Screening And Validation Of Leukemia-associated MicroRNAs And Phylogenetic Analysis Of Cyclin

The thesis consists of two parts. The first part focuses on the microRNAs (miRNAs) screening and their regulatory mechanism in leukemia. The second part is the phylogenetic analysis of cyclins in eukaryotes.The first part of this study focuses on the screening and validation of leukemia-associated microRNAs. MiRNAs are a family of endogenous small noncoding RNAs with approximately 22 nucleotides in length. They inhibit mRNA translation or mediating mRNA decay on post-transcriptional level. MiRNAs are involved in various developmental and physiological processes in animals and plants, and play vital roles in many diseases. Cancer studies have found that various miRNAs play different roles as oncogenes or tumor suppressor genes. Leukemia is a clonal disorder of the bone marrow, characterized by the abnormal proliferation of immature blood cells. With new chemotherapy treatments applied, patients’ outcomes have been significantly improved. However, the relapse of patients is hindering the treatment of leukemia. This study focuses on the regulatory mechanism of leukemia-miRNA in prior/post-treatment, it will provide a guideline for diagnosis and treatment.We performed the following studies to screen and analyze the regulatory mechanism of miRNAs in leukemia-associated pathways in B-cell acute lymphoblastic leukemia (B-ALL). Firstly, the high-throughput sequencing data of miRNA in acute lymphoblastic leukemia (ALL) were downloaded from the NCBI SRA database. The expression data of 88 miRNA were obtained from 7 samples by filtering process. Secondly, we collected B-ALL patient samples. Total RNAs were extracted from the blood of two periods in prior/post-treatment for small RNA-seq. We screened 62 differentially expressed miRNAs in two B-ALL patients in prior/post-treatment. Combining the two data of miRNAs, we obtained five highly expressed miRNAs both in ALL and in B-ALL in prior/post-treatment. The prominent miRNAs are miR-142-5p and miR-21-5p. Thirdly, the data of miRNA targets and signaling pathways were collected to explore the relationships of miRNA regulating the signaling pathways. We found that miR-142-5p potentially regulates a large number of genes (11/19) in the TGF-P pathway. Finally, we validated the regulatory mechanism of miRNAs in TGF-P pathway by experiments. The results showed that miR-142-5p regulates the TGF-β signaling pathway by inhibiting SMAD2 and SMAD3. Furthermore, it was reported that miR-21-5p inhibits TGFBRII and SMAD7. Based on these, we constructed a molecular model of miRNAs regulating TGF-β pathway and discussed their cross-talkings. TGF-β pathway was regulated by miR-142/21 inhibiting its different components. This suggests that miRNAs play a multidirectional role in the treatment of leukemia. Taken together, this study enhanced the understanding of regulatory mechanism of miRNAs in leukemia. It is a guideline for diagnosis and treatment of leukemia and shed light on combining bioinformatics and experiments in the research of leukemia.The second part of this study is a phylogenetic analysis of cyclins in eukaryotes. Cyclins are a family of diverse proteins that play fundamental roles in regulating cell cycle progression in eukaryotes. Cyclins have been identified from protists to higher eukaryotes with the development of high-throughput technologies, while its evolution remains vague. Current classification of cyclins is mainly based on their functions, which may not be appropriate for the systematic evolutionary analysis. It is necessary to carry out a comprehensive phylogenetic analysis of diverse cyclins in eukaryotes. Firstly, the Cyclin_N domain Pfam HMMER model was used to identify the cyclin-like proteins which serve as the candidate cyclin proteins. By filtering these candidate proteins,294 cyclins were collected in some representative species. Then, we performed comparative and phylogenetic analysis of cyclins to investigate the classification, origin and evolution. Cyclins may originate in early eukaryotes and evolved from protists to plants, fungi and animals. Based on the phylogenetic tree, cyclins can be divided into three major groups designated as the groups Ⅰ, Ⅱ and Ⅲ with different functions and features. Group Ⅰ plays key roles in cell cycle, group Ⅱ are kingdom (plant, fungi and animal) specific, and group Ⅲ functions in transcription regulation. Our results showed that the dominating cyclins (group Ⅰ) were diverged during the evolution of plants, fungi and animals, while the divergence of the other cyclins (groups Ⅱ and Ⅲ) has occurred in protists. This reclassification and evolutionary analysis of cyclins might facilitate understanding eukaryotic cell cycle control.