The Processing And Regulation Mechanism Of MiR-918and Its Function In The Erythroid Differentiation

Background: Hematopoietic process is the development and mature process of various types of bloodcells in the human body. The hematopoietic process involves three stages, the first is hematopoietic stemcells (hemopietic stem cells), they can through self replication (self renewal) to maintain the stability of thenumber itself, and can form departments directional differentiation of progenitor cells(committedprogenitors); The second stage is the directional progenitor cell stage, i.e., the red blood cellprecusors (CFU-E), grain of macrophage progenitor (CFU-GM), the giant nuclear progenitor cells (CFU-MK) and TB lymphoid progenitor cells (CFU-TB); Third stage is the form to identify precursor cells(precursors) stage, these cells further mature into with special features of all kinds of terminal blood cells,and then released into the blood. Erythroid differentiation is an important branch of hematopoieticdifferentiation.In recent years, the study found that microRNAs (miRNAs) regulated the process of erythroiddifferentiation. MiRNAs are18-25nucleotides, non-coding small RNA molecules, regulating geneexpression in the post-transcription level。Researchers found that these molecules involved in control allaspects of life and associated with a variety of disease.Objective:We mainly research the regulation processing of miR-918and the function and mechanismof erythroid differentiation.Methods: we screened a batch of micrornas associated with erythroid differentiation using the methodof real-timePCR, then designed these primary transcript， precusor，mature primers of the micrornas todetect their expression changes in K562cells induced by hemin.we collected k562cells from different timepoints.The results was the inconsistent expression from primary-918to mature-918in erythroiddifferentiation, that was the expression of pri-918rise and mature-918lower in K562cells. K562celllines were from a kind of chronic myelogenous leukemia, which were induced by chlorine in high iron redelement (hemin) showing erythroid differentiation characteristics. We speculated that the RNA bindingprotein may be involved in the process of miR-918. Then we made the5,-race and3,-race experiment andgained the length of the pri-918transcript. We have obtained over pri-918to predicte several candidates RNA binding protein in RBPDB web site. We transfected si-RNA of the prediction of RNA bindingprotein into k562cells by hemin induced to extract total RNA, by using the method of real-time PCR. wedetected the change of primary-918and mature-918and found that QKI-5played an important role in theprocessing of regulation of miR–918. To verify QKI-5interaction with miR-918, we used the RNA andprotein interactions such as RNA-IP, RNA-EMSA, MS2-MBP and a series of experiments to verify thecombination. In order to further understand the function of miR-918in erythroid differentiation,wesynthetized an artificial synthesis of miR-918in vitro, an oligonucleotide sequence, miR-918mimic, tooverexpress miR-918in K562cell lines by hemin induced. After collecting cells, we extracted total RNAby using real-timePCR to detect the change of gamma–globin and analysis the surface markers oferythroid differentiation CD235a/CD71expression via streaming antibody.At the same time, in order to further research the function of QKI-5in erythroid differentiation, webuild the knocked-down QKI-5plasmid to transfect K562cells, then extracted total RNA to detect gamma-globin and flow cytometry to detect the surface markers of erythroid differentiation.To confirm the mechanism of miR-918in erythroid differentiation, we used online software topredict the number of candidate target genes. We coped the candidate gene3’UTR region containing themiRNA binding site sequence into the sea renal luciferase reporter gene termination codon downstream,and then the candidate gene3’UTR region and miR-918mimic was transfected into293TN cells. Weadopted double fluorescence experiment and western blot experiments to determine the target genes ofmiR-918.Results: we verified a direct interaction QKI-5and miR–918in vitro by a series of experiments;Overexpression of miR-918in K562cells by hemin induced, real-timePCR revealed the hemoglobinsynthesis was significantly suppressed, the same results as flow cytometry. These all results showed thatmiR-918inhibits erythroid differentiation. At the same time, to confirm miR-918function in normalerythropoiesis, CD34+HSC cells was isolated from human umbilical cord blood and infected withrecombinant lentivirus encoding miR-918. It is consistent with the results of cell lines. QKI-5promotederythroid differentiation after knocked down in K562cells by realtimePCR and flow cytometry.Double fluorescence experiment suggsted that containing TAL1and c-MYB3’UTR report of geneexpression significantly inhibited by miR–918. We found that the inhibition disappear after the order of binding sites mutation. Western hybridization showed TAL1and c-MYB levels were significantly reducedafter overexpression of miR–918. These results determined the TAL1and c–MYB was direct target genesof miRNA–918. That is to say, miR-918regulated the mechanism of erythroid differentiation throughTAL1, c-MYB.Conclusion:1, QKI-5promoted miR-918processing;2, miR-918suppressed erythroid differentiation;3, QKI-5inhibited erythroid differentiation;4, TAL1and c-MYB was target genes of miR-918in erthroid differentiation.