Untranslated Region Of Bcl-2 mRNA Research Articles

Nucleolin’s Role in the Biogenesis of Pri‐miRNA 16 by the Microprocessor Complex: An In silico Approach

MicroRNAs (miRNAs) are short, non‐coding strands of RNA found in eukaryotes. They play a role in gene silencing through inhibiting translation or degrading mRNA. Primary miRNA (pri‐miRNA) processing is the initial step in producing a mature microRNA molecule. The microprocessor complex (MP) proteins, DROSHA and DGCR8, cleaves the pri‐miRNA molecule to a shorter hairpin structure. This cleaved RNA hairpin, precursor miRNA, is sent to the cytoplasm for further processing into the mature miRNA. Previous research has uncovered that the expression and cellular localization of the stress‐responsive protein, nucleolin (NCL) affects the processing of several miRNAs, including microRNA 16 (miR16). Nucleolin is an RNA binding protein and is primarily located in the nucleolus but can also be localized in various cellular compartments. When NCL is present in the nucleus, the MP complex works more efficiently in cleaving pri‐miR 16‐1 to its pre‐miRNA form resulting in an upregulation of miR 16‐1. It has been proposed that nucleolin binds to pri‐miR 16‐1 and stabilizes its confirmation for cleavage by the MP complex. However, the mechanistic and structural details on how nucleolin interacts with pri‐miRNA 16‐1 or the MP are still unclear. It is also unknown if NCL is similarly involved in miR 16‐2 biogenesis. NCL and miRNA 16 are also intricately involved in the regulation of the expression of bcl2, an anti‐apoptotic and oncogenic protein. When NCL is in the cytoplasm, it stabilizes the 3’ untranslated region of bcl2 mRNA leading to an increased expression of the protein. Unlike nucleolin, miR 16 represses the translation of bcl2 mRNA. At high levels of miR 16 when NCL is localized to the nucleus, bcl2 protein expression is low. When cellular stress occurs, NCL returns to the cytoplasm to stabilize bcl2 and this also results in less efficiency in pri‐miR 16 processing, resulting in an overall increase in bcl2 protein. It is evident that NCL balances the levels of bcl2 and miR16 through its cellular localization. Since the molecular mechanism underlying NCL’s role in the MP‐based processing of pri‐miR16‐1 remains largely unknown, in this study, we used computational approaches including structural alignments, protein‐protein, and protein‐RNA docking analyses to determine how pri‐miR 16‐1, nucleolin, and the MP complex interact. In addition, we have investigated if pri‐miR 16‐2 interacts with the NCL and the MP complex in a similar fashion. We show in our predictive models that the interaction interface of pri‐miR 16‐1 and pri‐miR 16‐2 with nucleolin and the MP complex nucleolin is distinct. For pri‐miR 16‐1, the interaction interface involves NCL and DGCR8 B and C, but in the case of pri‐miR 16‐2, it involves NCL and DROSHA. Additionally, in the presence of nucleolin, the hydrogen bond interactions are reduced between pri‐miR 16‐2 and the MP complex. Although the number of interactions between the different components of the MP complex is the same in both scenarios, the specific residues that form the interaction interface differ, suggesting distinct mechanisms. Since the levels of miR16 expression can lead to apoptosis of cancerous cells and prevent cell proliferation, the results of our study provide important clues in understanding how the NCL‐miR‐MP complex can be targeted in drug design for cancer therapy.

MicroRNA-21 regulates the viability and apoptosis of diffuse large B-cell lymphoma cells by upregulating B cell lymphoma-2.

Diffuse large B-cell lymphoma (DLBCL), one of the most frequently diagnosed non-Hodgkin lymphoma (NHL), is partly attributed to hereditary factors. MicroRNA-21 (miR-21) is an oncogenic substance that induces NHL and primarily targets tumor-suppressive molecules, such as B cell lymphoma-2 (Bcl-2). The present study explored whether Bcl-2, targeted by miR-21, would affect the development of NHL. Specimens were harvested from 55 patients with DLBCL who had undergone surgical treatment. Expression levels of miR-21 and Bcl-2 were evaluated through reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. Luciferase-reporter assays were performed to investigate the potential association between miR-21 and Bcl-2. MTT assays, flow cytometric analysis and caspase-3 activity assays were used to evaluate cell viability and apoptosis of DLBCL cells, respectively. Furthermore, statistical analysis was conducted using SPSS 19.0 software and the expression levels of miR-21 and Bcl-2 within DLBCL tissues were significantly upregulated when compared to those in normal tissues (P<0.01). As predicted by TargetScan, perfect base pairing was observed between the seed sequence of mature miR-21 and the 3' untranslated region of Bcl-2 mRNA. Dual luciferase reporter gene assays also revealed that miR-21 significantly facilitated the luciferase activity of Bcl-2 wild-type, with 61% upregulation (P<0.01) observed. MTT assays demonstrated that the viability of OCI-LY3 cells was decreased when cells were transfected with miR-21 inhibitor or Bcl-2 small interfering RNA and compared with those of control and negative control groups (all P<0.05). The apoptosis rate and caspase-3 activity level of the miR-21 group were 2.73±0.48 and 0.47±0.05, respectively, which were both significantly different from the groups with lower levels of miR-21 expression levels (all P<0.01). Since miR-21 may contribute to increased viability and decreased apoptosis of DLBCL cells through targeting Bcl-2, both Bcl-2 and miR-21 are likely to serve as effective targets for developing novel DLBCL treatments in the future.

PP2A inhibition from LB100 therapy enhances daunorubicin cytotoxicity in secondary acute myeloid leukemia via miR-181b-1 upregulation

Patients with secondary acute myeloid leukemia (sAML) arising from myelodysplastic syndromes have a poor prognosis marked by an increased resistance to chemotherapy. An urgent need exists for adjuvant treatments that can enhance or replace current therapeutic options. Here we show the potential of LB100, a small-molecule protein phosphatase 2 A (PP2A) inhibitor, as a monotherapy and chemosensitizing agent for sAML using an in-vitro and in-vivo approach. We demonstrate that LB100 decreases cell viability through caspase activation and G2/M cell-cycle arrest. LB100 enhances daunorubicin (DNR) cytotoxicity resulting in decreased xenograft volumes and improved overall survival. LB100 profoundly upregulates miR-181b-1, which we show directly binds to the 3′ untranslated region of Bcl-2 mRNA leading to its translational inhibition. MiR-181b-1 ectopic overexpression further diminishes Bcl-2 expression leading to suppression of sAML cell growth, and enhancement of DNR cytotoxicity. Our research highlights the therapeutic potential of LB100, and provides new insights into the mechanism of LB100 chemosensitization.

MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells

MicroRNAs (miRNAs) can regulate cell survival and death by targeting apoptosis-related gene expression. miR-210 is one of the most hypoxia-sensitive miRNAs. In this study, we evaluated the roles of miR-210 in hypoxia-induced insults to neural cells. Treatment of neuro-2a cells with oxygen/glucose deprivation (OGD) induced cell apoptosis in a time-dependent manner. In parallel, OGD time-dependently increased cellular miR-210 levels. Knocking down miR-210 expression using specific antisenses significantly attenuated OGD-induced neural apoptosis. Concurrently, OGD increased hypoxia-inducible factor (HIF)-1α mRNA and protein syntheses. Pretreatment with YC-1, an inhibitor of HIF-1α, reduced OGD-caused cell death. Sequentially, OGD specifically decreased antiapoptotic Bcl-2 mRNA and protein levels in neuro-2a cells. A search by a bioinformatic approach revealed that miR-210-specific binding elements exist in the 3'-untranslated region of Bcl-2 mRNA. Application of miR-210 antisenses simultaneously alleviated OGD-involved inhibition of Bcl-2 mRNA expression. In comparison, overexpression of miR-210 synergistically diminished OGD-caused inhibition of Bcl-2 mRNA expression and consequently induced greater cellular insults. Taken together, this study shows that OGD can induce miR-210 expression through activating HIF-1α. And miR-210 can mediate hypoxia-induced neural apoptosis by targeting Bcl-2.

Effect of Modulation of hnRNP L Levels on the Decay of bcl-2 mRNA in MCF-7 Cells

It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.

Regulation of Bcl-2 Expression by HuR in HL60 Leukemia Cells and A431 Carcinoma Cells

Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.

MiR-34a, a microRNA up-regulated in a double transgenic mouse model of Alzheimer's disease, inhibits bcl2 translation

MicroRNAs (miRNAs) are short noncoding regulatory RNA molecules that modulate protein expression by inhibiting mRNA translation or promoting mRNA degradation. However, little is understood about the roles of miRNAs in Alzheimer's disease. During a research for miRNAs that are differentially expressed in cerebral cortex of APPswe/PSΔE9 mice (a model for Alzheimer's disease) and age-matched controls, one candidate miRNA that is relatively highly expressed, miR-34a, was studied further because sequence analysis suggested a likely interaction with the 3′-untranslated region of bcl2 mRNA. We show that the expression of miR-34a is inversely correlated with the protein level of bcl2 in APPswe/PSΔE9 mice and age-matched controls, and miR-34a expression directly inhibits bcl2 translation in SH-SY5Y cells. No effect on bcl2 mRNA level was observed. Western blot analysis of active caspase-3 showed higher levels in APPswe/PSΔE9 mice and stable transfecant cell line of miR-34a than in controls. Consistently, miR-34a knockdown through antisense LNA oligonucleotides increased the level of bcl2 protein in SH-SY5Y cells, which was accompanied by a decrease of active caspase-3. These findings suggested that bcl2 is an important functional target for miR-34a, and the abnormal expression of miR-34a may contribute to the pathogenesis of Alzheimer's disease, at least in part by affecting the expression of bcl2.

Role of HuR in the regulation of bcl‐2 mRNA stability in human HL60 leukemia cells

High bcl‐2 expression in hematological tumors is frequently an obstacle to cancer chemotherapy. We have demonstrated that Bcl‐2 overexpression in some leukemia cells is a consequence of abnormal bcl‐2 mRNA stability. Previously, nucleolin was identified as a factor that stabilizes bcl‐2 mRNA in human HL60 leukemia cells. This interaction occurs via an AU‐rich element (ARE) in the 3’‐untranslated region of bcl‐2 mRNA. Our current study demonstrated that HuR also plays a role in the regulation of bcl‐2 mRNA stability in HL60 cells. Using EMSA we observed that HuR in HL60 cell extracts binds to the bcl‐2 mRNA ARE. Also, we found that such interactions occur in vivo. Taxol treatment induced proteolysis of cytoplasmic HuR, suggesting that modulation of HuR may play a role in the downregulation of bcl‐2 that precedes taxol‐induced apoptosis. Pull‐down assays indicate that nucleolin and HuR co‐immunoprecipitate in an RNA‐dependent manner, suggesting the two proteins are part of common bcl‐2 mRNP complexes. Moreover, nucleolin and HuR can bind independently and concurrently to bcl‐2 ARE RNA in vitro. This suggests HuR and nucleolin bind to separate, non‐overlapping sites on the bcl‐2 ARE. Sh‐RNA‐mediated knockdown of HuR in A431 epithelial cancer cells leads to reduced levels of bcl‐2 mRNA and protein. Taken together, our studies suggest that both HuR and nucleolin play a role in stabilization of bcl‐2 mRNA, which contributes to the overexpression of Bcl‐2 protein in some types of leukemia cells. Supported by NCI R01CA87553 (EKS) and Leukemia and Lymphoma Society Grant 6006‐06 (DJF).

Activity and Mechanism of Action of AS1411 in Acute Myeloid Leukemia Cells.

AS1411 is a 26 base unmodified DNA aptamer which is currently in a Phase II clinical trial for the treatment of acute myeloid leukemia (AML) and will shortly enter a Phase II trial in renal cell carcinoma (RCC). AS1411 has previously been shown to have cytostatic (a result of arrest in S phase of the cell cycle) and cytotoxic effects on tumor cells, but not normal cells. AS1411 binds to nucleolin, which is overexpressed in the cytoplasm and on the surface of tumor cells. Treatment of MV4-11 cells (an AML-derived cell line) with low micromolar concentrations of AS1411 results in cell growth inhibition and a reduction in viability; this effect becomes apparent after 48 hours incubation. Co-immunoprecipitation of nucleolin and AS1411 from MV4-11 cells treated with radiolabeled AS1411 confirmed previous work that AS1411 binds to nucleolin. In addition, confocal microscopy showed that nucleolin is present in the cytoplasm and the plasma membrane of MV4-11 cells. Incubation of these cells with AS1411 decreased nucleolin mRNA levels, and nucleolin protein was decreased in both cytoplasm and nucleus to approximately 10% of control levels after 96 hours. The reduction in nucleolin levels started to appear at around 48 hours and may reflect cells arresting in S phase of the cell cycle. Nucleolin binds to an ARE sequence in the 3′-untranslated region of Bcl-2 mRNA resulting in stabilization of the mRNA. Using gel shift assays with a radiolabeled fragment of Bcl-2 mRNA we show here that AS1411 interferes with this binding. In addition, levels of Bcl-2 mRNA in MV4-11 cells decrease to 10% of control levels after 48 hours incubation with AS1411 and the level of Bcl-2 protein also decreases in comparison to control cells. Finally, mononuclear cells isolated from blood samples of AML patients were incubated with AS1411 for 96 hours and viability determined. In all samples, AS1411 caused a reduction in cell viability at low micromolar concentrations, similar to those required to kill cell lines. This work therefore demonstrates that AS1411 has both cytostatic and cytotoxic effects on AML cells and underpins the dosing regime of AS1411 in the ongoing AML clinical trial.

CA Repeats in the 3′-Untranslated Region of bcl-2 mRNA Mediate Constitutive Decay of bcl-2 mRNA

An AU-rich element (ARE) in the 3'-untranslated region (UTR) of bcl-2 mRNA has previously been shown to be responsible for destabilizing bcl-2 mRNA during apoptosis through increasing AUF1 binding. In the present study, we investigated the effect of the region upstream of the ARE on bcl-2 mRNA stability using serial deletion constructs of the 3'-UTR of bcl-2. Deletion of 30 nucleotides mostly consisting of the CA repeats, located upstream of the ARE, resulted in the stabilization of bcl-2 mRNA abundance, in the absence or presence of the ARE. The specificity of the CA repeats in terms of destabilizing bcl-2 mRNA was proven by the substituting the CA repeats with other alternative repeats of purine/pyrimidine, but this had no effect on the stability of bcl-2 mRNA. CA repeats alone, however, failed to confer instability to bcl-2 or gfp reporter mRNAs, indicating a requirement for additional sequences in the upstream region of the 3'-UTR. Serial deletion and replacement of a part of the region upstream of the CA repeats revealed that the entire 131-nucleotide upstream region is an essential prerequisite for the CA repeat-dependent destabilization of bcl-2 mRNA. Unlike the ARE, CA repeat-mediated degradation of bcl-2 mRNA was not accelerated upon apoptotic stimulus. Moreover, the upstream sequences and CA repeats are conserved among mammals. Collectively, CA repeats contribute to the constitutive decay of bcl-2 mRNA in the steady states, thereby maintaining appropriate bcl-2 levels in mammalian cells.

Identification of TINO: A NEW EVOLUTIONARILY CONSERVED BCL-2 AU-RICH ELEMENT RNA-BINDING PROTEIN

Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.

Taxol- and okadaic acid-induced destabilization of bcl-2 mRNA is associated with decreased binding of proteins to a bcl-2 instability element

The observation that overexpression of the anti-apoptotic protein Bcl-2 is associated with both cancer development and anti-cancer drug resistance suggests that factors which regulate bcl-2 expression may be important therapeutic targets. We report here that taxol or okadaic acid (OA) treatment of HL-60 cells reduced bcl-2 mRNA steady state levels to 50% of control cell levels in 20–24 hr of treatment. The 3′-untranslated region of bcl-2 mRNA contains four potential A+U-rich elements (AREs), which are associated with mRNA destabilization. RNA gel mobility shift assays revealed that HL-60 cell extracts contain proteins that bind to RNA transcripts containing the first bcl-2 ARE (ARE 1). ARE 1 binding activity was substantially reduced in extracts of cells treated for 20 hr with taxol or OA and was abolished after 32 hr of treatment. UV-induced RNA cross-linking assays revealed that untreated HL-60 cell extracts contain approximately eight proteins, ranging in size from 32 to 100 kDa, that bind to ARE 1 RNA. Following 20 hr of taxol or OA treatment, RNA cross-linking to ∼70 and ∼38 kDa proteins was greatly reduced, and cross-linking to four proteins of 45–60 kDa sizes was progressively reduced with 10–34 hr of OA or taxol treatment. Collectively, these results suggest a novel action of taxol and OA on bcl-2 expression, which involves bcl-2 mRNA downregulation through inactivation of bcl-2 mRNA stabilizing factors.

AUF1 Is a bcl-2 A + U-rich Element-binding Protein Involved in bcl-2 mRNA Destabilization during Apoptosis

We previously identified a conserved A + U-rich element (ARE) in the 3'-untranslated region of bcl-2 mRNA. We have also recently demonstrated that the bcl-2 ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2 mRNA half-life reduction. Here we show that the AUBP AUF1 binds in vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1-257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to the bcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2-3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis.

BCL-2 Regulation Targeting the AU-Rich Domain

In an effort to identify potential upregulators of bcl-2 activity in t(14;18) follicular B lymphoma cells, we detected a hybrid bcl-2/IgH RNA transcribed in antisense orientation. This antisense transcript may contribute to upregulation of bcl-2 expression in t(14;18) cells, overlapping AU-rich motifs present in the 3′-untranslated region of bcl-2 mRNA. We have studied the enzymatic efficiency of a ribozyme directed towards the bcl-2 AU-rich region in a cell-free system determining its kinetic parameters.