MiR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level.
ABSTRACT: BACKGROUND: miR-15a and miR-16-1(miR-15a/16-1) have been implicated as tumor suppressors in chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemic cells. However the mechanism of inhibiting the proliferation of leukemic cells is poorly understood. METHODS: K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E, cell growth were measured by CCK-8 assay and direct cell count. Meanwhile WT1 protein and mRNA level were measured by Western blotting and quantitative real-time PCR. RESULTS: In this study we found that over-expression of miR-15a/16-1 significantly inhibited K562 and HL-60 cells proliferation. Enforced expression of miR-15a/16-1 in K562 and HL-60 cells significantly reduced the protein level of WT1 but not affected the mRNA level. However enforced expression of miR-15a/16-1 can not reduce the activity of a luciferase reporter carrying the 3'-untranslated region(3'UTR) of WT1. Silencing of WT1 by specific siRNA suppressed leukemic cells proliferation resembling that of miR-15a/16-1 over-expression. Anti-miR-15a/16-1 oligonucleotides (AMO) reversed the expression of WT1 in K562 and HL-60 cells. Finally, we found a significant inverse correlation between miR-15a or miR-16-1 expression and WT1 protein levels in primary acute myeloid leukemia (AML) blasts and normal controls. CONCLUSIONS: These data suggest that miR-15a/16-1 may function as a tumor suppressor to regulate leukemic cell proliferation potentially by down-regulating the WT1 oncogene. However WT1 is not directly targeted by miR-15a/16-1 through miRNA-mRNA base pairing, therefore more study are required to understand the mechanism by which miR-15a/16-1 downregulate WT1.
Project description:BACKGROUND: Pure curcumin has been reported to down-regulate the expression of WT1 in leukemic cells. However, the molecular mechanism underlying the down-regulation of WT1 by curcumin is not completely delineated. The purpose of this present study is to identify a new miRNA-mediated mechanism which plays an important role in the anti-proliferation effects of curcumin in leukemic cells. METHODS: K562 and HL-60 cells were treated with different concentrations of curcumin for 24 and 48 hours, the level of miR-15a/16-1 and WT1 were detected by qRT-PCR and Western blotting. WT1 expression and cell proliferation were detected by Western blotting and CCK-8, after curcumin treated-K562 and HL-60 cells were transfected with anti-miR-15a/16-1 oligonucleotides. RESULTS: We found that pure curcumin upregulated the expression of miR-15a/16-1 and downregulated the expression of WT1 in leukemic cells and primary acute myeloid leukemia (AML) cells. Overexpression of miR-15a/16-1 deduced the protein level of WT1 in leukemic cells, but downregulation of WT1 by siRNA-WT1 could not increase the expression of miR-15a/16-1 in leukemic cells. These results reveal that curcumin induced-upregulation of miR-15a/16-1 is an early event upstream to downregulation of WT1. Furthermore, anti-miR-15a/16-1 oligonucleotides (AMO) partly reversed the downregulation of WT1 induced by pure curcumin in leukemic cells and AMO promoted the growth of curcumin treated-K562 and HL-60 cells. CONCLUSION: Thus, these data suggest for the first time that pure curcumin downregulated the expression of WT1 partly by upregulating the expression of miR-15a/16-1 in leukemic cells. miR-15a/16-1 mediated WT1 downregulation plays an important role in the anti-proliferation effect of curcumin in leukemic cells.
Project description:MicroRNAs (miRNAs) are short noncoding RNAs regulating gene expression that play roles in human diseases, including cancer. Each miRNA is predicted to regulate hundreds of transcripts, but only few have experimental validation. In chronic lymphocytic leukemia (CLL), the most common adult human leukemia, miR-15a and miR-16-1 are lost or down-regulated in the majority of cases. After our previous work indicating a tumor suppressor function of miR-15a/16-1 by targeting the BCL2 oncogene, here, we produced a high-throughput profiling of genes modulated by miR-15a/16-1 in a leukemic cell line model (MEG-01) and in primary CLL samples. By combining experimental and bioinformatics data, we identified a miR-15a/16-1-gene signature in leukemic cells. Among the components of the miR-15a/16-1 signature, we observed a statistically significant enrichment in AU-rich elements (AREs). By examining the Gene Ontology (GO) database, a significant enrichment in cancer genes (such as MCL1, BCL2, ETS1, or JUN) that directly or indirectly affect apoptosis and cell cycle was found.
Project description:Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the presence of Bcr-abl, a hallmark of CML; stable expression of Bcr-abl elevated SMS activity in HL-60 cells while inhibition of the tyrosine kinase activity of Bcr-abl with Imatinib mesylate decreased SMS activity in K562 cells. The increased SMS activity was the result of up-regulation of the Sms1 isoform. Inhibition of SMS activity with D609 (a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA selectively inhibited the proliferation of Bcr-abl-positive cells. The inhibition was associated with an increased production of ceramide and a decreased production of DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNA-mediated down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate that Sms1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of Bcr-abl-positive cells.
Project description:While microRNAs (miRs) have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T cell activation are less clear. We found reduced levels of miR-15a/16 at 3-18 h post-T cell receptor (TCR) stimulation, suggesting a role in shaping T cell activation. An inducible miR15a/16 transgenic mouse model was developed to determine how elevating miR-15a/16 levels during early stages of activation would affect T cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycyclin (DOX) induced expression of miR-15a/16 from 0-18 h post-TCR stimulation decreased ex vivo proliferation as well as in vivo antigen-specific proliferation. Bioinformatic and proteomic approaches were combined to identify MEK1 as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased MEK1 containing the 3’-UTR target nucleotide sequence (UGCUGCUA) but did not decrease MEK1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules ERK1/2 and Elk1 were decreased with DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T cell activation to facilitate increased MEK1 and ERK1, and this promotes sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.
Project description:Kaffir lime (Citrus hystrix) is a plant member of family Rutaceae, and its leaves are commonly used in folk medicine. The present study explores antileukemic effects of the extracts and purified active compounds from the leaves. The antileukemic activity was investigated via inhibition of Wilms' tumor 1 (WT1), which is a protein that involves in leukemic cell proliferation. In addition, the compounds were investigated for their effects on WT1 gene expression using real time RT-PCR and Western blotting. Cell cycle arrest and total cell number were investigated using flow cytometry and trypan blue exclusion method, respectively. The results demonstrated that the hexane fractionated extract had the greatest inhibitory effect on WT1 gene expression of many leukemic cell lines and significantly decreased WT1 protein levels of K562 cells (representative of the leukemic cells), in a dose- and time-dependent manner. Subfraction No. 9 (F9) after partial purification of hexane fractioned extract showed the highest suppression on WT1 protein and suppressed cell cycle at G2/M. The organic compounds were isolated from F9 and identified as phytol and lupeol. The bioassays confirmed antiproliferative activities of natural products phytol and lupeol. The results demonstrated anticancer activity of the isolated phytol and lupeol to decrease leukemic cell proliferation.
Project description:Chronic lymphocytic leukemia (CLL) is the most common human leukemia and is characterized by predominantly nondividing malignant B cells overexpressing the antiapoptotic B cell lymphoma 2 (Bcl2) protein. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. Here, we demonstrate that miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and that both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors.
Project description:miR-302a have been reported to participate in various physiological and pathological processes, however, a role for miR-302a in etoposide (VP-16) resistance of acute myeloid leukemia (AML) has not been reported. In this study, the aberrant expression of miR-302a was analyzed in patients with AML and in the AML HL-60 and U937 cell lines. Overexpression of miR-302a, by targeting the 3'UTR of Rad52, enhanced VP-16 sensitivity in the HL-60 and U937 cell. Accordingly, knockdown of Rad52 sensitized the HL-60 and U937 cells to VP-16-induced apoptosis and proliferation suppression. In addition, miR-302a enhanced the tumor-suppressive effect of VP-16 in a xenograft model of human HL-60 and U937 cell lines. Moreover, miR-302a repressed the AKT/Gsk3?/?-catenin pathway after Rad52 inhibition. Reintroduction of Rad52 reversed miR-302a-induced signaling suppression. The results of the present study demonstrated that miR-302a may be a target for the treatment of AML and a potential indicator of the therapeutic sensitivity of AML to VP-16.
Project description:Two new ?-dihydroagarofuran-type sesquiterpenes (<b>1</b>-<b>2</b>) were isolated and identified from the fruit of <i>Celastrus orbiculatus</i> Thunb, together with seventeen known compounds (<b>3</b>-<b>19</b>). The structures of the isolated new compounds were elucidated based on extensive spectroscopic analyses. The cytotoxic activities of the 19 sesquiterpenes on three cell lines, human acute promyelocytic leukemia HL-60, human leukemic K562, and human colon cancer HCT-116 cells, were evaluated in vitro<i>.</i> Compound <b>4</b> exhibited potent cytotoxic activity against HL-60, K562, and HCT116 cell lines with IC<sub>50</sub> values of 3.61 ??, 17.13 ?? and 10.15 ??, respectively, and the other compounds displayed moderate activity.
Project description:Fatty acid synthase (FASN) is upregulated in breast cancer and correlates with poor prognosis. FASN contributes to mammary oncogenesis and serves as a bona fide target in cancer therapies. MicroRNAs inhibit gene expression through blocking mRNA translation or promoting mRNA degradation by targeting their 3'-UTRs. We identified four microRNAs in two microRNA clusters miR-15a-16-1 and miR-497-195 that share a common seed sequence to target the 3'-UTR of the FASN mRNA. In reporter assays, both of these microRNA clusters inhibited the expression of a reporter construct containing the FASN 3'-UTR. However, only ectopic miR-15a-16-1, but not miR-497-195, markedly reduced the levels of endogenous FASN in breast cancer cells. Both miR-15a and miR-16-1 contributes to inhibiting FASN expression and breast cancer cell proliferation. Consistently, a sponge construct consisting of eight repeats of the FASN 3'-UTR region targeted by these microRNAs could markedly increase endogenous FASN levels in mammary cells. When FASN expression was restored by ectopic expression in breast cancer cells, retarded cell proliferation caused by miR-15a-16-1 was partially rescued. In conclusion, we demonstrated that FASN expression is primarily downregulated by miR-15a and miR-16-1 in mammary cells and FASN is one of the major targets of these two tumor suppressive microRNAs.
Project description:The multiple cellular targets affected by proteasome inhibition implicate a potential role for bortezomib, a first-in-class proteasome inhibitor, in enhancing antitumor activities in hematologic malignancies. Here, we examined the antitumor activity and drug targets of bortezomib in leukemia cells. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis assays and associated molecular events assessed by Western Blot. Gene silencing was performed by small interference RNA. Drug was tested in vivo in xenograft models of human leukemia cell lines and in primary leukemia cells. Clinical samples were assessed by immunohistochemical staining. Bortezomib differentially induced apoptosis in leukemia cells that was independent of its proteasome inhibition. Cancerous inhibitor of protein phosphatase 2A, a cellular inhibitor of protein phosphatase 2A, mediated the apoptotic effect of bortezomib. Bortezomib increased protein phosphatase 2A activity in sensitive leukemia cells (HL-60 and KG-1), but not in resistant cells (MOLT-3 and K562). Bortezomib's downregulation of cancerous inhibitor of protein phosphatase 2A and phospho-Akt correlated with its drug sensitivity. Furthermore, cancerous inhibitor of protein phosphatase 2A negatively regulated protein phosphatase 2A activity. Ectopic expression of CIP2A up-regulated phospho-Akt and protected HL-60 cells from bortezomib-induced apoptosis, whereas silencing CIP2A overcame the resistance to bortezomib-induced apoptosis in MOLT3 and K562 cells. Importantly, bortezomib exerted in vivo antitumor activity in HL-60 xenografted tumors and induced cell death in some primary leukemic cells. Cancerous inhibitor of protein phosphatase 2A was expressed in leukemic blasts from bone marrow samples. Cancerous inhibitor of protein phosphatase 2A plays a major role in mediating bortezomib-induced apoptosis in leukemia cells.