Bcl-2 Promoter Research Articles

Carbazole Derivatives Binding to Bcl-2 Promoter Sequence G-quadruplex.

In this study, we used ultraviolet-visible (UV-Vis), fluorescence, and circular dichroism (CD) techniques, as well as molecular modeling, to probe the interactions between carbazole derivatives and the G-quadruplex structure formed in the promoter region of gene Bcl-2. This gene is a rational target for anticancer therapy due to its high expression in a variety of tumors as well as resistance to chemotherapy-induced apoptosis. We employed a sequence with a specific dual G-to-T mutation that may form a mixed-type hybrid G-quadruplex structure in the Bcl-2 P1 promoter region. The three tested carbazole compounds differing in substitution on the nitrogen atom of carbazole interact with the Bcl-2 G-quadruplex by the same binding mode with the very comparable binding affinities in the order of 105 M-1. During absorption and fluorescence measurements, large changes in the ligand spectra were observed at higher G4 concentrations. The spectrophotometric titration results showed a two-step complex formation between the ligands and the G-quadruplex in the form of initial hypochromicity followed by hyperchromicity with a bathochromic shift. The strong fluorescence enhancement of ligands was observed after binding to the DNA. All of the used analytical techniques, as well as molecular modeling, suggested the π-π interaction between carbazole ligands and a guanine tetrad of the Bcl-2 G-quadruplex. Molecular modeling has shown differences in the interaction between each of the ligands and the tested G-quadruplex, which potentially had an impact on the binding strength.

Oncogenic Role of SATB2 In Vitro: Regulator of Pluripotency, Self-Renewal, and Epithelial-Mesenchymal Transition in Prostate Cancer.

Special AT-rich sequence binding protein-2 (SATB2) is a nuclear matrix protein that binds to nuclear attachment regions and is involved in chromatin remodeling and transcription regulation. In stem cells, it regulates the expression of genes required for maintaining pluripotency and self-renewal and epithelial-mesenchymal transition (EMT). In this study, we examined the oncogenic role of SATB2 in prostate cancer and assessed whether overexpression of SATB2 in human normal prostate epithelial cells (PrECs) induces properties of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in prostate cancer cell lines and CSCs, but not in PrECs. Overexpression of SATB2 in PrECs induces cellular transformation which was evident by the formation of colonies in soft agar and spheroids in suspension. Overexpression of SATB2 in PrECs also resulted in induction of stem cell markers (CD44 and CD133), pluripotency-maintaining transcription factors (cMYC, OCT4, SOX2, KLF4, and NANOG), CADHERIN switch, and EMT-related transcription factors. Chromatin immunoprecipitation assay demonstrated that SATB2 can directly bind to promoters of BCL-2, BSP, NANOG, MYC, XIAP, KLF4, and HOXA2, suggesting SATB2 is capable of directly regulating pluripotency/self-renewal, cell survival, and proliferation. Since prostate CSCs play a crucial role in cancer initiation, progression, and metastasis, we also examined the effects of SATB2 knockdown on stemness. SATB2 knockdown in prostate CSCs inhibited spheroid formation, cell viability, colony formation, cell motility, migration, and invasion compared to their scrambled control groups. SATB2 knockdown in CSCs also upregulated the expression of E-CADHERIN and inhibited the expression of N-CADHERIN, SNAIL, SLUG, and ZEB1. The expression of SATB2 was significantly higher in prostate adenocarcinoma compared to normal tissues. Overall, our data suggest that SATB2 acts as an oncogenic factor where it is capable of inducing malignant changes in PrECs by inducing CSC characteristics.

Electroacupuncture regulates histone acetylation of Bcl-2 and Caspase-3 genes to improve ischemic stroke injury

BackgroundImbalances between Bcl-2 and caspase-3 are significant evidence of apoptosis, which is considered an influential factor in rapidly occurring neuronal cell death and the decline of neurological function after stroke. Studies have shown that acupuncture can reduce poststroke brain cell damage via either an increase in Bcl-2 or a reduction in caspase-3 exposure. The current study aimed to investigate whether acupuncture could modulate Bcl-2 and caspase-3 expression through histone acetylation modifications, which could potentially serve as a neuroprotective mechanism. MethodsThis study used TTC staining, Nissl staining, Clark neurological system score, and Evans Blue (EB) extravasation to evaluate neurological damage following stroke. The expression of Bcl-2/caspase-3 mRNA was detected by real-time fluorescence quantification of PCR (real-time PCR), whereas the protein expression levels of Bcl-2, Bax, caspase-3, and cleaved caspase-3 were assessed using western blotting. TUNEL staining of the ischemic cortical neurons determined apoptosis in the ischemic cortex. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, along with the protein performance of AceH3, H3K9ace, and H3K27ace, were detected to evaluate the degree of histone acetylation. The acetylation enrichment levels of H3K9 and K3K27 in the Bcl-2/caspase-3 gene were assessed using Chromatin Immunoprecipitation (ChIP) assay. ResultsOur data demonstrated that electroacupuncture (EA) exerts a significant neuroprotective effect in middle cerebral artery occlusion (MCAO) rats, as evidenced by a reduction in infarct volume, neuronal damage, Blood-Brain Barrier (BBB) disruption, and decreased apoptosis of ischemic cortical neurons. EA treatment can promote the mRNA and protein expression of the Bcl-2 gene in the ischemic brain while reducing the mRNA and protein expression levels of caspase-3 and effectively decreasing the protein expression levels of Bax and cleaved caspase-3. More importantly, EA treatment enhanced the level of histone acetylation, including Ace-H3, H3K9ace, and H3K27ace, significantly enhanced the occupancy of H3K9ace/H3K27ace at the Bcl-2 promoter, and reduced the enrichment of H3K9ace and H3K27ace at the caspase-3 promoter. However, the Histone Acetyltransferase inhibitor (HATi) treatment reversed these effects. ConclusionsOur data demonstrated that EA mediated the expression levels of Bcl-2 and caspase-3 in MCAO rats by regulating the occupancy of acetylated H3K9/H3K27 at the promoters of these two genes, thus exerting a cerebral protective effect in ischemic reperfusion (I/R) injury.

Exploring the DNA2-PNA heterotriplex formation in targeting the Bcl-2 gene promoter: A structural insight by physico-chemical and microsecond-scale MD investigation

Peptide Nucleic Acids (PNAs) represent a promising tool for gene modulation in anticancer treatment. The uncharged peptidyl backbone and the resistance to chemical and enzymatic degradation make PNAs highly advantageous to form stable hybrid complexes with complementary DNA and RNA strands, providing higher stability than the corresponding natural analogues. Our and other groups’ research has successfully shown that tailored PNA sequences can effectively downregulate the expression of human oncogenes using antigene, antisense, or anti-miRNA approaches. Specifically, we identified a seven bases-long PNA sequence, complementary to the longer loop of the main G-quadruplex structure formed by the bcl2midG4 promoter sequence, capable of downregulating the expression of the antiapoptotic Bcl-2 protein and enhancing the anticancer activity of an oncolytic adenovirus. Here, we extended the length of the PNA probe with the aim of including the double-stranded Bcl-2 promoter among the targets of the PNA probe. Our investigation primarily focused on the structural aspects of the resulting DNA2-PNA heterotriplex that were determined by employing conventional and accelerated microsecond-scale molecular dynamics simulations and chemical-physical analysis. Additionally, we conducted preliminary biological experiments using cytotoxicity assays on human A549 and MDA-MB-436 adenocarcinoma cell lines, employing the oncolytic adenovirus delivery strategy.

Characterization of a Very Short Meq Protein Isoform in a Marek's Disease Virus Strain in Japan.

Marek's disease virus (MDV) causes malignant lymphoma (Marek's disease; MD) in chickens. The Meq protein is essential for tumorigenesis since it regulates the expression of host and viral genes. Previously, we reported that the deletion of the short isoform of Meq (S-Meq) decreases the pathogenicity of MDV. Recently, we identified a further short isoform of Meq (very short isoform of Meq, VS-Meq) in chickens with MD in Japan. A 64-amino-acid deletion was confirmed at the C-terminus of VS-Meq. We measured the transcriptional regulation by VS-Meq in three gene promoters to investigate the effect of VS-Meq on protein function. Wild-type VS-Meq decreased the transrepression of the pp38 promoter but did not alter the transactivation activity of the Meq and Bcl-2 promoters. The deletion in VS-Meq did not affect the activity of the pp38 promoter but enhanced the transactivation activities of the Meq and Bcl-2 promoters. Collectively, the deletion of VS-Meq potentially enhanced the activity of the Meq promoter, while other amino acid sequences in wild-type VS-Meq seemed to affect the weak transrepression of the pp38 promoter. Further investigation is required to clarify the effects of these changes on pathogenicity.

Exploring the Dispersion and Electrostatic Components in Arene-Arene Interactions between Ligands and G4 DNA to Develop G4-Ligands.

G-Quadruplex (G4) DNA structures are important regulatory elements in central biological processes. Small molecules that selectively bind and stabilize G4 structures have therapeutic potential, and there are currently >1000 known G4 ligands. Despite this, only two G4 ligands ever made it to clinical trials. In this work, we synthesized several heterocyclic G4 ligands and studied their interactions with G4s (e.g., G4s from the c-MYC, c-KIT, and BCL-2 promoters) using biochemical assays. We further studied the effect of selected compounds on cell viability, the effect on the number of G4s in cells, and their pharmacokinetic properties. This identified potent G4 ligands with suitable properties and further revealed that the dispersion component in arene-arene interactions in combination with electron-deficient electrostatics is central for the ligand to bind with the G4 efficiently. The presented design strategy can be applied in the further development of G4-ligands with suitable properties to explore G4s as therapeutic targets.

Invivo and invitro evaluation of antitumor effects of iron oxide and folate core shell-iron oxide nanoparticles.

Nanoparticles are considered viable options in the treatment of cancer. This study was conducted to investigate the effect of magnetite nanoparticles (MNPs) and magnetite folate core shell (MFCS) on leukemic and hepatocarcinoma cell cultures as well as their effect on the animal model of acute myelocytic leukemia (AML). Through current study nanoparticles were synthesized, characterized by various techniques, and their properties were studied to confirm their nanostructure. Invivo study, nanoparticles were evaluated to inspect their cytotoxic activity against SNU-182 (human hepatocellular carcinoma), K562 (human leukemia), and THLE2 (human normal epithelial liver) cells via MTT test. Apoptotic signaling proteins Bcl-2 and Caspase-3 expression were inspected through RT-PCR method. A cytotoxic effect of MNPs and MFCS was detected in previous cell cultures. Moreover, the apoptosis was identified through significant up-regulation of caspase-3, with Bcl-2 down-regulation. Invitro study, AML was induced in rats by N-methyl-N-nitrosourea followed by oral treatment with MNPS and MFCS. Biochemical indices such as aspartate and alanine amino transferases, and lactate dehydrogenase activities, uric acid, complete blood count, and Beta -2-microglubulin were assessed in serum. Immunophenotyping for CD34 and CD38 detection was performed. Liver, kidney, and bone marrow were microscopically examined. Bcl-2 promoter methylation, and mRNA levels were examined. Although, both MNPs and MFCS depict amelioration in biochemical parameters, MFCS alleviated them toward normal control. Anticancer activity of MNPs and MFCS was approved especially for AML. Whenever, administration of MFCS was more effective than MNPs. The present work is one of few studies used MFCS as anticancer agent.

Phosphorylation of RelA/p65 Ser536 inhibits the progression and metastasis of hepatocellular carcinoma by mediating cytoplasmic retention of NF-κB p65.

Intrahepatic and extrahepatic metastases contribute to the high recurrence rate and mortality of hepatocellular carcinoma (HCC). Constitutive activation of nuclear factor-κB (NF-κB) is a crucial feature of HCC. NF-κB p65 (p50-p65) is the most common dimeric form. Ser536 acts as an essential phosphorylation site of RelA/p65. However, the effect of RelA/p65 Ser536 phosphorylation on progression and metastases during intermediate and advanced HCC has not been reported. Phosphorylation of RelA/p65 (p-p65 Ser536) and NF-κB p65 were detected by using immunohistochemical staining in HCC tissue samples. The biological effects of RelA/p65 Ser536 phosphorylation were evaluated by using xenograft and metastasis models. NF-κB p65 nuclear translocation was detected by using Western blotting. The binding of NF-κB p65 to the BCL2, SNAIL, and MMP9 promoters was detected by using chromatin immunoprecipitation. The biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition were assessed by using tetrazolium-based colorimetry, colony formation, EdU incorporation, flow cytometry, cell wound healing, and transwell assay. NF-κB p65 is highly expressed, while p-p65 Ser536 is not well expressed in intermediate and advanced HCC tissues. In vivo experiments demonstrated that a phosphorylation-mimetic mutant of RelA/p65 Ser536 (p65/S536D) prevents tumor progression and metastasis. In vitro experiments showed that p65/S536D inhibits proliferation, migration, and invasion. Mechanistically, RelA/p65 Ser536 phosphorylation inhibits NF-κB p65 nuclear translocation and reduces NF-κB p65 binding to the BCL2, SNAIL, and MMP9 promoters. RelA/p65 Ser536 phosphorylation was detrimental to NF-κB p65 entry into the nucleus and inhibited HCC progression and metastasis by reducing BCL2, SNAIL, and MMP9. The phosphorylation site of RelA/p65 Ser536 has excellent potential to be a promising target for NF-κB-targeted therapy in HCC.

Targeted Activation of HNF4α by AMPK Inhibits Apoptosis and Ameliorates Neurological Injury Caused by Cardiac Arrest in Rats

Previous studies have shown that AMPK plays an important role in cerebral ischemia–reperfusion injury by participating in apoptosis, but the exact mechanism and target of action remains unclear. This study aimed to investigate the protective mechanism of AMPK activation on brain injury secondary to cardiac arrest. HE, Nills and TUNEL assays were used to evaluate neuronal damage and apoptosis. The relationships between AMPK, HNF4α and apoptotic genes were verified by ChIP-seq, dual-luciferase and WB assays. The results showed that AMPK improved the 7-day memory function of rats, and reduced neuronal cell injury and apoptosis in the hippocampal CA1 region after ROSC, while the use of HNF4α inhibitor weakened the protective effect of AMPK. Further research found that AMPK positively regulated the expression of HNF4α, and AMPK could promote the expression of Bcl-2 and inhibit the expression of Bax and Cleaved-Caspase 3. In vitro experiments showed that AMPK ameliorated neuronal injury by inhibiting apoptosis through the activation of HNF4α. Combined with ChIP-seq, JASPAR analysis and Dual-luciferase assay, the binding site of HNF4α to the upstream promoter of Bcl-2 was found. Taken together, AMPK attenuates brain injury after CA by activating HNF4α to target Bcl-2 to inhibit apoptosis.

DNA G-Quadruplex-Binding Protein Developed Using the RGG Domain of Translocated in Liposarcoma/Fused in Sarcoma Inhibits Transcription of bcl-2.

The G-quadruplexes (G4s) in the genome are important drug targets because they regulate gene expression and the genome structure. Several small molecules that bind the G4 have been developed, but few artificial G4 binding proteins have been reported. We previously reported a novel DNA G4 binding protein (RGGF) engineered using the Arg-Gly-Gly repeat (RGG) domain of TLS (translocated in liposarcoma), also known as FUS (fused in sarcoma) protein (TLS/FUS). Here, we show that RGGF recognizes DNA loops in the G4 and preferentially binds DNA G4 with long loops in vitro. Furthermore, RGGF binds to the DNA G4 of the bcl-2 promoter in vitro. RGGF overexpression in HeLa cells represses bcl-2 transcription. On the basis of these findings, G4 binding protein engineered from the RGG domain will be useful for investigating G4 transcriptional function in the genome.

Astilbin attenuates neonatal postnatal immune activation-induced long-lasting cognitive impairment in adult mice

Postnatal immune activation (PIA) often induces neuroinflammation and disrupts neural functions, ultimately causing cognitive impairment. Among them, neuronal dysfunction is one of the key causes of many neurological diseases. It is particularly important to develop effective neuroprotectants to prevent inflammatory brain diseases. Recently, the protective effect of astilbin (ASB) on nervous system diseases has attracted much attention. However, whether ASB can resolve neuronal dysfunction and cognitive decline triggered by PIA remains unclear. In this study, we used the lipopolysaccharide (LPS)-induced PIA model and found that ASB improved PIA-induced cognitive impairment and neuronal dysfunction in adult mice. Moreover, ASB inhibited PIA-induced neurogenesis deficits and synaptic damage. Finally, we found that ASB treatment inhibited the levels of EZH2/H3K27me3 and decreased the enrichment level of H3K27me3 at the promoter of BCL2 and BDNF. Together, our results demonstrated that ASB may protect against PIA-induced cognitive impairment through EZH2-mediated histone methylation. This study established the role of ASB and epigenetic factor EZH2 regulatory network on the PIA model, which provided new clues and theoretical guidance for the clinical treatment of PIA-induced cognitive impairment.

Long noncoding RNA HOTAIR facilitates pulmonary vascular endothelial cell apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD

BackgroundTo study the regulatory effect of Long non-coding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) on pulmonary vascular endothelial cell (HPVEC) apoptosis and determine whether the HOTAIR facilitate HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in chronic obstructive pulmonary disease (COPD).MethodsLncRNA array was used to measure the differentially expressed lncRNAs in COPD and non-COPD lung tissues. Expression of HOTAIR in COPD patient lungs and cigarette smoke extract (CSE)-induced HPVEC was assessed by qRT-PCR. The location of HOTAIR was determined in COPD patient lungs and HPVEC by RNA in situ hybridization (RNA-ISH). The emphysema mouse model and HOTAIR knockdown mice were each established by inhaling cigarette smoke or intratracheal lentiviral vectors instillation. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Cleaved-caspase 3 protein expression were detected by Western blotting. HOTAIR, DNMT1, Bcl-2 and Bax mRNA expression were measured by quantitative real-time polymerase chain reaction. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to assess apoptotic ratio in mice and CSE-induced HPVEC. Methylation-specific PCR (MSP) assay was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. RNA pull-down assay was used for analysis of the correlation between HOTAIR and DNMT1.ResultsThe expression levels of the HOTAIR were up-regulated in COPD patient lungs and CSE-induced HPVEC. HPVEC apoptosis with down-regulated Bcl-2 expression, increased promoter methylation, DNMT1, Bax and Cleaved-caspase 3 expression was found in emphysema mouse model and CSE-induced HPVEC. Knockdown HOTAIR can attenuate cell apoptosis and emphysema via DNMT1 mediated hypermethylation of Bcl-2 promoter in mice. In vitro, HOTAIR can aggravate the apoptosis of CSE-exposed HPVEC. DNMT1 was a target of HOTAIR and had a positive correlation with HOTAIR.ConclusionHOTAIR facilitates HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD, and attenuating the expression of HOTAIR may be a new therapy to prevent COPD.

Preferential stabilization of c-MYC and BCL-2 promoter G-quadruplexes by a natural betaine-type alkaloid

In this study, we demonstrate for the first time that the betaine-type alkaloid Lycobetaine ( LYC ) preferentially binds to c-MYC and BCL-2 promoter G-quadruplexes (G4s). Biophysical experiments including absorption titrations, CD melting and NMR studies showed that LYC had the ability to bind and stabilize both c-MYC and BCL-2 G4s through an end-stacking mode. Additional biological assays including RT-PCR and Western blotting indicated that LYC simultaneously inhibited the expression of c-MYC and BCL-2 oncogenes in A549/DDP cells, which thereby led to cell cycle arrest, apoptosis, as well as inhibition on cell proliferation and migration. Taken together, these results revealed a new potential anticancer mechanism of LYC with potential application of LYC in drug-resistant cancers. • The betaine-type alkaloid lycobetaine ( LYC ) preferentially binds to c-MYC and BCL-2 promoter G-quadruplexes. • LYC simultaneously inhibits the expression of c-MYC and BCL-2 oncogenes. • This study uncovers a new potential anticancer mechanism of LYC . • This work extended the application of LYC in drug-resistant cancers.

BCL6-Mediated Escape from Negative Selection Enables RAS-Driven B-Cell Transformation

Background: In B-cell acute lymphoblastic leukemia (B-ALL), RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) are found in ~40% of cases (Irving et al., 2014; Jerchel et al., 2018; Zhang et al., 2011). Evidence suggests that RAS-pathway mutations in B-ALL subclones are selected for during therapy and are associated with relapse, which has a poor prognosis and remains one of the leading causes of death in children with malignancy (Irving et al., 2014; Jerchel et al., 2018). Beyond its role as a driver of B-ALL, ERK-activation can induce expression of PRDM1, which functions as a tumor suppressor and triggers negative B-cell selection and cell death (Yasuda et al., 2011; Hug et al., 2014; Setz et al., 2018). During B-cell development, PRDM1 and the proto-oncogene BCL6 are reciprocal antagonists (Shaffer et al., 2002). Here, we examined the mechanism by which oncogenic RAS-ERK signaling in B-ALL avoids negative selection and cell death to promote leukemogenesis. Results: Mimicking survival signals from pre-B cell receptor (pre-BCR), constitutive Erk activation upon oncogenic RAS (NRASG12D) expression in mouse B-cell precursors massively induced the mRNA (~390-fold) and protein (~50-fold) levels of the proto-oncogene Bcl6. Highlighting an essential role of Erk-signaling in upregulation of Bcl6 expression downstream of oncogenic RAS, pharmacological Erk-inhibition markedly reduced NRASG12D-mediated Bcl6 induction. Both the pre-BCR linker molecule Blnk and the surrogate light chains (SLCs) of the pre-BCR are required for Erk-mediated induction of Bcl6. Genetic ablation of Blnk or the SLC component λ5 abrogated RAS-mediated Bcl6-induction. Hence, oncogenic expression of Bcl6 in RAS-driven B-ALL cells depended on structural elements of the pre-BCR. Additionally, ERK-activation in patient-derived B-ALL cells had the same effect on BCL6 expression. Pharmacological reactivation of ERK (BCI-215) markedly induced BCL6 expression, whereas small molecule inhibition of ERK-activity (trametinib) reduced BCL6 levels in patient-derived B-ALL cells. Consistent with BCL6-transactivation downstream of ERK-activation, ChIP-seq analysis revealed binding of ERK-dependent transcription factors (CREB1, ELK1, EGR1, JUND and C-JUN) to the BCL6 promoter in human B cells. Altogether, our findings illuminated a critical role of ERK-signaling in positive regulation of BCL6 expression. Using a genetic model for Cre-mediated deletion of Bcl6 in NRASG12D B-ALL cells, we found that Bcl6 was required for leukemia-initiation in transplant recipient mice (P=0.007). Patient-derived RAS-driven B-ALL cells were highly sensitive to pharmacological inhibition of BCL6 using peptide (RI-BPI) and small molecule (FX1) inhibitors. Furthermore, pharmacological inhibition of BCL6 delayed onset of fatal disease and prolonged survival of transplant recipient mice bearing patient-derived RAS-driven B-ALL cells (P=0.009). Taken together, our findings demonstrated that suppressing BCL6 function is a promising option for targeted therapeutics in RAS-driven B-ALL. Mechanistically, genetic ablation or pharmacological inhibition of BCL6 increased PRDM1 expression. Gene expression analysis revealed that ectopic expression of Prdm1 in B-cell precursors promoted gene expression programs of apoptosis and cell cycle arrest (GSE111692). While Bcl6-deletion resulted in cell death of B-ALL cells, this was largely reversed by shRNA-mediated silencing of Prdm1. Altogether, our results showed that compromised leukemogenesis was a result of aberrant PRDM1 expression in BCL6-deficient RAS-driven B-ALL cells. Conclusions: We previously investigated targeted engagement of negative selection in B-ALL and showed that hyperactivation of SYK or AKT functionally mimics autoreactive pre-BCR signaling and results in negative selection and cell death (Chen et al., 2015; Shojaee et al., 2016). Here, we reinforced this concept by providing a mechanistic basis on how negative selection is downregulated in RAS-driven B-ALL and how it can be reactivated for therapeutic benefit - namely, BCL6 curbs ERK-mediated induction of PRDM1 expression to promote leukemogenesis and this mechanism can be exploited as synthetic lethality in the treatment of this disease.

HBV-upregulated Lnc-HUR1 inhibits the apoptosis of liver cancer cells

Lnc-HUR1 is an HBV-related long non-coding RNA, which can promote the proliferation of hepatoma cells and the occurrence and development of liver cancer. In this study we explored the effect of lnc-HUR1 on the apoptosis of hepatocellular carcinoma cells by taking the approach of immunoblotting, quantitative real time PCR, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and flow cytometry. We found that overexpression of lnc-HUR1 significantly reduced the activity of caspase3/7 and the cleavage of PARP-1, while knocking down of lnc-HUR1 significantly increased the activity of caspase3/7 and promoted the cleavage of PARP-1 in HepG2 cells treated with TGF-β, pentafluorouracil or staurosporine. Consistently, the data from Annexin-V/PI staining showed that overexpression of lnc-HUR1 inhibited apoptosis, while knockdown of lnc-HUR1 promoted apoptosis. Moreover, overexpression of lnc-HUR1 up-regulated the apoptosis inhibitor Bcl-2 and down-regulated the pro-apoptotic factor BAX at both RNA and protein levels. In the CCL4-induced acute liver injury mice model, the expression of Bcl-2 in the liver tissue of lnc-HUR1 transgenic mice was higher than that of the control mice. The data from ChIP assay indicated that lnc-HUR1 reduced the enrichment of p53 on Bcl-2 and BAX promoters. All these results indicated that lnc-HUR1 inhibited the apoptosis by promoting the expression of apoptosis inhibitor Bcl-2 and inhibiting the expression of apoptosis promoting factor BAX. Further studies showed that lnc-HUR1 regulated the transcription of Bcl-2 and BAX in HCT116 cells, but had no effect on the expression of Bcl-2 and BAX in HCT116 p53-/- cells, indicating that lnc-HUR1 regulates the transcription of Bcl-2 and BAX dependent upon the activity of p53. In conclusion, HBV upregulated lnc-HUR1 can inhibit the apoptosis of hepatoma cells. Lnc-HUR1 inhibits apoptosis by inhibiting the transcriptional activity of p53. These results suggest that lnc-HUR1 plays an important role in the occurrence and development of HBV-related hepatocellular carcinoma.

DNA G-Quadruplex in Human Telomeres and Oncogene Promoters: Structures, Functions, and Small Molecule Targeting.

DNA G-quadruplex secondary structures formed in guanine-rich human telomeres and oncogene promoters are functionally important and have emerged as a promising new class of cancer-specific drug targets. These globular intramolecular structures are stabilized by K+ or Na+ and form readily under physiological solution conditions. Moreover, G-quadruplexes are epigenetic features and can alter chromatin structure and function together with interactive proteins. Here, we discuss our efforts over the last two decades to understand the structures and functions of DNA G-quadruplexes formed in key oncogene promoters and human telomeres and their interactions with small molecules. Using high-field NMR spectroscopy, we determined the high-resolution structures of physiologically relevant telomeric G-quadruplexes in K+ solution with a major form (hybrid-2) and a minor form (hybrid-1), as well as a two-tetrad intermediate. The intrinsic structural polymorphism of telomeric DNA may be important for the biology of human telomeres, and we proposed a model for the interconversion. More recently, we have worked on G-quadruplexes of MYC, BCL2, PDGFR-β, VEGF, and k-RAS oncogene promoters. We determined the structure of the major G-quadruplex formed in the MYC promoter, a prototype for parallel G-quadruplexes. It is the first example of the parallel-stranded G3NG3 structure motif with a 1-nt loop, which is prevalent in promoter sequences and likely evolutionarily selected to initiate folding. Remarkably, the parallel MYC promoter G-quadruplexes are highly stable. Additionally, we determined the molecular structures of G-quadruplexes formed in human BCL2, VEGF, and PDGFR-β promoters, each adopting a unique structure. For example, the BCL2 promoter contains distinct interchangeable G-quadruplexes in two adjacent regions, suggesting precise regulation by different proteins. The PDGFR-β promoter adopts unique "broken-strand" and vacancy G-quadruplexes, which can be recognized by cellular guanine metabolites for a potential regulatory role.Structural information on G-quadruplexes in complex with small-molecules is critical for understanding specific recognition and structure-based rational drug design. Our studies show that many G-quadruplexes contain unique structural features such as capping and loop structures, allowing specific recognition by drugs and protein. This represents a paradigm shift in understanding DNA as a drug target: Rather than a uniform, nonselective binding site in duplex DNA, the G-quadruplex is being pursued as a new class of selectively targetable drug receptors. We focus on targeting the biologically relevant MYC promoter G-quadruplex (MycG4) with small molecules and have determined its first and additional drug complex structures. Very recently, we have discovered clinically tested indenoisoquinolines as strong MycG4 binders and potent MYC inhibitors. We have also discovered drugs targeting the unique dGMP-bound-vG4 formed in the PDGFR-β promoter. Moreover, we determined the complex structures of the first small molecules that specifically recognize the physiologically relevant human telomeric G-quadruplexes. Unlike the previously recognized dogma that the optimal G-quadruplex ligands are large aromatic or cyclic compounds, our results suggest that smaller asymmetric compounds with appropriate functional groups are better choices to specifically bind G-quadruplexes. This body of work lays a strong foundation for future work aimed at understanding the cellular functions of G-quadruplexes and G-quadruplex-targeted drug design.

LncRNA SNHG1 promotes sepsis-induced myocardial injury by inhibiting Bcl-2 expression via DNMT1.

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.

IL-7 promoted the development of thymic DN3 cells in aged mice via DNA demethylation of Bcl2 and c-Myc genes

IL-7 promotes the development of thymic double negative (DN) T cells during β-selection, which might contribute to the remission of aging-associated thymic involution. Methylation levels of CpG sites is correlated with aging and modulates the development. To determine the involvement of DNA methylation/demethylation instructed by IL-7 signaling during the expansion of double negative (DN) T cells, the aged mice were treated with recombinant Adeno-Associated Virus 2-mediated IL-7 (rAAV2-IL-7) and the DNA methylation/demethylation modifications in this process were analyzed. The results showed that rAAV2-IL-7 increased the number of thymocytes, especially the DN3 thymocytes during β-selection in aged mice. With aging, the methylation levels of Bcl2 and c-Myc promoter regions were increased in DN3 cells. Following rAAV2-IL-7 treatment, DNA methyltransferase Dnmt3a and Dnmt3b decreased, DNA demethylation factors TET2 and TET3 increased, and the methylation levels of Bcl2 and c-Myc in DN3 cells were reduced during DN3 stage in aged mice, consequently, resulting in the upregulation of Bcl2 and c-Myc and the larger increase of DN3 cells in thymus. In conclusion, these findings showed that Bcl2 and c-Myc genes of DN3 cells had an increased DNA methylation levels in aged mice compared to the young, and the hypermethylation in aged mice could be restored following rAAV2-IL-7 treatment.

Evaluation of Bax and BCL 2 Genes Polymorphisms in Iraqi Women with Breast Cancer.

The present study aimed to examine the polymorphism -938C > A of BCL-2 gene and promoter -248G>A in the Bax gene, as well as their relationship with specific clinical-pathological characteristics, in patients with breast cancer. Blood samples were obtained from 70 patients who had been diagnosed with breast cancer and 34 healthy women as the control group. Polymorphic analysis was performed using thepolymerase chain reaction-restriction fragment length polymorphism assay. Anthropometric data were assessed. Estrogen receptor (ER), human epidermal growth factor receptor 2 (Her-2), and progesterone receptor (PR) were measured by immunohistochemistry. The data of age and body mass index (BMI) demonstrated no significant variations between the two groups (P>0.05). The results of HER-2 revealed that 42.86% of breast cancer patients reflected positively for Her-2/neu expression, while 24.29% reflected negative results of Her-2/neu. Moreover, the results of ER revealed that 42.86% and 28.57% of subjects were positive and negative ER, respectively; moreover, the missing data was 28.57%. In addition, the results of PR indicated that 35.71% of patients (25/70) were positive for PR, while 28.57% reflected negative results, and the missing results were 35.71%. The genotype and allele frequencies of BCL-2(-938C>A) were not statistically significant in women with breast cancer and the control group (P=0.574, P=0.533) for heterozygous and recessive models, respectively. The genotype of BCL-2(-938C>A) in control and patients in codominant, dominant, recessive, and additive models demonstrated no significant variations of all genotypes in all groups. Genotypes and allele frequencies for Bax (-248G>A) in patientswith breast cancer and control indicated that the frequencies of GG, AG, and AA genotypes in cases were 16.67%, 3.33%, and 80 %, while in controls, these values were 3.23 %, 58.06 %, and 3.23 %, respectively. The heterozygous genotype (AG) in the codominant model was OR=36.00 (95% CI 4.5608- 284.1608; P=0.0007). In comparison with the wild type (GG), there was a 36-fold increase in the risk of breast cancer. Furthermore, the findings of this study revealed a significant correlation between Bax (-248G>A) polymorphism and breast cancer risk under the dominant and overdominant (OR=6.33; 95% CI 2.2604 -17.7452; P=0.0004, and OR=40.154; 95% CI 5.1365 - 313.8949; P=0.0004, respectively. The recessive model revealed that there was a decreased risk of breast cancer (OR= 0.167; 95% CI 0.0303to0.9168; P=0.039). Based on the results, it can be concluded that there were no significant variations in BCL-2 (-938C>A) polymorphism of all genotypes models when breast cancer women are compared with healthy ones. In a similar vein, there was no significant association between the BCL-2 (-938C>A) polymorphism and breast cancer risk under dominant, codominant, or recessive models.

Contrasting roles for G-quadruplexes in regulating human Bcl-2 and virus homologues KSHV KS-Bcl-2 and EBV BHRF1

Herpesviruses are known to acquire several genes from their hosts during evolution. We found that a significant proportion of virus homologues encoded by HSV-1, HSV-2, EBV and KSHV and their human counterparts contain G-quadruplex motifs in their promoters. We sought to understand the role of G-quadruplexes in the regulatory regions of viral Bcl-2 homologues encoded by KSHV (KS-Bcl-2) and EBV (BHRF1). We demonstrate that the KSHV KS-Bcl-2 and the EBV BHRF1 promoter G-quadruplex motifs (KSHV-GQ and EBV-GQ) form stable intramolecular G-quadruplexes. Ligand-mediated stabilization of KS-Bcl-2 and BHRF1 promoter G-quadruplexes significantly increased the promoter activity resulting in enhanced transcription of these viral Bcl-2 homologues. Mutations disrupting KSHV-GQ and EBV-GQ inhibit promoter activity and render the KS-Bcl-2 and the BHRF1 promoters non-responsive to G-quadruplex ligand. In contrast, promoter G-quadruplexes of human bcl-2 gene inhibit promoter activity. Further, KS-Bcl-2 and BHRF1 promoter G-quadruplexes augment RTA (a virus-encoded transcription factor)-mediated increase in viral bcl-2 promoter activity. In sum, this work highlights how human herpesviruses have evolved to exploit promoter G-quadruplexes to regulate virus homologues to counter their cellular counterparts.