BCL2L1 Research Articles

In silico analysis highlighting the prevalence of BCL2L1 gene and its correlation to miRNA in human coronavirus (HCoV) genetic makeup.

The ongoing pandemic that resulted from coronavirus disease (COVID-19), which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), had been spiraling out of control with no known antiviral drugs or vaccines. Due to the extremely serious nature of the disease, it has claimed many lives, with a mortality rate of 3.4% declared by the World Health Organization (WHO) on March 3, 2020. The aim of this study is to gain an understanding of the regulatory nature of the proteins involved in COVID-19 and to explore the possibility that microRNA (miRNA) could become a major component in the decoding of the virus. In the study, we were able to correlate the host protein gene BCL2L1 with miRNA miR-23b via network analysis. MiRNAs have previously been associated with the antiviral properties of various viral diseases, such as enterovirus 71 and hepatitis. They have been reported to act as antiviral regulators, since they are an integral component in the direct regulation of viral genes. MiRNAs are also capable of enabling the virus to avoid the host immune response by suppressing the IFN-α/β signaling pathway or increasing the production of IFN-α/β and as a result, inhibiting the viral infection. Here, we explain and shed light on the various correlations in the miRNA-gene-disease association that are seen in the host proteins of COVID-19.

Role of long noncoding RNA KCNQ1 overlapping transcript 1/microRNA-124-3p/BCL-2-like 11 axis in hydrogen peroxide (H2O2)-stimulated human lens epithelial cells

ABSTRACT Age-related cataract (ARC) is one of the most common causes of vision loss in aging people. This research analyzed the functions and mechanism of long noncoding RNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) in hydrogen peroxide (H2O2)-stimulated human lens epithelial cells (SRA01/04 cells) in ARC. SRA01/04 cells were stimulated with 200 µM H2O2 to establish oxidative damage in the ARC model. A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry analysis were conducted to evaluate cell growth and apoptosis. The relevance between KCNQ1OT1 and microRNA (miR)-124-3p or miR-124-3p and BCL-2-like 11 (BCL2L11) was measured through Starbase and a dual luciferase reporter gene assay. The levels of KCNQ1OT1 and miR-124-3p were assessed via quantitative real-time polymerase chain reaction (qRT-PCR). We observed that KCNQ1OT1 was over-expressed and miR-124-3p was low-expressed in H2O2-stimulated SRA01/04 cells. KCNQ1OT1 interacted with miR-124-3p and negatively mediated its levels. In addition, KCNQ1OT1-siRNA reversed the effects of H2O2 on SRA01/04 cells, evidenced by enhanced cell viability, inhibited apoptotic cells, promoted Bcl-2 expression, and reduced Bax levels. Nevertheless, these observations were inverted after miR-124-3p inhibitor treatment. Likewise, miR-124-3p mimic had a protective effect on H2O2-stimulated SRA01/04 cells. Our data suggested that BCL2L11 targeted miR-124-3p directly. In summary, the data indicated that lncRNA KCNQ1OT1 down-regulation protected SRA01/04 cells from oxidative stress stimulated damage via the miR-124-3p/BCL2L11 pathway.

The utility of plasma circulating cell-free messenger RNA as a biomarker of glioma: a pilot study

BackgroundResearch into the potential utility of plasma-derived circulating cell-free nucleic acids as non-invasive adjuncts to radiological imaging have been occasioned by the invasive nature of brain tumour biopsy. The objective of this study was to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and TCGA glioma-derived RNA.MethodsBlood samples were collected from twenty glioma patients prior to tumour resection. Plasma ccfmRNAs and glioma-derived RNA were extracted and profiled.ResultsBCL2L1, GZMB, HLA-A, IRF1, MYD88, TLR2, and TP53 genes were significantly over-expressed in glioma patients (p < 0.001, versus control). GZMB and HLA-A genes were significantly over-expressed in high-grade glioma patients (p < 0.001, versus low-grade glioma patients). Moreover, the fold change of the BCL2L1 gene was observed to be higher in patients with high-grade glioma (p = 0.022, versus low-grade glioma patients). There was positive correlation between the magnitude of fold change of differentially expressed genes in plasma- and glioma-derived RNA (Spearman r = 0.6344, n = 14, p = 0.017), and with the mean FPKM in TCGA glioma-derived RNA samples (Spearman r = 0.4614, n = 19, p < 0.05). There was positive correlation between glioma radiographic tumour burden and the magnitude of fold change of the CSF3 gene (r = 0.9813, n = 20, p < 0.001).ConclusionWe identified significant differential expression of genes involved in cancer inflammation and immunity crosstalk among patients with different glioma grades, and there was positive correlation between their transcriptomic profile in plasma and tumour samples, and with TCGA glioma-derived RNA.

SchizandrinA can inhibit non‑small cell lung cancer cell proliferation by inducing cell cycle arrest, apoptosis and autophagy.

Schizandrin A (SchA) can be extracted from the vine plant Schisandra chinensis and has been reported to confer various biologically active properties. However, its potential biological effects on non-small cell lung cancer (NSCLC) remain unknown. Therefore, the present study aims to address this issue. NSCLC and normal lung epithelial cell lines were first treated with SchA. Cell viability and proliferation were measured using CellTiter-Glo Assay and colony formation assays, respectively. PI staining was used to measure cell cycle distribution. Cell cycle-related proteins p53, p21, cyclin D1, CDK4, CDK6, cyclin E1, cyclin E2, CDK2 and DNA damage-related protein SOX4 were detected by western blot analysis. Annexin V-FITC/PI staining, DNA electrophoresis and Hoechst 33342/PI dual staining were used to detect apoptosis. JC-1 and DCFH-DA fluorescent dyes were used to measure the mitochondrial membrane potential and reactive oxygen species concentrations, respectively. Apoptosis-related proteins caspase-3, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP), cleaved PARP, BimEL, BimL, BimS, Bcl2, Bax, caspase-9 and cleaved caspas-9 were measured by western blot analysis. Dansylcadaverine was used to detect the presence of the acidic lysosomal vesicles. The expression levels of the autophagy-related proteins LC3-I/II, p62/SQSTM and AMPKα activation were measured using western blot analysis. In addition, the autophagy inhibitor 3-methyladenine was used to inhibit autophagy. SchA treatment was found to reduce NSCLC cell viability whilst inhibiting cell proliferation. Low concentrations of SchA (10-20 µM) mainly induced G1/S-phase cell cycle arrest. By contrast, as the concentration of SchA used increases (20-50 µM), cells underwent apoptosis and G2/M-phase cell cycle arrest. As the treatment concentration of SchA increased from 0 to 50 µM, the expression of p53 and SOX4 protein also concomitantly increased, but the expression of p21 protein was increased by 10 µM SchA and decreased by higher concentrations (20-50 µM). In addition, the mRNA and protein expression levels of Bcl-like 11 (Bim) EL, BimL and BimS increased following SchA application. SchA induced the accumulation of acidic vesicles and induced a marked increase in the expression of LC3-II protein, suggsting that SchA activated the autophagy pathway. However, the expression of the p62 protein was found to be increased by SchA, suggesting that p62 was not degraded during the autophagic flux. The 3-methyladenine exerted no notable effects on SchA-induced apoptosis. Taken together, results from the present study suggest that SchA exerted inhibitory effects on NSCLC physiology by inducing cell cycle arrest and apoptosis. In addition, SchA partially induced autophagy, which did not result in any cytoprotective effects.

1095 The Utility of Plasma Circulating Cell-Free Messenger RNA as A Biomarker of Glioma: A Pilot Study

Abstract Introduction Research into the potential utility of plasma-derived circulating-cell-free nucleic acids as non-invasive adjuncts to radiological imaging has been occasioned by the invasive nature of brain tumour biopsy. Circulating-cell-free messenger RNAs are short fragments of RNA present in blood. The objective of this study was to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and TCGA glioma derived RNA. Method Blood samples were collected from twenty-nine patients prior to tumour resection. Plasma-ccfmRNA and glioma derived RNA were extracted and profiled. Results BCL2L1, CXCL5, GZMB, HLA-A, HLA-C, IRF1, MYD88, TGFB1, TLR2, and TP53 genes were significantly over-expressed in glioma (high-grade-glioma-HGG and low-grade-glioma-LGG) patients (p &amp;lt; 0.05, versus control). BCL2L1, GZMB and HLA-A genes were significantly over-expressed in HGG patients (p &amp;lt; 0.05, versus LGG patients). There was positive correlation between the magnitude of fold change of differentially expressed genes in plasma and glioma derived RNA (Spearman r = 0.6344, n = 14, p = 0.017), and with the mean FPKM of TCGA glioma derived RNA samples (Spearman r = 0.4614, n = 19, p = 0.047). There was positive correlation between glioma radiographic tumour burden and the magnitude of fold change of CSF3 gene (r = 0.9813, n = 20, p &amp;lt; 0.001). Conclusions We identified significant differential expression of genes involved in cancer inflammation and immunity among patients with different glioma grades, and we identified positive correlation between the plasma transcriptomic profile and tumour samples, and with TCGA glioma derived RNA.

OAB-018: A BCL2L1 armoured BCMA-targeting CAR T cells to overcome exhaustion and enhance persistence in multiple myeloma

<h3>Background</h3> Chimeric antigen receptor (CAR) T cells targeting BCMA have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. <h3>Methods</h3> Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with anti BCMA CAR T cells. <h3>Results</h3> CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BclxL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD). <h3>Conclusions</h3> Cognizant of the role BclxL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BclxL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BclxL armored CAR (BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and in vivo studies. While BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCL2L1_CAR viability and cytotoxic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions, where CAR T cells were stimulated every 2 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCL2L1_CAR and BCMA_CAR with respect to the composition of Tem cells (CCR7–CD45RO+CD45RA–) or Tcm cells (CCR7+CD45RO+CD45RA–). However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation and anti-MM cytotoxic activities of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Therefore BCL2L1 blockade of AICD not only enhanced the viability and cytotoxicity of CAR-T cells but surprisingly also reduced their functional exhaustion. Our findings provide a novel approach for CAR-T optimization and overcoming relapse resulting from lack of persistence.

TAZ is indispensable for c-MYC-induced hepatocarcinogenesis

Mounting evidence implicates the Hippo downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in hepatocellular carcinoma (HCC). We investigated the functional contribution of YAP and/or TAZ to c-MYC-induced liver tumor development. The requirement for YAP and/or TAZ in c-Myc-driven hepatocarcinogenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout (KO) mice. An hepatocyte-specific inducible TTR-CreERT2 KO system was applied to evaluate the role of YAP and TAZ during tumor progression. Expression patterns of YAP, TAZ, c-MYC, and BCL2L12 were analyzed in human HCC samples. We found that the Hippo cascade is inactivated in c-Myc-induced mouse HCC. Intriguingly, TAZ mRNA levels and activation status correlated with c-MYC activity in human and mouse HCC, but YAP mRNA levels did not. We demonstrated that TAZ is a direct transcriptional target of c-MYC. In c-Myc induced murine HCCs, ablation of Taz, but not Yap, completely prevented tumor development. Mechanistically, TAZ was required to avoid c-Myc-induced hepatocyte apoptosis during tumor initiation. The anti-apoptotic BCL2L12 gene was identified as a novel target regulated specifically by YAP/TAZ, whose silencing strongly suppressed c-Myc-driven murine hepatocarcinogenesis. In c-Myc murine HCC lesions, conditional knockout of Taz, but not Yap, led to tumor regression, supporting the requirement of TAZ for c-Myc-driven HCC progression. TAZ is a pivotal player at the crossroad between the c-MYC and Hippo pathways in HCC. Targeting TAZ might be beneficial for the treatment of patients with HCC and c-MYC activation. The identification of novel treatment targets and approaches for patients with hepatocellular carcinoma is crucial to improve survival outcomes. We identified TAZ as a transcriptional target of c-MYC which plays a critical role in c-MYC-dependent hepatocarcinogenesis. TAZ could potentially be targeted for the treatment of patients with c-MYC-driven hepatocellular carcinoma.

O51 The utility of plasma circulating cell-free messenger RNA as a biomarker of glioma: a pilot study

Abstract Introduction Research into the potential utility of plasma-derived circulating cell-free nucleic acids as non-invasive adjuncts to radiological imaging has been occasioned by the invasive nature of brain tumour biopsy. Circulating-cell-free messenger RNAs are short fragments of RNA present in blood. The objective of this study was to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and TCGA glioma derived RNA. Method Blood samples were collected from twenty-nine patients prior to tumour resection. Plasma ccfmRNA and glioma derived RNA were extracted and profiled. Result BCL2L1, CXCL5, GZMB, HLA-A, HLA-C, IRF1, MYD88, TGFB1, TLR2, and TP53 genes were significantly over-expressed in glioma (high-grade-glioma-HGG and low-grade-glioma-LGG) patients (P &amp;lt; 0.05, versus control). BCL2L1, GZMB and HLA-A genes were significantly over-expressed in HGG patients (P &amp;lt; 0.05, versus LGG patients). There was positive correlation between the magnitude of fold change of differentially expressed genes in plasma and glioma derived RNA (Spearman r = 0.6344, n = 14, P = 0.017), and with the mean FPKM of TCGA glioma derived RNA samples (Spearman r = 0.4614, n = 19, P = 0.047). There was positive correlation between glioma radiographic tumour burden and the magnitude of fold change of CSF3 gene (r = 0.9813, n = 20, P &amp;lt; 0.001). Conclusion We identified significant differential expression of genes involved in cancer inflammation and immunity among patients with different glioma grades, and we identified positive correlation between the plasma transcriptomic profile and tumour samples, and with TCGA glioma derived RNA. Take-home Message The plasma transcriptomic profile of glioma patients appears to be representative of synchronously obtained glioma samples.

FOXM1-AKT Positive Regulation Loop Provides Venetoclax Resistance in AML.

Forkhead box protein M1 (FOXM1) is a crucial regulator of cancer development and chemoresistance. It is often overexpressed in acute myeloid leukemia (AML) and is associated with poor survival and reduced efficacy of cytarabine therapy. Molecular mechanisms underlying high FOXM1 expression levels in malignant cells are still unclear. Here we demonstrate that AKT and FOXM1 constitute a positive autoregulatory loop in AML cells that sustains high activity of both pro-oncogenic regulators. Inactivation of either AKT or FOXM1 signaling results in disruption of whole loop, coordinated suppression of FOXM1 or AKT, respectively, and similar transcriptomic changes. AML cells with inhibited AKT activity or stable FOXM1 knockdown display increase in HOXA genes expression and BCL2L1 suppression that are associated with prominent sensitization to treatment with Bcl-2 inhibitor venetoclax. Taken together, our data indicate that AKT and FOXM1 in AML cells should not be evaluated as single independent regulators but as two parts of a common FOXM1-AKT positive feedback circuit. We also report for the first time that FOXM1 inactivation can overcome AML venetoclax resistance. Thus, targeting FOXM1-AKT loop may open new possibilities in overcoming AML drug resistance and improving outcomes for AML patients.

LncRNA TUG1 regulates human pulmonary microvascular endothelial cell apoptosis via sponging of the miR-9a-5p/BCL2L11 axis in chronic obstructive pulmonary disease.

The aim of the present study was to investigate the function of long non-coding RNA taurine-upregulated gene 1 (lncRNA TUG1) in chronic obstructive pulmonary disease and further assess the underlying molecular mechanisms. Flow cytometry analysis was performed to detect cell apoptosis of human pulmonary microvascular endothelial cells (HPMECs) treated with 1% cigarette smoke extract (CSE). The activity of caspase-3 was measured using a Caspase-3 Activity assay kit and the protein expression of cleaved caspase-3, caspase-3 and Bcl-2 like 11 (BCL2L11) were measured using western blotting. Reverse transcription-quantitative PCR (RT-qPCR) was performed to measure the expression of TUG1 mRNA levels in the treated cells. The association between TUG1, the miR-9a-5p/BCL2L11 axis and with miR-9a-5p were predicted and verified using a dual luciferase reporter assay system. The mRNA expression of miR-9a-5p and BCL2L11, and the transfection efficiency were measured by RT-qPCR. The results showed that CSE induced cell apoptosis and increased lncRNA TUG1 expression in HPMECs. CSE significantly reduced the expression of miR-9a-5p in HPMECs compared with the control group. TUG1-short hairpin RNA relieved cell apoptosis induced by CSE by upregulating miR-9a-5p in HPMECs. The present study predicted and verified that BCL2L11 is a direct target of miR-9a-5p. The mRNA expression of BCL2L11 was increased in HPMECs following CSE treatment compared with the control group. miR-9a-5p mimic and BCL2L11-plasmid markedly increased the expression of miR-9a-5p and BCL2L11, respectively. miR-9a-5p mimic reversed the increase in cell apoptosis induced by CSE by inhibiting BCL2L11 expression in HPMECs. To conclude, the present study demonstrated that lncRNA TUG1 exerted roles in cell apoptosis induced by CSE through modulating the miR-9a-5p/BCL2L11 axis.

A Three-Genes Signature of Anti-Apoptotic Genes Showed Different Mechanisms in Preventing Apoptosis in Acute Myeloid Leukemia Cells and Could Provide an Useful Model to Establish the Proper BH3-Mimetich Drug or Combination

BACKGROUNDSeveral biological mechanisms sustain leukemic development, but the inhibition of apoptotic death, which should be induced by the genetic instability of the disease, is surely one the key neoplastic alterations. Apoptotic regulation involves complex pathways of positive and negative regulators which may and do represent a target for personalized therapy. Furthermore, the overexpression of specific anti-apoptotic genes likely represent a useful prognostic instrument. Nowadays, BCL-2 inhibitors have successfully passed preliminary phases of clinical experimentation, however the involvement of other essential genes, such as MCL-1 and BCL2L1 (coding for bcl-xL), has been proposed as resistance mechanism to BCL-2 inhibitors and as novel therapeutic target in Acute Myeloid Leukemia (AML).Our study aims to investigate the expression of anti-apoptotic genes in AML patients to cluster them in different categories and study their impact on prognosis, in order to reveal possible mechanism of resistance to BCL-2 inhibitors and stratify patients that are more likely to take advantage of administration or co-administration of one between MCL-1 and BCL-2/BCL2L2 inhibitors. METHODSWe performed Human Transcriptome Array 2.0 (Affymetrix) in a cohort of 59 new diagnosed AML. Gene Expression Profiling (GEP) analysis has been conducted with Transcriptome Array V3.0 Software (Affymetrix). K-means clustering has been used to categorize different cluster of patients basing on BCL-2, MCL-1 and BCL2L1 gene expression. A pool of 7 healthy donor was used to normalize expression data. Survival analysis was conducted with Kaplan-Meyer method and differences in survival were assessed with Log-Rank test. RESULTSAnalyzing BCL-2, MCL-1 and BCL2L1 gene expression values in 59 AML patients at diagnosis we established 5 categories where patients are almost uniformly assigned (Figure 1a): 11/59 patients in Cluster 1, characterized by the only one overexpression of MCL1; 27/59 patients in Cluster 2, which presents low expression of all analyzed genes; 2/59 patients in Cluster 3, where BCL2L1 is strongly overexpressed; 6/59 patients in Cluster 4, distinguished by BCL-2 overexpression and 13/59 patients in Cluster 5, which high values of expression of all 3 genes. Thus, Clusters 2 and 5, where no gene is overexpressed in comparison with the others, include the majority of patients (40/59, approximately 68%), suggesting that apoptotic death control is realized thanks to an intricate debate between these 3 considered proteins and BCL-2 should not be considered the unique oncogenic anti-apoptotic mechanism. Furthermore, the 3-D Scatter Plot (Figure 1A) allows us to observe how the patients out of the central cloud (Clusters 2-5) always present one of the three anti-apoptotic genes overexpressed, and when BCL-2 level is progressively low (Cluster 1-2-3), MCL-1 and BCL2L1 values are high, respectively. Therefore, this documented behavior may indicate the presence of many ways used by leukemic cell to prevent apoptosis, with one of them almost always activated.In term of Overall Survival (OS), unsurprisingly no statistically significant difference arised between Cluster 1-3-4 where one different anti-apoptotic gene is overexpressed (Figure 1b), underling the probable equal effect of each mechanism in inhibiting apoptotic death and in sustaining leukemic progression. Finally, analyzing the impact only of BCL-2 expression on OS in a cohort of 36 young patients treated with chemotherapy it is possible to observe a trend close to statistically significance (p=0.07)(Figure 1c), unreached probably because of the relative limitation of the sample, confirming the BCL-2 induced chemoresistance. CONCLUSIONSApoptotic death regulation is precisely controlled equally by BCL-2, MCL-1 and BCL2L2, their expression is quite balanced in AML patients evaluated. One out of these three genes is almost always overexpressed, reaffirming the key role of anti-apoptotic oncogenes in leukemogenesis. Even though BCL-2 expression influence patients prognosis, MCL-1 and BCL2L2 overexpression has been documented when BCL-2 is normally or underexpressed. In conclusion, BCL-2 inhibitors resistance may be caused by MCL-1 and BCL2L2 overexpression detected in patients BCL-2 independents. Moreover, a synthetic lethality approach should be realized to switch-off leukemic anti-apoptotic multiple mechanisms. [Display omitted] DisclosuresMartinelli:Celgene: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy; Roche: Consultancy.

Alpha lipoic acid priming enhances the hepatoprotective effect of adipose derived stem cells in CCl4 induced hepatic injury in-vitro

Mesenchymal stem cells are known to support hepatic defense against liver fibrosis. However, the fibrosis induced oxidative microenvironment affects the proliferative, regenerative, and angiogenic properties of mesenchymal stem cells. Alpha lipoic acid (ALA) is a strong anti-oxidant which has been shown to ameliorate the adverse effects of fibrosis that otherwise can lead to severe liver problems like cirrhosis and liver failure. Here, we studied the protective role of ALA primed adipose derived stem cells (ADSCs) against carbon tetrachloride (CCl4) induced hepatotoxicity in primary hepatocytes in-vitro. Priming of ADSCs helped to abrogate the damaging effects of fibrosis induced oxidative stress as evidenced by significantly reduced levels of alkaline phosphatase (ALP), Alanine Aminotransferase (ALAT) along with decreased lactate dehydrogenase (LDH) release and improved superoxide dismutase (SOD) activity. ALA and ADSCs synergistically down-regulated the expression of Bax gene, an apoptosis regulator while enhancing cell proliferation by up-regulating the expression of Bcl2l1 gene. This treatment improved the expression of albumin (Alb), cytokeratin-8 (Ck8), and hepatic nuclear factor alpha (Hnf4α). Cytochrome P450 2E1 (Cyp2e1) and Alpha fetoprotein (Afp) were down-regulated to lessen the damage caused by CCl4 treatment. Furthermore, paracrine release of several growth factors like hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and insulin growth factor (IGF) reinforced the improved response of primary hepatocytes against CCl4 induced hepatotoxicity in the presence of ALA primed ADSCs. This study suggests that ALA priming may improve the therapeutic potential of ADSCs against chronic liver problems by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant factors heme oxygenase 1 (HO-1) and quinone acceptor oxidoreductase-1 (NQO1).

Plasma circulating cell free messenger RNA as a potential biomarker of melanoma

Background Blood borne cell free nucleic acids are increasingly emerging as significant non-invasive adjuncts to current methods of disease status evaluation in cancer patients. In this study, we sought to examine whether significant differences exist in the plasma transcriptomic profile of advanced melanoma patients with a high disease burden compared to patients with a low disease burden or therapeutic response. Methods Pathway focussed gene expression analysis was performed using cDNA derived from the plasma circulating cell free messenger ribonucleic acid (ccfmRNA) samples of twenty-two patients with advanced melanoma. Patients were assessed with paired blood sample collection and CT scan assessments at baseline and at 3 months follow up. Results We identified several genes which were significantly over-expressed in patients with a low disease burden or therapeutic response; BCL2L1, CXCL9, IDO1, IL13, MIF, MYD88 and TLR4 (p ≤ 0.001, versus high disease burden). There was an increase in the magnitude of fold change (2^ (-dd CT)) of BCL2L1 (p = 0.031), CCL4 (p = 0.001), CCL5 (p = 0.043), CXCL9 (p = 0.012), GZMB (p = 0.023) and TNFSF10 (p = 0.039) genes in patients with therapeutic response at 3 months follow up assessment relative to baseline assessment. Moreover, in stage IV melanoma patients with brain metastases, CCL18, CCR1, CCR4, CD274, CSF2, EGF, and PTGS2 genes were significantly over-expressed (p < 0.001, versus patients without melanoma brain metastasis). Conclusion Significant differences were observed in the plasma transcriptomic profile between the various melanoma patient groups, and we postulate that these differences may be exploited to identify novel therapeutic targets or biomarkers relevant to melanoma.

Gene Expression Alterations Associated with Sanguinarine‐Induced Antiproliferative effects and Apoptosis in Triple‐Negative Breast Cancer Cells

Sanguinarine (SAN) is a plant alkaloid found in various plants, mainly Sanguinaria canadensis. SAN has various pharmacological properties, including anti-bacterial, anti-fungal, anti-inflammatory, and anti-cancer effects. The current study investigated the mechanisms that mediated the anti-cancer effects of SAN in two triple-negative breast cancer (TNBC) cell lines; MDA-MB-231 and MDA-MB-468 cells. The data obtained suggested SAN is a potent compound in reducing cell viability and proliferation. Moreover, the results show that MDA-MB-468 cells were more sensitive than MDA-MB-231 cells to SAN. Also, SAN induced apoptosis in both cell lines by arresting cell growth. The compound arrested MDA-MB-231 cells at the Sub-G1 phase and MDA-MB-468 cells at the G2/M phase. Gene expression profiling indicated that MDA-MB-468 cells had significant transcription activation (2-4-fold) in eighteen genes. The genes activated include two members of the caspase family (caspase 1 and caspase 10), seven members of the TNF receptor superfamily (TNFRSF) such as TNFRSF10A, TNFRSF10B, TNFRSF11B, TNFRSF21, and TNFRSF25), in addition to TNFRSF1A Associated Via Death Domain (TRADD) and Fas-associated via death domain (FADD). On the other hand, in MDA-MB-231 cells, SAN was found to alter twelve five genes. The upregulated genes include Lymphotoxin Alpha (LTA; 15-fold), growth arrest and DNA damage-inducible 45 alpha (GADD45A), BCL2 Related Protein A1 (BCL2A1), BCL2-Like 11 (BCL2L11), and Bcl-2-Binding Protein Bis (BAG3). These results provide evidence that genetically different cells may respond differently to treatments, explaining the inadequate therapeutic response of some patients with advanced TNBC. The data also suggest that this plant alkaloid SAN could have therapeutic potential for TNBC patients.

Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation.

Simple SummaryOocyte-secreted factors play an essential role in oogenesis and fertility through bidirectional crosstalk between oocytes and somatic cells. Interleukin-7, known as an oocyte-secreted factor, has recently been shown to improve oocyte developmental competence through interaction with cumulus cells around the oocytes. This study aimed to investigate the effects of interleukin-7 on porcine cumulus-oocyte complexes during in vitro maturation. Our results showed that supplementation with interleukin-7 during in vitro maturation exerted beneficial effects on porcine oocyte meiotic maturation by upregulating antioxidant-related genes and enhanced the subsequent developmental potential of porcine embryos after parthenogenetic activation.Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

Single-Nucleotide Variants in microRNAs Sequences or in their Target Genes Might Influence the Risk of Epilepsy: A Review.

Single-nucleotide variant (SNV) is a single base mutation at a specific location in the genome and may play an import role in epilepsy pathophysiology. The aim of this study was to review case-control studies that have investigated the relationship between SNVs within microRNAs (miRs) sequences or in their target genes and epilepsy susceptibility from January 1, 2010 to October 31, 2020. Nine case-control studies were included in the present review. The mainly observed SNVs associated with drug-resistant epilepsy (DRE) risk were SNVs n.60G > C (rs2910164) and n.-411A > G (rs57095329), both located at miR-146a mature sequence and promoter region, respectively. In addition, the CC haplotype (rs987195-rs969885) and the AA genotype at rs4817027 in the MIR155HG/miR-155 tagSNV were also genetic susceptibility markers for early-onset epilepsy. MiR-146a has been observed as upregulated in human astrocytes in epileptogenesis and it regulates inflammatory process through NF-κB signaling by targeting tumor necrosis factor-associated factor 6 (TRAF6) gene. The SNVs rs2910164 and rs57095329 may modify the expression level of mature miR-146a and the risk for epilepsy and SNVs located at rs987195-rs969885 haplotype and at rs4817027 in the MIR155HG/miR-155 tagSNV could interfere in the miR-155 expression modulating inflammatory pathway genes involved in the development of early-onset epilepsy. In addition, SNVs rs662702, rs3208684, and rs35163679 at 3'untranslated region impairs the ability of miR-328, let-7b, and miR-200c binding affinity withpaired box protein PAX-6 (PAX6), BCL2 like 1 (BCL2L1), and DNA methyltransferase 3 alpha (DNMT3A) target genes. The SNV rs57095329 might be correlated with DRE when a larger number of patients are evaluated. Thus, we concluded that the main drawback of most of studies is the small number of individuals enrolled, which lacks sample power.

Impact of Genetic Polymorphism of Myeloid Differentiation Primary Response Gene 88, Enhancer of Zeste Homolog 2, and B-cell Lymphoma 2 like 11 in Patients with Diffuse Large B Cell Lymphoma Treated with Rituximab, Cyclophosphamide, Doxorubicin, Vincristin

BACKGROUND: Despite the growing landscape of genetic drivers in Diffuse Large B-cell Lymphoma, yet their clinical implication is still unclear and R-CHOP regimen remains a “one size fits all” therapy. We aimed in this study to examine the prevalence of EZH2, BCL211 and MYD 88 genetic polymorphisms in DLBCL patients and correlate the results with various clinical and survival outcomes.&#x0D; METHODS: Genotyping of MYD88 (rs387907272 T/C), EZH2 (rs3757441 C/T), and BCL2L11 (rs3789068 A/G) polymorphisms were conducted using real time polymerase chain reaction analysis in a total of 75 DLBCL patients.&#x0D; RESULTS: Most of our cases carried the wild TT genotype of MYD88 gene (64%), the mutant TT genotype of EZH2 gene (52%) and the wild AA genotype of BCL2L11 gene (48%). Regarding cell of origin, Germinal Centre (GC) phenotype was present in 56% of cases while 44% expressed the Post-GC (PGC) phenotype. Poor response outcome to first line R-CHOP was significantly correlated with the mutated CC genotype of MYD 88 (p=0.02), while better response to R-CHOP was significantly associated with younger age &lt;50 years (p &lt;0.0001), good PS (p=0.046), normal LDH level (p=0.003), earlier stage (p &lt;0.0001), good IPI score (p=0.009), absence of extranodal disease (p &lt;0.0001) and absence of bulky disease (p=0.004). The median PFS and the 2 year OS were significantly higher in younger age, earlier stage, good IPI score, absence of extranodal disease, absence of bulky disease and in GC phenotype.&#x0D; CONCLUSIONS: Our results emphasized that the mutated genotype of MYD 88 gene polymorphism is significantly associated with poor response to R-CHOP therapy.

Identification of Crucial Genes and Pathways Associated with Atherosclerotic Plaque in Diabetic Patients.

BackgroundPatients with diabetes have more calcification in atherosclerotic plaque and a higher occurrence of secondary cardiovascular events than patients without diabetes. The objective of this study was to identify crucial genes involved in the development of diabetic atherosclerotic plaque using a bioinformatics approach.MethodsMicroarray dataset GSE118481 was downloaded from the Gene Expression Omnibus (GEO) database; the dataset included 6 patients with diabetic atherosclerotic plaque (DBT) and 6 nondiabetic patients with atherosclerotic plaque (Ctrl). Differentially expressed genes (DEG) between the DBT and Ctrl groups were identified and then subjected to functional enrichment analysis. Based on the enriched pathways of DEGs, diabetic atherosclerotic plaque-related pathways were screened using the comparative toxicogenomics database (CTD). We then constructed a protein–protein interaction (PPI) network and transcription factor (TF)–miRNA–mRNA network.ResultsA total of 243 DEGs were obtained in the DBT group compared with the Ctrl group, including 85 up-regulated and 158 down-regulated DEGs. Functional enrichment analysis showed that up-regulated DEGs were mainly enriched in isoprenoid metabolic process, DNA-binding TF activity, and response to virus. Additionally, DEGs participating in the toll-like receptor signaling pathway were closely related to diabetes, carotid stenosis, and insulin resistance. The TF–miRNA–mRNA network showed that toll-like receptor 4 (TLR4), BCL2-like 11 (BCL2L11), and glutamate-cysteine ligase catalytic subunit (GCLC) were hub genes. Furthermore, TLR4 was regulated by TF signal transducer and activator of transcription 6 (STAT6); BCL2L11 was targeted by hsa-miR-24-3p; and GCLC was regulated by nuclear factor, erythroid 2 like 2 (NFE2L2).ConclusionIdentification of hub genes and pathways increased our understanding of the molecular mechanisms underlying the atherosclerotic plaque in patients with or without diabetes. These crucial genes (TLR4, BC2L11, and GCLC) might function as molecular biomarkers for diabetic atherosclerotic plaque.

Human Cytomegalovirus UL7, miR-US5-1, and miR-UL112-3p Inactivation of FOXO3a Protects CD34+ Hematopoietic Progenitor Cells from Apoptosis.

Human cytomegalovirus (HCMV) infection of myeloid lineage cells, such as CD34+ hematopoietic progenitor cells (HPCs) or monocytes, results in the upregulation of antiapoptotic cellular proteins that protect the newly infected cells from programmed cell death. The mechanisms used by HCMV to regulate proapoptotic cellular proteins upon infection of CD34+ HPCs have not been fully explored. Here, we show that HCMV utilizes pUL7, a secreted protein that signals through the FLT3 receptor, and miR-US5-1 and miR-UL112-3p to reduce the abundance and activity of the proapoptotic transcription factor FOXO3a at early times after infection of CD34+ HPCs. Regulation of FOXO3a by pUL7, miR-US5-1, and miR-UL112 results in reduced expression of the proapoptotic BCL2L11 transcript and protection of CD34+ HPCs from virus-induced apoptosis. These data highlight the importance of both viral proteins and microRNAs (miRNAs) in protecting CD34+ HPCs from apoptosis at early times postinfection, allowing for the establishment of latency and maintenance of viral genome-containing cells.IMPORTANCE Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals and is a significant problem during transplantation. The virus can establish a latent infection in CD34+ hematopoietic progenitor cells (HPCs) and periodically reactivate to cause disease in the absence of an intact immune system. What viral gene products are required for successful establishment of latency is still not fully understood. Here, we show that both a viral protein and viral miRNAs are required to prevent apoptosis after infection of CD34+ HPCs. HCMV pUL7 and miRNAs miR-US5-1 and miR-UL112-3p act to limit the expression and activation of the transcription factor FOXO3a, which in turn reduces expression of proapoptotic gene BCL2L11 and prevents virus-induced apoptosis in CD34+ HPCs.

Stingless Bee Propolis: New Insights for Anticancer Drugs.

Natural products are important sources of biomolecules possessing antitumor activity and can be used as anticancer drug prototypes. The rich biodiversity of tropical and subtropical regions of the world provides considerable bioprospecting potential, including the potential of propolis produced by stingless bee species. Investigations of the potential of these products are extremely important, not only for providing a scientific basis for their use as adjuvants for existing drug therapies but also as a source of new and potent anticancer drugs. In this context, this article organizes the main studies describing the anticancer potential of propolis from different species of stingless bees with an emphasis on the chemical compounds, mechanisms of action, and cell death profiles. These mechanisms include apoptotic events; modulation of BAX, BAD, BCL2-L1 (BCL-2 like 1), and BCL-2; depolarization of the mitochondrial membrane; increased caspase-3 activity; poly (ADP-ribose) polymerase (PARP) cleavage; and cell death induction by necroptosis via receptor interacting protein kinase 1 (RIPK1) activation. Additionally, the correlation between compounds with antioxidant and anti-inflammatory potential is demonstrated that help in the prevention of cancer development. In summary, we highlight the important antitumor potential of propolis from stingless bees, but further preclinical and clinical trials are needed to explore the selectivity, efficacy, and safety of propolis.