BESST: a novel LncRNA knockout strategy with less genome perturbance.

Long noncoding RNAs (lncRNAs) are >200 nt RNA transcripts without protein-coding potential. LncRNAs can be categorized into intergenic, intronic, bidirectional, sense, and antisense lncRNAs based on the genomic localization to nearby protein-coding genes. The current CRISPR-based lncRNA knockout strategy works efficiently for lncRNAs distant from the protein-coding gene, whereas it causes genomic perturbance inevitably due to technical limitations. In this study, we introduce a novel lncRNA knockout strategy, BESST, by deleting the genomic DNA fragment from the branch point to the 3' splicing site in the last intron of the target lncRNA. The BESST knockout exhibited comparable or superior repressive efficiency to RNA silencing or conventional promoter-exon1 deletion. Significantly, the BESST knockout strategy minimized the intervention of adjacent/overlap protein-coding genes by removing an average of ∼130 bp from genomic DNA. Our data also found that the BESST knockout strategy causes lncRNA nuclear retention, resulting in decapping and deadenylation of the lncRNA poly(A) tail. Further study revealed that PABPN1 is essential for the BESST-mediated decay and subsequent poly(A) deadenylation and decapping. Together, the BESST knockout strategy provides a versatile tool for investigating gene function by generating knockout cells or animals with high specificity and efficiency.

The Emerging Role of Autophagy-Associated lncRNAs in the Pathogenesis of Neurodegenerative Diseases.

Neurodegenerative diseases (NDDs) have become a significant global public health problem and a major societal burden. The World Health Organization predicts that NDDs will overtake cancer as the second most common cause of human mortality within 20 years. Thus, it is urgently important to identify pathogenic and diagnostic molecular markers related to neurodegenerative processes. Autophagy is a powerful process for removing aggregate-prone proteins in neurons; defects in autophagy are often associated with the pathogenesis of NDDs. Long non-coding RNAs (lncRNAs) have been suggested as key regulators in neurodevelopment; aberrant regulation of lncRNAs contributes to neurological disorders. In this review, we summarize the recent progress in the study of lncRNAs and autophagy in the context of neurodegenerative disorders, especially Alzheimer's disease (AD) and Parkinson's disease (PD). The information presented here should provide guidance for future in-depth investigations of neurodegenerative processes and related diagnostic molecular markers and treatment targets.

Canagliflozin Attenuates Lipotoxicity in Cardiomyocytes by Inhibiting Inflammation and Ferroptosis through Activating AMPK Pathway.

Diabetic cardiomyopathy (DCM) is a myocardial disease independent of other cardiovascular diseases, such as coronary heart disease, hypertension, etc. Lipotoxicity is closely related to DCM. In this study, we investigated the mechanism of lipid metabolism disturbance in DCM in HL-1 cells. Through bioinformatics and Western blotting analysis, we found that canagliflozin (CAN) significantly inhibited the expression of inflammatory factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Ferroptosis is mediated by lipid peroxidation. We demonstrated the presence of ferroptosis in cardiomyocytes by detecting intracellular Fe2+ content and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), and mitochondrial membrane potential (MMP). CAN could significantly regulate the indicators of ferroptosis. By using specific inhibitors celecoxib (coxib), S-methylisothiourea sulfate (SMT), Ferrostatin-1 (Fer-1), and Compound C, we further found that CAN regulated inflammation and ferroptosis through AMP-activated protein (AMPK), and inflammation interacted with ferroptosis. Our study indicated that CAN attenuated lipotoxicity in cardiomyocytes by regulating inflammation and ferroptosis through activating the AMPK pathway. This study provides a new direction of myocardial lipotoxicity and some new information for the treatment of DCM.

USP30-AS1 contributes to mitochondrial quality control in glioblastoma cells.

Glioblastoma is the most serious type of brain cancer with poor prognosis. Here, using the publicly available glioma database, we identified that USP30-AS1, an antisense lncRNA locating on the opposite strand of USP30 locus, is upregulated in human gliomas, particularly in high grade glioma. High level of USP30-AS1 is correlated with poor survival in both primary and recurrent glioma patients. USP30-AS1 regulates mitochondrial homeostasis and mitophagy in glioblastoma cells. Knockdown of USP30-AS1 decreases mitochondrial protein expression and mitochondrial mass, promotes mitochondrial uncoupler-induced mitophagy. However, USP30-AS1 does not regulate USP30 expression in a cis-regulatory manner. In summary, this study proposed that USP30-AS1 may serve as a valuable prognostic marker for gliomas. USP3-AS1 is a negative regulator of mitophagy and the regulatory effect is USP30-independent. USP30-AS1 mediated repression of mitophagy may contribute to the loss of mitochondrial homeostasis and tumor development in glioma.

Upregulation of the APOBEC3 Family Is Associated with a Poor Prognosis and Influences Treatment Response to Raf Inhibitors in Low Grade Glioma.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) has been identified as a group of enzymes that catalyze cytosine deamination in single-stranded (ss) DNA to form uracil, causing somatic mutations in some cancers. We analyzed the APOBEC3 family in 33 TCGA cancer types and the results indicated that APOBEC3s are upregulated in multiple cancers and strongly correlate with prognosis, particularly in low grade glioma (LGG). Then we constructed a prognostic model based on family expression in LGG where the APOBEC3 family signature is an accurate predictive model (AUC of 0.85). Gene mutation, copy number variation (CNV), and a differential gene expression (DEG) analysis were performed in different risk groups, and the weighted gene co-expression network analysis (WGCNA) was employed to clarify the role of various members in LGG; CIBERSORT algorithm was deployed to evaluate the landscape of LGG immune infiltration. We found that upregulation of the APOBEC3 family expression can strengthen Ras/MAPK signaling pathway, promote tumor progression, and ultimately reduce the treatment benefits of Raf inhibitors. Moreover, the APOBEC3 family was shown to enhance the immune response mediated by myeloid cells and interferon gamma, as well as PD-L1 and PD-L2 expression, implying that they have immunotherapy potential. Therefore, the APOBEC3 signature enables an efficient assessment of LGG patient survival outcomes and expansion of clinical benefits by selecting appropriate individualized treatment strategies.

MBNL2 Regulates DNA Damage Response via Stabilizing p21.

RNA-binding proteins are frequently dysregulated in human cancer and able to modulate tumor cell proliferation as well as tumor metastasis through post-transcriptional regulation on target genes. Abnormal DNA damage response and repair mechanism are closely related to genome instability and cell transformation. Here, we explore the function of the RNA-binding protein muscleblind-like splicing regulator 2 (MBNL2) on tumor cell proliferation and DNA damage response. Transcriptome and gene expression analysis show that the PI3K/AKT pathway is enriched in MBNL2-depleted cells, and the expression of cyclin-dependent kinase inhibitor 1A (p21CDKN1A) is significantly affected after MBNL2 depletion. MBNL2 modulates the mRNA and protein levels of p21, which is independent of its canonical transcription factor p53. Moreover, depletion of MBNL2 increases the phosphorylation levels of checkpoint kinase 1 (Chk1) serine 345 (S345) and DNA damage response, and the effect of MBNL2 on DNA damage response is p21-dependent. MBNL2 would further alter tumor cell fate after DNA damage, MBNL2 knockdown inhibiting DNA damage repair and DNA damage-induced senescence, but promoting DNA damage-induced apoptosis.

Canagliflozin Modulates Hypoxia-Induced Metastasis, Angiogenesis and Glycolysis by Decreasing HIF-1α Protein Synthesis via AKT/mTOR Pathway.

The microenvironment plays a vital role in tumor progression, and hypoxia is a typical microenvironment feature in nearly all solid tumors. In this study, we focused on elucidating the effect of canagliflozin (CANA), a new class of antidiabetic agents, on hepatocarcinoma (HCC) tumorigenesis under hypoxia, and demonstrated that CANA could significantly inhibit hypoxia-induced metastasis, angiogenesis, and metabolic reprogramming in HCC. At the molecular level, this was accompanied by a reduction in VEGF expression level, as well as a reduction in the epithelial-to-mesenchymal transition (EMT)-related proteins and glycolysis-related proteins. Next, we focused our study particularly on the modulation of HIF-1α by CANA, which revealed that CANA decreased HIF-1α protein level by inhibiting its synthesis without affecting its proteasomal degradation. Furthermore, the AKT/mTOR pathway, which plays an important role in HIF-1α transcription and translation, was also inhibited by CANA. Thus, it can be concluded that CANA decreased metastasis, angiogenesis, and metabolic reprogramming in HCC by inhibiting HIF-1α protein accumulation, probably by targeting the AKT/mTOR pathway. Based on our results, we propose that CANA should be evaluated as a new treatment modality for liver cancer.

NEAT1 regulates neuroglial cell mediating Aß clearance via the epigenetic regulation of endocytosis-related genes expression.

The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.

NEAT1 modulates herpes simplex virus-1 replication by regulating viral gene transcription.

Nuclear paraspeckle assembly transcript 1 (NEAT1) is the crucial structural platform of paraspeckles, which is one type of nuclear bodies. As a stress-induced lncRNA, the expression of NEAT1 increases in response to viral infection, but little is known about the role of NEAT1 or paraspeckles in the replication of herpes simplex virus-1 (HSV-1). Here, we demonstrate that HSV-1 infection increases NEAT1 expression and paraspeckle formation in a STAT3-dependent manner. NEAT1 and other paraspeckle protein components, P54nrb and PSPC1, can associate with HSV-1 genomic DNA. By binding with STAT3, PSPC1 is required for the recruitment of STAT3 to paraspeckles and facilitates the interaction between STAT3 and viral gene promoters, finally increasing viral gene expression and viral replication. Furthermore, thermosensitive gel containing NEAT1 siRNA or STAT3 siRNA effectively healed the skin lesions caused by HSV-1 infection in mice. Our results provide insight into the roles of lncRNAs in the epigenetic control of viral genes and into the function of paraspeckles.

Loss of autophagy causes a synthetic lethal deficiency in DNA repair.

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.

Aloin Suppresses Lipopolysaccharide-Induced Inflammatory Response and Apoptosis by Inhibiting the Activation of NF-κB.

Numerous herbal-derived natural products are excellent anti-inflammatory agents. Several studies have reported that aloin, the major anthraquinone glycoside obtained from the Aloe species, exhibits anti-inflammatory activity. However, the molecular mechanism of this activity is not well understood. In this report, we found that aloin suppresses lipopolysaccharide-induced pro-inflammatory cytokine secretion and nitric oxide production, and downregulates the expression of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Aloin inhibits the phosphorylation and acetylation of the NF-κB p65 subunit by suppressing the upstream kinases p38 and Msk1, preventing LPS-induced p65 translocation to the nucleus. We have also shown that aloin inhibits LPS-induced caspase-3 activation and apoptotic cell death. Collectively, these findings suggest that aloin effectively suppresses the inflammatory response, primarily through the inhibition of NF-κB signaling.

Crosslinked Aspartic Acids as Helix-Nucleating Templates.

Described is a facile helix-nucleating template based on a tethered aspartic acid at the N-terminus [terminal aspartic acid (TD)]. The nucleating effect of the template is subtly influenced by the substituent at the end of the side-chain-end tether as indicated by circular dichroism, nuclear magnetic resonance, and molecular dynamics simulations. Unlike most nucleating strategies, the N-terminal amine is preserved, thus enabling further modification. Peptidomimetic estrogen receptor modulators (PERMs) constructed using this strategy show improved therapeutic properties. The current strategy can be regarded as a good complement to existing helix-stabilizing methods.

MiR-378a-3p enhances adipogenesis by targeting mitogen-activated protein kinase 1.

Previous studies showed that miR-378a plays important roles in adipogenesis and obesity; however, the precise mechanisms of action remain unknown. Here, we found that miR-378a-3p expression is up-regulated in adipose tissues of high fat diet-induced obese mice, as well as during the differentiation of 3T3-L1 preadipocytes. Mir-378a-3p induced adipogenesis by targeting mitogen-activated protein kinase 1 (MAPK1). Overexpression of miR-378a-3p or silencing MAPK1 reduced MAPK1 expression and enhanced adipogenesis, whereas blockage of endogenous miR-378a-3p had the opposite effect, suggesting that miR-378a-3p promotes the adipogenesis of 3T3-L1 cells by targeting MAPK1.

DNA damage and ER stress contribute to oblongifolin C-induced cell killing in Bax/Bak-deficient cells.

A key clinical problem in oncology is the treatment of apoptosis-resistant tumors. Tumor cells deficient in both of the proapoptotic proteins Bax and Bak are protected against most chemotherapeutic drug-induced apoptosis. We report here that a natural compound, oblongifolin C (OC), effectively eliminates Bax/Bak-deficient murine embryonic fibroblasts and colon carcinoma HCT116 cells. OC not only triggers DNA double-strand breaks and DNA damage response, but also inhibits repair of DNA damage. In addition, OC induces ER stress through upregulation of the transcription factor CHOP and activation of JNK kinases. Upon treatment with OC, cells undergo Bax/Bak-independent, caspase-mediated apoptosis. Taken together, our data establish a rationale for the broad use of OC to treat apoptosis deficient tumors.

Identification and characterization of anticancer compounds targeting apoptosis and autophagy from Chinese native Garcinia species.

Natural compounds from medicinal plants are important resources for drug development. Active compounds targeting apoptosis and autophagy are candidates for anti-cancer drugs. In this study, we collected Garcinia species from China and extracted them into water or ethanol fractions. Then, we performed a functional screen in search of novel apoptosis and autophagy regulators. We first characterized the anti-proliferation activity of the crude extracts on multiple cell lines. HeLa cells expressing GFP-LC3 were used to examine the effects of the crude extracts on autophagy. Their activities were confirmed by Western blots of A549 and HeLa cells. By using bioassay guided fractionation, we found that two caged prenylxanthones from Garcinia bracteata, neobractatin and isobractatin, can significantly induce apoptosis and inhibit autophagy. Our results suggest that different Garcinia species displayed various degrees of toxicity on different cancer cell lines. Furthermore, the use of a high content screening assay to screen natural products was an essential method to identify novel autophagy regulators.

MiR-210 disturbs mitotic progression through regulating a group of mitosis-related genes.

MiR-210 is up-regulated in multiple cancer types but its function is disputable and further investigation is necessary. Using a bioinformatics approach, we identified the putative target genes of miR-210 in hypoxia-induced CNE cells from genome-wide scale. Two functional gene groups related to cell cycle and RNA processing were recognized as the major targets of miR-210. Here, we investigated the molecular mechanism and biological consequence of miR-210 in cell cycle regulation, particularly mitosis. Hypoxia-induced up-regulation of miR-210 was highly correlated with the down-regulation of a group of mitosis-related genes, including Plk1, Cdc25B, Cyclin F, Bub1B and Fam83D. MiR-210 suppressed the expression of these genes by directly targeting their 3'-UTRs. Over-expression of exogenous miR-210 disturbed mitotic progression and caused aberrant mitosis. Furthermore, miR-210 mimic with pharmacological doses reduced tumor formation in a mouse metastatic tumor model. Taken together, these results implicate that miR-210 disturbs mitosis through targeting multi-genes involved in mitotic progression, which may contribute to its inhibitory role on tumor formation.

MicroRNA-188 suppresses G1/S transition by targeting multiple cyclin/CDK complexes.

BACKGROUND: Accelerated cell cycle progression is the common feature of most cancers. MiRNAs can act as oncogenes or tumor suppressors by directly modulating cell cycle machinery. It has been shown that miR-188 is upregulated in UVB-irradiated mouse skin and human nasopharyngeal carcinoma CNE cells under hypoxic stress. However, little is known about the function of miR-188 in cell proliferation and growth control. RESULTS: Overexpression of miR-188 inhibits cell proliferation, tumor colony formation and G1/S cell cycle transition in human nasopharyngeal carcinoma CNE cells. Using bioinformatics approach, we identify a series of genes regulating G1/S transition as putative miR-188 targets. MiR-188 inhibits both mRNA and protein expression of CCND1, CCND3, CCNE1, CCNA2, CDK4 and CDK2, suppresses Rb phosphorylation and downregulates E2F transcriptional activity. The expression level of miR-188 also inversely correlates with the expression of miR-188 targets in human nasopharyngeal carcinoma (NPC) tissues. Moreover, studies in xenograft mouse model reveal that miR-188 is capable of inhibiting tumor initiation and progression by suppressing target genes expression and Rb phosphorylation. CONCLUSIONS: This study demonstrates that miR-188 exerts anticancer effects, via downregulation of multiple G1/S related cyclin/CDKs and Rb/E2F signaling pathway.

CDK1 switches mitotic arrest to apoptosis by phosphorylating Bcl-2/Bax family proteins during treatment with microtubule interfering agents.

Microtubule interfering agents (MIAs), that can stabilise or depolymerise microtubules, are an important class of cancer chemotherapeutic drugs. They can lead to mitotic arrest and subsequent apoptosis. We demonstrate that cell cycle-dependent kinase 1 (CDK1) is important in switching cells from mitotic arrest to apoptosis during MIAs treatment. Overexpression of non-degradable cyclin B1 sustained CDK1 activation and mitotic arrest, followed by caspase-3 dependent apoptosis. CDK1 is responsible for the phosphorylation of several pro- and anti-apoptotic Bcl-2 family proteins during MIAs treatment. CDK1-mediated Bcl-2 serine 70 phosphorylation enhances its pro-apoptotic function, whereas CDK1-mediated Bad serine 128 phosphorylation promotes apoptosis. Blockage of CDK1 activity with a specific pharmacological inhibitor suppresses Mcl-1 phosphorylation, degradation and its anti-apoptotic function. Therefore, the death of cancer cells under MIAs treatment was caused by imbalance between CDK1-induced alterations in the pro-apoptotic and anti-apoptotic functions of phosphorylated Bcl-2 family proteins.

Simple, Visual, Point-of-Care SARS-CoV-2 Detection Incorporating Recombinase Polymerase Amplification and Target DNA-Protein Crosslinking Enhanced Chemiluminescence.

The ongoing COVID-19 pandemic, driven by persistent SARS-CoV-2 transmission, threatens human health worldwide, underscoring the urgent need for an efficient, low-cost, rapid SARS-CoV-2 detection method. Herein, we developed a point-of-care SARS-CoV-2 detection method incorporating recombinase polymerase amplification (RPA) and DNA-protein crosslinking chemiluminescence (DPCL) (RPADPCL). RPADPCL involves the crosslinking of biotinylated double-stranded RPA DNA products with horseradish peroxidase (HRP)-labeled streptavidin (SA-HRP). Modified products are captured using SA-labeled magnetic beads, and then analyzed using a chemiluminescence detector and smartphone after the addition of a chemiluminescent substrate. Under optimal conditions, the RPADPCL limit of detection (LOD) was observed to be 6 copies (within the linear detection range of 1-300 copies) for a plasmid containing the SARS-CoV-2 N gene and 15 copies (within the linear range of 10-500 copies) for in vitro transcribed (IVT) SARS-CoV-2 RNA. The proposed method is convenient, specific, visually intuitive, easy to use, and does not require external excitation. The effective RPADPCL detection of SARS-CoV-2 in complex matrix systems was verified by testing simulated clinical samples containing 10% human saliva or a virus transfer medium (VTM) spiked with a plasmid containing a SARS-CoV-2 N gene sequence or SARS-CoV-2 IVT RNA. Consequently, this method has great potential for detecting targets in clinical samples.

Universal smartphone-assisted label-free CRISPR/Cas12a-DNAzyme chemiluminescence biosensing platform for on-site detection of nucleic acid and non-nucleic acid targets.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) system enables sensitive and specific detection of biomolecules, thanks to its programmability, high fidelity, and powerful signal amplification capabilities. Herein, a universal smartphone-assisted label-free G-quadruplex (G4) DNAzyme-based chemiluminescence CRISPR/Cas12a biosensing platform (G4CLCas) is firstly described that achieves on-site, ultrasensitive visual detection of nucleic acid and non-nucleic acid targets. The G4CLCas-based sensing platform relies on Cas12a trans-cleavage activation that triggers the cleavage of the G4 DNAzyme, resulting in chemiluminescence signals off/on compared to that of the control. Chemiluminescence signals are captured as images that are quantitatively analyzed and visualized using a smartphone-assisted imaging cartridge. Under optimal conditions, G4CLCas achieves a low limit of detection (LOD) of 8.6 aM (∼5.2 copies/µL) for monkeypox virus (MPXV) DNA within the linear concentration range of 10-300 aM and can accurately quantify viral DNA in spiked samples. G4CLCas can also detect non-nucleic acid targets, whereby it achieves a low LOD value of 84.3 nM for adenosine triphosphate (ATP) within the linear concentration range of 2-2000 µM. Here, a label-free, portable, on-site CRISPR/Cas12a chemiluminescence biosensing platform based on the G4 DNAzyme substrates is proposed with potential applications in clinical detection and bioanalytical chemistry research.