Systematic Validation Of Predicted MicroRNAs Targeting Cyclin D1 And Profiling Of MicroRNA Expression Patterns In Human Breast Cancer Cells Treated With American Ginseng Extract

This study includes two parts which were separately conducted in China and USA. As a primary work for master degree, the first part focused on the optimization of a novel blastospore transformation system of Beauveria bassiana for higher efficiency of genetic manipulation of fungal biocontrol agents. The second part was a collaborative work with Dr. Yinyuan Mo (Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, USA) under a joint PhD plan of China Scholarship Council. The latter study sought to systematically validate predicted microRNAs (miRNAs) for the regulation of cellular cyclin D1 gene (CCND1) and to analyze miRNA expression pattern changes in cancer cells after treatment with American ginseng. The results are summarized as follows.Validation of predicted miRNAs to target CCND1 in dual-luciferease reporter system. MiRanda (http://www.microRNA.org) was chosen as a primary prediction tool to predict CCND1-targeting miRNAs. Of 58 predicted miRNA precursors,51 were cloned into pCDH-CMV-MCS-EF1-copGFP. Based on real-time PCR analysis,45 of the cloned miRNA precursors expressed relatively high levels of exogenous miRNAs. These 45 miRNA precursors were then tested for their effects on the luciferase reporter carrying the 3'-untranslated region (3'-UTR) of CCND1. By an arbitrary cut off of 35% reduction, we identified 7 miRNAs that were able to suppress Luc-CCND1-UTR activity. Thus, the positive ratio for the tested miRNAs is about 16%. Four of them (miR-16, miR-34a, miR-195 and miR-424a) were previously validated targets while miR-19a, miR-155 and miR-503 were validated to be positive in this study. Comparison of the CCND1-targeting miRNAs predicted from miRanda, TargetScan and PicTar indicates that miRNAs in all the three lists gave rise to the highest possibility of positivity. However, there was still a possibility that functional miRNA might be found in only miRanda list, highlighting the necessity of experimental validation. The ratio of true/predicted targets was relatively low in our system, further suggesting the complexity of the miRNA/mRNA interactions.Systematic validation of CCND1-targeting miRNAs. Three new miRNAs for CCND1 (miR-19a, miR-155 and miR-503) validated in the dual-luciferease reporter system were subjected to Western blot and real-time RT-PCR analysis for the suppression of CCND1 protein and mRNA. Of those, miR-503 significantly reduced the expression of endogenous CCND1 protein and mRNA in human head and neck carcinoma cell line UMSCC10B. To confirm the specificity of the suppressed CCND1 expression to miR-503, immortalized kidney epithelial cells were transfected with anti-miR-503 along with the luciferease reporter. In contrast to miR-503, anti-miR-503 caused an increase in the luciferase activity compared to the scrambled oligo. Two putative miR-503 binding sites were then disrupted to validate their suppressive activity. As a result, the suppression activity of miR-503 was substantially impaired by the disruption, indicating that the two binding sites are important for miR-503-mediated suppression. Furthermore, miR-503 was capable of reducing S phase cell populations and causing cell growth inhibition in the UMSCC10B cells. This implies that miR-503 could be a putative tumor suppressor.MiRNA expression profiling of breast cancer cells treated with American ginseng extract. The cells of MDA-MB-231 breast cancer cell line were morphologically changed over time after respective treatments with 0.5,0.75 and 1.0 mg/mL American ginseng extract. The treated cells were shrunk in shape and their cytoplasm became coarse. Total RNA was extracted from the cells after 24-h treatment with the extract of 0.5 mg/mL and examined by RT-PCR and real time PCR. As a result of examining 95 cancer related miRNAs in the treated cells, miR-145 and miR-205 were upreglated by 3.2 fold and 4.5 fold, respectively, while miR-196a was downreglated by 82%.Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation. The techniques of restriction enzyme-mediated integration (REMI) and electroporation (EP) were applied for the first time to improving the blastospore transformation of B. bassiana for higher frequency. The blastospores from≤24 h incubation in glucose-mineral medium after shaking conidia for 48 h in Subouraud dextrose broth were found most competent for integrating 1μg plasmid DNA vectoring the phosphinothricin (PPT) resistance gene bar mediated by 100 U HindⅢor XbaI. Such blastospores were also most suitable for EP transformation at the optimized field strength of 10 kV/cm. The optimized REMI and EP generated 39 (±4.4) and 53 (±4.8) transformants/μg DNA whereas polyethylene glycol (PEG) integration yielded only 22 (±1.7). Compared to the PEG integration, REMI and EP enhanced the frequency of the blastospore transformation by 73 and 137%, respectively. All transformants grew well on Czapek's agar containing 400μg PPT/mL after three rounds of cultivation on the same agar excluding PPT but their parental strain showed no resistance. The target gene inserted into the genomes of 10 transformants randomly taken from REMI or EP transformation was consistently detected by both PCR and Southern blotting.In summary, the present study substantially improved the efficiency of B. bassiana blastospore transformation by REMI and EP. Systematic validation of predicted miRNAs for CCND1 indicates that miR-503 specifically targets the carcinogenic gene CCND1 and functions as a tumor suppressor and highlights a necessity of experimental validation for the miRNAs predicted by online software analysis. The expression patterns of miR-145, miR-205 and miR-196a in the MDA-MB-231 cells treated with the ginseng extract were well differentiated by the profiling of real time PCR. The results provide new insights into the genetic manipulation of fungal biocontrol agents and the validation of intracellular miRNA/mRNA interaction at molecular level.