| Section 1 MicroRNAs expression signatures are associated with lineage and survival in acute leukemiasObjective:The aim of this section was to assess expression of precursors of 23 miRNAs in 85 Chinese adolescent and adult de novo acute leukemia bone marrow specimens and to investigate microRNAs expression signatures in patients with lineage and survival.Methods:Quantitative real-time PCR was used to assess expression of precursors of 23 miRNAs in 85 Chinese adolescent and adult de novo acute leukemia bone marrow specimens.All patients were followed up.Results:①We identified 16 miRNAs differentially expressed between 85 ALL and AML samples. Nine (i.e., miR-128a, miR-128b, miR-155, miR-150, miR-17, miR-29a/c, miR-29b, miR-146a, and miR-20a) were expressed at a significantly higher level in ALL than in AML; In contrast, seven (i.e., miR-223, miR-221, miR-222, miR-27a/b, miR-23a, miR-196b and let-7b) were expressed at a significantly higher level in AML compared to ALL.②We found that three miRNAs including miR-146a, miR-181a/c, and miR-221 were significantly associated with overall survival of the 32 ALL patients. Expression level of the first two precursor miRNAs was associated with a poor outcome whereas that of the latter was associated with a good outcome.③We found that five miRNAs including miR-25, miR-26a, miR-29b, miR-146a, and miR-196b were significantly associated with overall survival of the 53 AML patients. Expression level of the first miRNA was positively whereas that of the latter four was negatively associated with a good outcome. We also found that only three miRNAs including miR-26a, miR-29b, and miR-146a were significantly associated with overall survival of the 40 non-M3 AML patients. Expression level of all three miRNAs was negatively associated with a good outcome.④Because miR-146a is the only miRNA whose expression is significantly associated with overall survival of both AML (including or excluding AML-M3) and ALL patients, we confirmed that the expression signature of miR-146a was significantly inversely associated with overall survival of 61 additional AML patients.⑤Through searching five common human miRNA-target prediction programs/databases, we found that miR-146a has a total of 7,200 predicted targets. We focused on the 622 miR-146a targets that are predicted by at least 3 programs/databases for the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. Particularly, in GO BP (biological process) terms, genes related to "negative regulation of biology process", "negative regulation of cellular process", "apoptosis", "cell cycle", "cell cycle checkpoint", "cell cycle arrest", and "DNA damage checkpoint" were significantly more enriched in the 622 miR-146a putative target list than in the 27,901 total putative miRNA target gene list. This might contribute to the association of miR-146a with poor survival.Conclusions:①Nine (i.e., miR-128a, miR-128b, miR-155, miR-150, miR-17, miR-29a/c, miR-29b, miR-146a, and miR-20a) were expressed at a significantly higher level in ALL than in AML, In contrast, seven (i.e., miR-223, miR-221, miR-222, miR-27a/b, miR-23a, miR-196b and let-7b) were expressed at a significantly higher level in AML compared to ALL.②Expression level of miR-146a and miR-181a/c was associated with a poor outcome whereas that of miR-221 was associated with a good outcome in ALL patients.③Expression level of miR-25 was positively whereas that of 4 miRNAs (i.e., miR-26a, miR-29b, miR-146a, miR-196b) was negatively associated with a good outcome in AML patients. Expression level of miR-26a, miR-29b, and miR-146a was negatively associated with a good outcome in non-M3 AML patients.④In GO BP terms, genes related to "negative regulation of biology process" "negative regulation of cellular process", "apoptosis", "cell cycle", "cell cycle checkpoint", "cell cycle arrest", and "DNA damage checkpoint" were significantly more enriched in the 622 miR-146a putative target list than in the 27,901 total putative miRNA target gene list. This might contribute to the association of miR-146a with poor survival.Section 2:The effects and mechanisms of micro RNAs to apoptosis induced by homoharringtonine in acute myeloid leukemia cells Objective: The aim of this section was to compare the miRNA expression profiles of HHT-treated AML cells with those of untreated and to study these mechanisms of microRNAs to apoptosis induced by homoharringtonine in acute myeloid leukemia cell.Methods:We used MTT assay to investigate the effects of HHT on AML cells (i.e., K562, HL60,U937). PI staining was used for cell cycle analysis by flow cytometry. AV-PI staining was used for detecting apoptosis. We used microarray to compare the miRNA expression profiles of HHT-treated K562 cells with those of untreated cells. We then compared miRNA expression in HHT-treated vs untreated AML cells (i.e., K562, HL60, U937) by using real-time PCR. The expression level of miR-17-92 in AML cells (i.e., K562, HL60, U937 and three primary AML patient blasts) was detected by real-time PCR. We compared c-Myc and p21 mRNA expression in HHT-treated vs untreated K562 and U937 cells by using real-time PCR. Western blot was used for detecting expression level of miR-17-92-associated proteins in K562 and U937 cells. We also detected the expression level of P210 bcr/abl and Bcl-2 family proteins in K562 cells by western blot.Results:①HHT could inhibit growth of AML cells (i.e., K562, HL60, U937). The cell growth was inhibited in a time-and dose-dependent manner, corresponding to the reduced cell viability.②HHT could not evidently induce K562 cell cycle arrested. HHT induced apoptosis of K562 and U937 cells in a dose-dependent manner.③From more than 600 miRNAs, we identified that the expression level of 13 miRNAs(i.e.,miR-17, miR-17*, miR-18a, miR-18b, miR-20a, miR-20b, miR-93, miR-106a, miR-126, miR-142-5p, miR-144, miR-233 and miR-320) was downregulated (changed more than 1.5 times vs untreated cells) whereas none was upregulated in 50ng/ml HHT-treated K562 cells in all times(i.e.,3h,12h,24h). The results were confirmed in HHT treated K562, HL60 and U937 cells by using real-time PCR.④The expression level of miR-17-92 pri-miRNA was downregulated in AML cells (i.e., K562, HL60, U937 and three primary AML patient blasts) treated with 50ng/ml HHT.⑤the expression level of C-Myc mRNA and protein was decreased in 50ng/ml HHT-treated K562 and U937 cells. The expression level of P210 bcr/abl was specifically decreased only in 24-hour-HHT-treated K562 cells.⑥The expression level of P-AKT protein was downregulated whereas that of PTEN, P-PTEN, p21 and E2F1 protein was upregulated, and that of BIM and total AKT protein was stable in K562 and U937 cells which were treated with 50ng/ml HHT.⑦The expression level of apopotosis-induced protein Bak and Bax was increased whereas that of Bcl-2 and Mcl-1 proteins was decreased in 50ng/ml HHT treated K562 cells at different time.Conclusions:①HHT inhibited cell growth and induced apoptosis in human AML cells (i.e., K562, HL60, U937).②The expression level of 13 miRNAs was downregulated whereas none that of miRNAs was upregulated in human AML cells (i.e., K562, HL60, U937) treated by HHT. Firsrly, we thought maybe HHT downregulated expression level of miR-17-92 pri-miRNA through downregulated expression level of miR-17-92 activator protein c-Myc. Secondly, some miRNAs expression level of miR-17-92 clusters was downregulated. Expression level of bcr/abl P210 protein was specifically decreased only in 24-hour-HHT-treated K562 cells. So we thought HHT downregulated expression of c-Myc, which bypassed bcr/abl P210 protein.③HHT downregulated expression of p21, PTEN and E2F1, which were target genes of miR-17-92 and its thomologous cluster in AML cells. HHT maybe downregulated expression level of those proteins through miR-17-92 and its two thomologous clusters. The enhanced expression level of p21 protein maybe resulted from enhanced p21 mRNA.HHT maybe inhibited expression of P-AKT through PTEN.④The expression level of apopotosis-induced protein Bak and Bax was increased whereas that of Bcl-2 and Mcl-1 was decreased in HHT-treated K562 cells.Section 3:The study on effcts of cells transfected with miR-17-92 or mimic and inhibitor of miR-17 and miR-20aObjective:To study the effects of miR-17-92, miR-17 and miR-20a to apoptosis induced by homoharringtonine in acute myeloid leukemia cells.Methods:The miR-17-92 gene was transfected into K562 and U937 cells by the retroviral-mediated gene transfer. The mimic and inhibitor of miR-17 and miR-20a was transfected into K562 cells by electroblot. We used real-time PCR to detect p21 expression of mRNA and western blot to detect expression of those targeted proteins. AV-PI staining was used for detecting apoptosis in HHT-treated and HHT-untreated K562 or U937 cells after miR-19-92 gene transfer. We also used AV-PI staining to detect apoptosis in HHT-treated K562 cells after mimic and inhibitor of miR-17 and miR-20a was transfected.Results:①The expression level of some proteins(i.e., E2F1, PTEN and p21) and p21 mRNA was decreased in K562 and U937 cells overexpressing miR-17-92 compared with controls.The percentage of apoptosis cells was deceased in 50ng/ml HHT-treated K562 and U937 cells overexpressing miR-17-92 compared with controls by using AV/PI staining.②The expression level of some proteins(i.e., E2F1, PTEN and p21) and p21 mRNA was decreased in K562 cells overexpressing miR-17 or miR-20a compared with controls.The percentage of apoptosis cells was deceased in 50ng/ml HHT-treated K562 cells overexpressing miR-17 or miR-20a compared with controls. The expression level of some proteins (i.e., E2F1, PTEN and p21) and p21 mRNA was upregulated in K562 cells transfected with miR-17 or miR-20a inhibitor compared with controls.The percentage of apoptosis cells was upregulated in 50ng/ml HHT-treated K562 cells transfected with miR-17 or miR-20a inhibitor compared with controls.Concludions:①In K562 and U937 cells, some genes (i.e., E2F1, PTEN and p21) were real target genes of miR-17-92.②In K562 cells, some gene (i.e., E2F1, PTEN and p21) were real target gene of miR-17 and miR-20a.③Incorporated with the results of second section, at first, HHT downregulated the expression of miR-17/miR-20a through miR-17-92, secondly, HHT downregulated the expression of some proteins (i.e., E2F1, PTEN and p21) through miR-17/miR-20a in AML cells.④Overexpressing miR-17-92 or miR-17/miR-20a decreased sensitivity to HHT induced K562 cells apoptosis, and inhibiting miR-17/miR-20a increased sensitivity to HHT induced K562 cells apoptosis.Summary:Our study shows that a group of microRNAs (e.g., miR-223, miR-128a and miR-128b) are discriminatory microRNAs between AML and ALL in Chinese patients. More importantly, our study also shows that expression of some microRNAs is associated with overall survival of patients with acute leukemias (e.g., miR-146a, miR-181a/c and miR-221 in ALL, and miR-25, miR-26a, miR-29b, miR-146a, and miR-196b in AML), which has not been reported previously. Furthermore, results from the GO and KEGG analysis of potential targets of miR-146a implicate some potential functions of miR-146a (e.g., negatively regulating genes that inhibit cell growth and promote apoptosis), which may be related to the association of miR-146a with poor survival.HHT induced apoptosis and downregulated expression of 13 mature miRNAs in human AML cells. Firstly, HHT downregulated expression of miR-17-92 pri-miRNA through downregulated expression of miR-17-92 activator protein c-Myc. Scondly, the expression of miR-17 and miR-20a were decreased by HHT through miR-17-92. At last some proteins (i.e., p21, PTEN and E2F1) were downregulated through some miRNA of miR-17-92 and its thomologous cluster. So we thought HHT induced human AML cells apotosis through real target protein (i.e., p21, PTEN and E2F1) of some miRNA of miR-17-92 and its thomologous cluster. This maybe one of the mechanisms that HHT induced AML cells apoptosis, which has not been reported previously.
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