Study Of Myelogram Characteristics And Related MiRNA Of ATD-induced Agranulocytosis

PartⅠ Analysis of myelogram characteristics and clinical data ofantithyroid drug induced agranulocytosis patientsObjective Analysis of myelogram characteristics and some related clinical data to explorethe relationship between bone marrow characteristics and clinical prognosis of antithyroiddrugs induced agranulocytosis patients.Methods From January2008to December2011,42ATD-induced agranulocytosis patientswere treated at three affiliated hospitals of South China university (The first affiliatedhospital,The second affiliated hospital and Chenzhou affiliated hospital).33of themreceived a bone marrow examination during agranulocytosis. Medical records of33patients were reviewed for duration of the offending ATDs, clinical features, bone marrowcharacteristics and outcomes.Results The bone marrow characteristics of ATD-induced agranulocytosis patients can bedivided into two types as granulocytes aplastic type and dysmaturity type.There were13cases (39.4%)with myelogram charactered as dysmaturity and20cases (60.6%) asaregeneratory. The median duration of neutrophil recovery and fever were (4.7±1.0) and（3.6±2.5）days in dysmaturity type while (8.0±2.8)and (8.6±3.1) days in aplastic type.Two cases in aplastic type group died of infection.Conclusion The myelogram charactered mainly as aplastic type in antithyroid druginduced agranulocytosis patients. Patients in dysmaturity type group are usually with goodclinical prognosis for quickly increased neutrophil and short duration of fever. PartⅡ Screening and identification of plasma differential expressionmiRNA in antithyroid drug induced agranulocytosisObjective To screen and identify the plasma miRNA differential expression profile inATD induced agranulocytosis by the miRNA array technique.Methods Ten plasma samples were obtained from5ATD induced agranulocytosis patientsduring agranulocytosis and after recovery of neutrophil count. The5patients were treatedwith same ATD and matched with gender, age and duration of drug administration. TotalRNA was extracted from10plasma samples and labeled with fluorescence. The humanmiRNA Array (v.16.0)(Agilent) was used to screen the differentially expressed miRNA.Target genes and predicted function of the distinct miRNA were analysed by bioinformaticsoftware online. Lower expressive hsa-miR-20a, hsa-miR-223, hsa-miR-106b andhsa-miR-19b were indentified by real-time quantitative PCR and the reliability of miRNAarray results was comfirmed. The relationship between variation of these miRNA and thebone marrow characteristics classification was analysed as well.Results Compared to neutrophil count recoveried group,32miRNA were differentiallyexpressed in plasma samples during agranulocytosis, including9up-regulated miRNA (FC≥2) and23down-regulated (FC≤0.5). The most significantly up-regulated miRNA werehsa-miR-200, hsa-miR-756, hsa-miR-320a, hsa-miR-21, hsa-miR-30a, hsa-miR-483-5pand hsa-miR-320e (FC≥5), while hsa-miR-20a, hsa-miR-223,hsa-miR-106b, hsa-miR-19b,hsa-miR-1234, hsa-miR-342-3p, hsa-miR-15a, hsa-miR-19and hsa-miR-144weresignificantly down regulated (FC≤0.2). Bioinformatics analysis showed lower expressivemiRNA was closely related to apoptosis and cell cycle regulation while higher expressivemiRNA participate in the regulation of transcriptional, cell proliferation and inflammation.The expression level of hsa-miR-20a, hsa-miR-223, hsa-miR-106b and hsa-miR-19b wasvalidated by qRT-PCR,which was confirmed to the result of miRNA chip array.There wasno significant difference of4miRNA expression level between the two groups of patientstaking PTU or MMI and two types of bone marrow characteristics (p>0.05). Conclusion1、Plasma miRNA profile of ATD induced agranulocytosis was acquired by miRNAmicroarray.2、 The miRNA profile of ATD induced agranulocytosis was consistent in two drugsadministration groups (PTU/MMI) and two types of bone marrow characteristics.3、The lower expressive miRNA was associated with the procedure of cell apoptosis. Thisresult prompted granulocyte apoptosis may be a cause of ATD-inducedagranulocytosis. PartⅢ The biological effects of hsa-miR-20a on HL-60、U-937cellsObjective The significantly lower expressed hsa-miR-20a obtained from miRNAmicroarray was selected as the object for further study. A series of experiments weredesigned to observe the biological effects of hsa-miR-20a expression level change onHL-60, U-937cell.Methods Construct a lentiviral expression vector to lower or over expression the level ofhsa-miR-20a in the aimed cells. The titer of lentiviral was determined by fluorescencedetection after infected293T cells. HL-60and U-937were recovered and cultured inRPMI-1640medium, Lentiviral was added to logarithmic growth phase cells according toMOI (multiplicity of infection) value in three groups, they are hsa-miR-20a overexpressive group（hsa-miR-20a up）, hsa-miR-20a lower expressive group(hsa-miR-20adown) and empty vector group (control). Changes in cell morphology were observed underthe microscope and cell proliferation states were dectected by MTT,48and72hours afterlentiviral infection,flow cytometry was used to detect cell apoptosis. Meanwhile, qRT-PCRwas performed to detect the change of hsa-miR-20a level.Result Hsa-miR-20a up/down lentiviral expression vectors were successfully constructed.The titer of hsa-miR-20a up lentiviral expression vectors was4×108TU/ml while the valueof hsa-miR-20a down was8×108TU/ml. Three groups of lentiviral expression vectors were successfully infected HL-60, U-937cells, the changes of hsa-miR-20a expressionlevel in those cells was confirmed by qRT-PCR. Hsa-miR-20a down can inhibit HL-60,U-937cell growth and promote apoptosis. Moreover, Cell growth inhibition and apoptosislevel were more pronounced in HL-60cells compared with U-937cells (p <0.05). Therewas no significant difference of cell proliferation and apoptosis in hsa-miR-20a up groupthan control.Conclusion Lentiviral expression vectors of hsa-miR-20a were successfully constructed.Lower expression of hsa-miR-20a can inhibit HL-60, U-937cell growth and promoteapoptosis. These biological effects were more obvious in HL-60cells than in U-937cells. Part Ⅳ Regulatory mechanism of hsa-miR-20a on neutrophilapoptosisObjective To investigate the target genes of hsa-miR-20a and clarify the potentialmechanism in the occurrence of agranulocytosisMethods The biological information of hsa-miR-20a such as seed sequence, apoptosisrelated target genes, targeting sites of3’UTR and binding free energy with target sequencewere analysed online by bioinformatics softwares. Dual luciferase reporter gene vector wasconstructed to detect the effectiveness of predicted sites. The mRNA and proteinexpression level of target gene were detected by RT-PCR and western-blot respectively,thechange of related downstream proteins was detected as well. The protein expression levelof target gene was also examined in bone marrow granulocytes of ATD inducedagranulocytosis patients by immunohistochemical.Result The prediction results of multiple bioinformatics software showed that hsa-miR-20ahad a theoretical effect binding sites with PTEN3’UTR. The data of dual luciferasereporter gene examination indicated that mRNA level of PTEN was significantly decreasedwhile hsa-miR-20a up and psiCHECK-2-PTEN-WT3’UTR plasmid was cotransfectedinto293T cells, but the effcet was not found in psiCHECK-2-PTEN-Mut3’UTR plasmid.The mRNA level of PTEN in cells of miR-20a up group was significantly reducedcompared with control and the change trend of PTEN protein was consistent with the trendof mRNA. The expression level of p-Akt and Bcl-2was decreased while Bax wasincreased according to the increase of PTEN protein.Conclusion PTEN was the target gene of hsa-miR-20a, PTEN may play a major role inATD induced agranulocytosis by regulating the mitochondrial apoptotic signal ofp-Akt/Bcl-2/Bax pathway.