Identification And Function Study Of Human Embryo Development-related MicroRNAs

Precisely and accurately regulation of gene expression is of vital significance for human embryo development. In a certain sense, tumorigenesis is the result of abnormally expression of embryonic genes. Over the last decade, microRNAs (miRNAs), approximately19~25-nucleotide non-coding RNA molecules, have emerged as fundamental actors in regulation of gene expression at the posttranscriptional level and suggest a possible involvement in a variety of pivotal physiological or pathological processes, including embryonic development, tumorigenesis and hematopoiesis. Recently, miRNA expression pattern of during the embryonic development of some model animals (e.g., zebrafish, mouse, etc) have been exhaustively studied. After obtaining the whole genome gene expression profiling of human early embryogenesis executed by our laboratory, we further explored the expression pattern of miRNAs during this process. We identified some miRNAs associated with human embryo development or tumorigenesis. Of them, hsa-miR-638, was further tested for its potential function in proliferation and differentiation of myeloid leukemia cells. These findings provided scientific evidence for the function study of miRNAs. Similar to gene expression profiling, we investigated the expression profile of all known human miRNAs (miRBase10.1) in embryogenesis. We analyzed the miRNA microarray data of human embryo samples at gestation from4to6weeks and50miRNAs were identified as human embryogenesis-related miRNAs. Moreover, we revealed the commonness and specialty in human and mouse embryos by comparing the miRNA expression pattern at the same developmental stage. This study paved a way towards our comprehensive understanding of the regulatory functions of miRNAs in human embryogenesis. In addition, we speculated that miRNA might also be involved in tumorigenesis due to the similarity embryogenesis and tumorigenesis in many ways. Thus, we selected three non-conserved or primate-specific miRNAs, hsa-miR-638, hsa-miR-720, and hsa-miR-1280, and qualitatively examined their expression levels in various normal and human tumor tissues by in situ hybridization. Our studies demonstrated that these miRNAs was deregulated in various solid tumors including gastric cancer, skin cancer, and cervical cancer. These results exhibited new evidence to reveal the correlation of early human embryo development and tumorigenesis.Hematopoiesis is part of development and abnormal hematopoiesis leads to leukemia. Numerous studies indicated that miRNAs was involved in normal and abnormal hematopoiesis. Herein, we observed a significant upregulation of miR-638expression in normal myeloid lineages but significant down regulation in myeloid leukemic cells. These observations suggest miR-638might play a role in myeloid differentiation and its dysregualtion may contribute to leukemogenesis. Therefore, we investigated the role of miR-638in proliferation and differentiation of myeloid leukemic cells. We observed that miR-638expression was dramatically upregulated upon differentiation in primary leukemic cells and established leukemic cell lines induced by chemotherapy drugs (phorbol12-myristate13-acetate and all-trans retinoic acid). These results further suggest that miR-638may participate in the terminal differentiation of myeloid cells. Indeed, ectopic expression of miR-638promoted differentiation in leukemic cell lines and primary acute myeloid leukemia (AML) blasts, while antagonizing miR-638caused the opposite phenotype. Consistently, overexpression of miR-638led to cell cycle arrest at Gl phase and reduced the capacity of proliferation and colony-forming ability in soft agar. These findings demonstrate miR-638promotes forced myeloid terminal differentiation in myeloid cells and its downregulation contributes to leukemogenesis.miRNAs regulate multiple physiological or pathological processes mainly by fine-tuning their target genes. We predicted and identified that cyclin-dependent kinase2(CDK2) was one of target genes of miR-638by site-directed mutagenesis and dual-luciferase reporter assay. Moreover, the CDK2protein and miR-638expression was negatively correlated in primary AML blasts. Function study revealed that CDK2inhibition by RNA interference phenotypically mimicked the phenotype of miR-638overexpression to promote myeloid terminal differentiation. Forced expression of CDK2rescued the colony-forming ability suppressed by miR-638. These findings indicate that aberrant downregulation of miR-638that lead to CDK2upregulation may facilitate leukemogenesis.In summary, we investigated the miRNA expression profile in human early embryogenesis and discovered that many miRNAs may be closely related to malignant transformation or tumorigenesis including AML. Among them, miR-638was found to be an important regulator in myeloid terminal myeloid differentiation by targeting CDK2and its downregulation contributed to the development of leukemia. Our studies not only help us reveal the molecular mechanism underlying the human embryogenesis but also provide potential biological marker or target for diagnosis, prognosis, and differentiation therapy of leukemia.