Discovery and mechanistic study of Imperatorin that inhibits HBsAg expression and cccDNA transcription.

Chronic hepatitis B virus (HBV) infection remains a significant global health challenge due to its link to severe conditions like HBV-related cirrhosis and hepatocellular carcinoma (HCC). Although current treatments effectively reduce viral levels, they have limited impact on certain HBV elements, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA). This highlights the urgent need for innovative pharmaceutical and biological interventions that can disrupt HBsAg production originating from cccDNA. In this study, we identified a natural furanocoumarin compound, Imperatorin, which markedly inhibited the expression of HBsAg from cccDNA, by screening a library of natural compounds derived from Chinese herbal medicines using ELISA assay and qRT-PCR. The pharmacodynamics study of Imperatorin was explored on HBV infected HepG2-NTCP/PHHs and HBV-infected humanized mouse model. Proteome analysis was performed on HBV infected HepG2-NTCP cells following Imperatorin treatment. Molecular docking and bio-layer interferometry (BLI) were used for finding the target of Imperatorin. Our findings demonstrated Imperatorin remarkably reduced the level of HBsAg, HBV RNAs, HBV DNA and transcriptional activity of cccDNA both in vitro and in vivo. Additionally, Imperatorin effectively restrained the actions of HBV promoters responsible for cccDNA transcription. Mechanistic study revealed that Imperatorin directly binds to ERK and subsequently interfering with the activation of CAMP response element-binding protein (CREB), a crucial transcriptional factor for HBV and has been demonstrated to bind to the PreS2/S and X promoter regions of HBV. Importantly, the absence of ERK could nullify the antiviral impact triggered by Imperatorin. Collectively, the natural compound Imperatorin may be an effective candidate agent for inhibiting HBsAg production and cccDNA transcription by impeding the activities of HBV promoters through ERK-CREB axis.

Deubiquitinase BAP1 regulates stability of BRCA1 protein and inactivates the NF-κB signaling to protect mice from sepsis-induced acute kidney injury.

Sepsis and its associated organ dysfunction syndrome is a leading cause of death in critically ill patients. Breast cancer susceptibility protein 1 (BRCA1)-associated protein 1 (BAP1) is a potential regulator in immune regulation and inflammatory responses. This study aims to investigate the function of BAP1 in sepsis-induced acute kidney injury (AKI). A mouse model with sepsis-induced AKI was induced by cecal ligation and puncture, and renal tubular epithelial cells (RTECs) were treated with lipopolysaccharide (LPS) to mimic an AKI condition in vitro. BAP1 was significantly poorly expressed in the kidney tissues of model mice and the LPS-treated RTECs. Artificial upregulation of BAP1 ameliorated the pathological changes, tissue injury and inflammatory responses in kidney tissues of the mice, and it reduced the LPS-induced injury and apoptosis of the RTECs. BAP1 was found to interact with BRCA1 and enhance stability of BRCA1 protein through deubiquitination modification. Further downregulation of BRCA1 activated the nuclear factor-kappa B (NF-κB) signaling pathway and blocked the protective roles of BAP1 in sepsis-induced AKI. In conclusion, this study demonstrates that BAP1 protects mice from sepsis-induced AKI through enhancing stability of BRCA1 protein and inactivating the NF-κB signaling.

Mechanism of lnRNA-ICL involved in lung cancer development in COPD patients through modulating microRNA-19-3p/NKRF/NF-κB axis.

The incidence of lung cancer (LC) in chronic obstructive pulmonary disease (COPD) patients is dozens of times higher than that in patients without COPD. Elevated activity of nuclear factor-k-gene binding (NF-κB) was found in lung tissue of patients with COPD, and the continuous activation of NF-κB is observed in both malignant transformation and tumor progression of LC, suggesting that NF-κB and its regulators may play a key role in the progression of LC in COPD patients. Here, we report for the first time that a key long non-coding RNA (lncRNA)-ICL involved in the regulation of NF-κB activity in LC tissues of COPD patients. The analyses showed that the expression of ICL significantly decreased in LC tissues of LC patients with COPD than that in LC tissues of LC patients without COPD. Functional experiments in vitro showed that exogenous ICL only significantly inhibited the proliferation, invasion and migration in primary tumor cells of LC patients with COPD compared to LC patients without COPD. Mechanism studies have shown that ICL could suppress the activation of NF-κB by blocking the hsa-miR19-3p/NKRF/NF-κB pathway as a microRNA sponge. Furthermore, In vivo experiments showed that exogenous ICL effectively inhibited the growth of patient-derived subcutaneous tumor xenografts (PDX) of LC patients with COPD and significantly prolonged the survival time of tumor-bearing mice. In a word, our study shows that the decrease of ICL is associated with an increased risk of LC in patients with COPD, ICL is not only expected to be a new therapeutic target for LC in COPD patients, but also has great potential to be used as a new marker for evaluating the occurrence, severity stratification and prognosis of LC in patients with COPD.

Long non-coding RNA CHRF accelerates LPS-induced acute lung injury through microRNA-146a/Notch1 axis.

BACKGROUND: The present study sought to investigate the regulatory role of the long non-coding RNA (lncRNA) cardiac hypertrophy-related factor (CHRF) in a mouse model of acute lung injury (ALI) and in primary mouse pulmonary microvascular endothelial cells (MPVECs) treated with lipopolysaccharide (LPS). METHODS: C57BL/6 mice were given adenovirus (Ad) sh-CHRF or negative control (NC) before undergoing cecal ligation and perforation. MPVECs transfected with Adsh-CHRF or NC were treated with LPS. Double luciferase assay was used to detect the binding of miR-146a to CHRF or Notch1. Subsequently, MPVECs were co-transfected with miR-146a inhibitor and sh-CHRF for 24 hours, and then treated with LPS. RESULTS: High expression of CHRF was detected in septic mice. Cecal ligation and perforation induced ALI and apoptosis in mice, whereas, CHRF knockout could inhibit ALI. The protein expression levels of TNF-α, IL-1ß and IL-6 in the lung and bronchoalveolar lavage fluid of the CLP group were up-regulated, whereas the expression of IL-4 and IL-10 was down-regulated. CHRF inhibition reduced the production of proinflammatory cytokines in septic mice. The inhibitory effect of CHRF gene knockdown on lung inflammation and apoptosis was confirmed in the septic cell model. Mechanistic investigation showed that CHRF up-regulated the level of Notch1 by sponging miR-146a. Additionally, the low expression of miR-146a reversed the inhibitory effect of CHRF gene knockout on LPS-induced inflammatory response and apoptosis. Together, in vivo and in vitro results demonstrated that CHRF enhanced sepsis-induced ALI by targeting miR-146a and up-regulating Notch1. CONCLUSIONS: CHRF can induce inflammation and apoptosis caused by sepsis by miR-146a/Notch1 axis. Therefore, it may serve as a potential drug target for treating sepsis-induced ALI.

Novel cytosensor for accurate detection of circulating tumor cells based on a dual-recognition strategy and BSA@Ag@Ir metallic-organic nanoclusters.

Herein, based on a dual-recognition strategy and BSA@Ag@Ir metallic-organic nanoclusters (BSA@Ag@Ir MONs), a highly specific and sensitive cytosensor was developed for detecting circulating tumor cells (CTCs). To amplify current signal, novel BSA@Ag@Ir MONs with outstanding catalytic activity and huge specific surface area were synthesized, and conjugated with hairpin DNA strands as signal probes. Orion carbon black 40 (Ocb40)//AuNPs were firstly used to modify electrode to increase its conductivity and surface area. Moreover, the dual recognition strategy based on DNA proximity effect was designed to improve the specificity of cytosensor. When two capture probes respectively bound to two adjacent membrane markers of target cells, the probes could form the associative toehold through the proximity effect to capture the signal probes. Only CTCs simultaneously expressing two membrane markers could be captured and generate current responses. The developed cytosensor could detect CTCs in the range of 3 - 3 × 106 cells mL-1 with a detection limit of 1 cell mL-1. Notably, the cytosensor could accurately identify CTCs even in whole blood. Therefore, this cytosensor has great potential for application in biological science, biomedical engineering and personalized medicine.

Selective leukemia cell death by activation of the double-stranded RNA-dependent protein kinase PKR.

Deregulated activity of the BCR-ABL tyrosine kinase encoded by the Bcr-Abl oncogene represents an important therapeutic target for all the chronic myelogenous leukemia (CML) phases. In this study, we sought to identify targeted PKR activation by Bcr-Abl AS RNA, an anti-sense RNA complementary to the unique mRNA fragments flanking the fusion point of Bcr-Abl, which can be used as an effective anti-leukemia strategy in K562 cells. Moreover, we observed expression of Bcr-Abl AS RNA in K562 cells which resulted in selective apoptosis induction through specific activation of PKR, leading to phosphorylation of eIF2α, global inhibition of protein synthesis, caspase-8 activation and BAX up-regulation. The targeted PKR activation and induced apoptosis were reversed by the PKR inhibitor 2-aminopurine. Taken together, our results indicate that targeted PKR activation led to selective apoptosis induction in K562 cells, which correlated with caspase-8 activity and enhanced expression of BAX.

Targeted blockage of signal transducer and activator of transcription 5 signaling pathway with decoy oligodeoxynucleotides suppresses leukemic K562 cell growth.

The protein signal transducer and activator of transcription 5 (STAT5) of the JAK/STAT pathway is constitutively activated because of its phosphorylation by tyrosine kinase activity of fusion protein BCR-ABL in chronic myelogenous leukemia (CML) cells. This study investigated the potential therapeutic effect of STAT5 decoy oligodeoxynucleotides (ODN) using leukemia K562 cells as a model. Our results showed that transfection of 21-mer-long STAT5 decoy ODN into K562 cells effectively inhibited cell proliferation and induced cell apoptosis. Further, STAT5 decoy ODN downregulated STAT5 targets bcl-xL, cyclinD1, and c-myc at both mRNA and protein levels in a sequence-specific manner. Collectively, these data demonstrate the therapeutic effect of blocking the STAT5 signal pathway by cis-element decoy for cancer characterized by constitutive STAT5 activation. Thus, our study provides support for STAT5 as a potential target downstream of BCR-ABL for CML treatment and helps establish the concept of targeting STAT5 by decoy ODN as a novel therapy approach for imatinib-resistant CML.

[Effect of targeted activation of protein kinase PKR on proliferation of leukemia cell line K562 and its mechanism].

BACKGROUND & OBJECTIVE: The bcr-abl fusion gene induced by reciprocal translocation of t(9; 22)(q34; q11) plays an important role in pathogenesis of chronic myeloid leukemia (CML). Using the strategy of activating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) by the dsRNA formed between the CML-specific bcr/abl fusion gene mRNA and the exogenous recombinant antisense RNA, this study was to investigate the effect of the activated PKR on the proliferation of leukemia cell line K562, and explore its possible mechanisms. METHODS: dsRNA analogue polyriboinosinic polyribocytidylic acid (PolyIC), retroviral vector containing 40 bp of bcr/abl fusion gene sequence (RV-40AS), RV-40AS and 2-aminopurine (2-AP), and retroviral vector containing green fluorescent protein sequence (RV-GFP) were transfected or infected into K562 cells respectively; ECV304 cells were used as control. Cell proliferation was determined by cell counting, MTT assay, and semisolid clone formation experiment. Cell cycle was analyzed by flow cytometry (FCM). The expression of PKR, phosphated PKR (p-PKR), eukaryotic initiation factor-2alpha (eIF2alpha), and phosphated eIF2alpha (p-eIF2alpha) was detected by Western blot. Total protein synthesis was studied by 3H-leucine incorporation. RESULTS: polyIC inhibited the proliferation of K562 cells and ECV304 cells unspecifically, while RV-40AS only inhibited the proliferation of K562 cells specifically. 2-AP blocked the inhibitory effect of RV-40AS on the proliferation of K562 cells. The S phase proportion was significantly lower in polyIC-and RV-40AS-treated K562 cells than in untreated cells [(37.26+/-2.35)% and (31.48+/-3.65)% vs. (58.53+/-5.42)%, P<0.05], while the G0/G1 phase proportion was significantly higher in polyIC-and RV-40AS-treated cells than in untreated cells [(50.97+/-2.18)% and (57.47+/-3.61)% vs. (36.44+/-4.20)%, P<0.05]. The expression of p-PKR and p-eIF2alpha in polyIC-and RV-40AS-treated K562 cells and polyIC-treated ECV304 cells was obviously up-regulated. The total protein synthesis level was significantly lower in RV-40AS-treated K562 cells than in untreated K562 cells [(3.5+/-1.9) cpm/ng vs. (26.8+/-2.6) cpm/ng, P<0.05]. CONCLUSION: Targeted activation of PKR could inhibit the proliferation of K562 cells through inhibiting protein synthesis, and arresting progression of cell cycle.

[Effect and possible mechanism of HnRNP E2 decoy RNA on proliferation of K562 leukemia cells].

BACKGROUND & OBJECTIVE: The abnormal expression of poly(rC)-binding protein E2 (hnRNP E2) induced by BCR/ABL plays an important role in blast crisis of chronic myeloid leukemia (CML). The present study was to investigate the effect of hnRNP E2 decoy RNA on the cell proliferation in K562 leukemia cells, and further elucidate the possible underlying mechanisms. METHODS: Decoy hnRNP E2 plasmid was constructed and transfected into K562 cells using cationic liposome. Stably transfected cells were selected with G418. The cell proliferation rate was determined by cell growth curve using trypan blue staining, and the cell cycle was analyzed by flow cytometry. The changes of CCAAT/enhancer-binding protein alpha (C/EBP alpha) and c-Myc gene expression were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: The proliferation rate of stably transfected K562 cells was inhibited by (62.73+/-12.92)%. The cell cycle was arrested at S phase [stably transfected K562 cell group: (55.59+/-4.67)%, control group: (44.70+/-4.21)%, P<0.05]; C/EBPalpha mRNA level remained unchanged. However the 42 ku-C/EBPalpha protein expression was elevated by (49.72+/-5.58)%; c-Myc mRNA and protein expression was inhibited by (58.27+/-7.23)% and (57.26+/-6.52)%, respectively. CONCLUSION: HnRNP E2 decoy RNA could inhibit the proliferation of K562 cells, and this may be caused by the blockage of the binding between hnRNP E2 and C/EBPalpha mRNA and subsequent elevation of 42 ku-C/EBPalpha by decoy RNA.