliferation and promoted apoptosis which could be partially rescued by exogenous OAA/NAC, but did not decrease ATP. This prompted glutamine metabolism supported colorectal cell survival and proliferation to a certain extent but may not be the exclusive nutrient.