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PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗

PubMed2006

The stable 2-kb LAT intron of herpes simplex stimulates the expression of heat shock proteins and protects cells from stress.

During latency of herpes simplex virus type 1 (HSV-1), the major viral transcript that is detected is the latency-associated transcript (LAT) 2-kb intron. This intron is excised from a larger (approximately 10 kb) primary transcript. Infection of cells with HSV expressing LATs showed that their presence increased accumulation of Hsp70 protein specifically during cold shock. Transfection of cells with a plasmid, expressing the 2-kb LAT intron, showed increased translation compared to cells transfected with plasmids deleted in regions of the intron. Cold shock of cells expressing the intron resulted in an up-regulation of Hsp70 protein, but not Hsp70 mRNA. Furthermore, these cells showed increased cell viability. Using plasmid deletion mutants in the LAT gene sequence, we have shown that the effect requires full-length LAT intron. These findings show that a function of the stable 2-kb LAT intron is to protect cells from cold-induced stress and that this may be mediated via Hsp70.

Cold Temperature↗

PubMed2004

RNA interfering approach for clarifying the PPARgamma pathway using lentiviral vector expressing short hairpin RNA.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARgamma also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARgamma expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARgamma-knockdown method will serve to clarify the PPARgamma pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.

3T3-L1 Cells↗

PubMed2004

World of small RNAs: from ribozymes to siRNA and miRNA.

RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation. This article reviews our contributions to the understanding of structure, the function of small RNAs, their use in biotechnology, and the understanding of phenotypes such as apoptosis, metastasis, and differentiation.

Animals↗

PubMed2006

Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans.

C. elegans contains numerous small RNAs of ~21-24 nt in length. The microRNAs (miRNAs) are small noncoding RNAs produced by DCR-1- and ALG-dependent processing of self-complementary hairpin transcripts. Endogenous small interfering RNAs (endo-siRNAs), associated with ongoing silencing of protein-coding genes in normal worms, are produced by mechanisms that involve DCR-1 but, unlike miRNAs, also involve RDE-2, RDE-3, RDE-4, RRF-1, and RRF-3. The tiny noncoding (tncRNAs) are similar to endo-siRNAs in their biogenesis except that they are derived from noncoding sequences. These endo-siRNA- and tncRNA-based endogenous RNAi pathways involve some components, including DCR-1 and RDE-4, that are shared with exogenous RNAi, and some components, including RRF-3 and ERI-1, that are specific to endogenous RNAi. rrf-3 and eri-1 mutants are enhanced for some silencing processes and defective for others, suggesting cross-regulatory interactions between RNAi pathways in C. elegans. Microarray expression profiling of RNAi-defective mutant worms further suggests diverse endogenous RNAi pathways for silencing different sets of genes.

Animals↗

PubMed2006

Mutation screening of brain-expressed X-chromosomal miRNA genes in 464 patients with nonsyndromic X-linked mental retardation.

MiRNAs are small noncoding RNAs that control the expression of target genes at the post-transcriptional level and have been reported to modulate various biological processes. Their function as regulatory factors in gene expression renders them attractive candidates for harbouring genetic variants with subtle effects on IQ. In an attempt to investigate the potential role of miRNAs in the aetiology of X-linked mental retardation, we have examined all 13 known, brain-expressed X-chromosomal miRNAs in a cohort of 464 patients with non-syndromic X-linked MR and found four nucleotide changes in three different pre-miRNA hairpins. All the observed changes appear to be functionally neutral which, taken together with the rarity of detected nucleotide changes in miRNA genes, may reflect strong selection and thus underline the functional importance of miRNAs.

Base Sequence↗

PubMed2005

A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing.

In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins that are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional processes we depleted P-body or essential pathway components from Drosophila cells and analyzed the effects of these depletions on the expression of reporter constructs, allowing us to monitor specifically NMD, RNAi, or miRNA function. We show that the RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. Our analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila.

Animals↗

PubMed2006

Clusters of internally primed transcripts reveal novel long noncoding RNAs.

Non-protein-coding RNAs (ncRNAs) are increasingly being recognized as having important regulatory roles. Although much recent attention has focused on tiny 22- to 25-nucleotide microRNAs, several functional ncRNAs are orders of magnitude larger in size. Examples of such macro ncRNAs include Xist and Air, which in mouse are 18 and 108 kilobases (Kb), respectively. We surveyed the 102,801 FANTOM3 mouse cDNA clones and found that Air and Xist were present not as single, full-length transcripts but as a cluster of multiple, shorter cDNAs, which were unspliced, had little coding potential, and were most likely primed from internal adenine-rich regions within longer parental transcripts. We therefore conducted a genome-wide search for regional clusters of such cDNAs to find novel macro ncRNA candidates. Sixty-six regions were identified, each of which mapped outside known protein-coding loci and which had a mean length of 92 Kb. We detected several known long ncRNAs within these regions, supporting the basic rationale of our approach. In silico analysis showed that many regions had evidence of imprinting and/or antisense transcription. These regions were significantly associated with microRNAs and transcripts from the central nervous system. We selected eight novel regions for experimental validation by northern blot and RT-PCR and found that the majority represent previously unrecognized noncoding transcripts that are at least 10 Kb in size and predominantly localized in the nucleus. Taken together, the data not only identify multiple new ncRNAs but also suggest the existence of many more macro ncRNAs like Xist and Air.

Animals↗

PubMed2025

Short-term diet intervention comprising of olive oil, vitamin D, and omega-3 fatty acids alters the small non-coding RNA (sncRNA) landscape of human sperm.

Offspring health outcomes are often linked with epigenetic alterations triggered by maternal nutrition and intrauterine environment. Strong experimental data also link paternal preconception nutrition with pathophysiology in the offspring, but the mechanism(s) routing effects of paternal exposures remain elusive. Animal experimental models have highlighted small non-coding RNAs (sncRNAs) as potential regulators of paternal effects. Here, we characterised the baseline sncRNA landscape of human sperm and the effect of a 6-week dietary intervention on their expression profile. This study involves sncRNAseq profiling, that was performed on a subset (n = 17) of the participants enrolled in the PREPARE trial: 9 from the control group and 8 from the intervention group. 5'tRFs, miRNAs and piRNAs were the most abundant sncRNA subtypes identified; their expression was associated with age, BMI, and sperm quality. Nutritional intervention with olive oil, vitamin D and omega-3 fatty acids altered expression of 3 tRFs, 15 miRNAs and 112 piRNAs, targeting genes involved in fatty acid metabolism and transposable elements in the sperm genome. PREPARE Trial registration number: ISRCTN50956936, Trial registration date: 10/02/2014.

Humans↗

PubMed2026

miR-197 Targets NLRP3 3' UTR and Correlates with NLRP3/Caspase-1/IL-18 Signaling in Hyperoxia-Stimulated Neonatal BPD Mouse Model.

Reduced circulating miR-197 was previously observed in preterm infants who later developed bronchopulmonary dysplasia (BPD), but its relationship with NLRP3 inflammasome signaling remains unclear. This study examined miR-197 expression, NLRP3 inflammasome-related markers, and the interaction between miR-197 and the NLRP3 3' UTR in a neonatal hyperoxia model. Neonatal C57BL/6J mice were exposed to 60% oxygen or room air from birth, and lung tissues were collected on postnatal days 1, 7, 14, and 21. Lung injury and alveolar development were assessed by histology, radial alveolar count, mean linear intercept, and lung wet-to-dry ratio. miR-197 and NLRP3 expression and NF-κB-, caspase-1-, and IL-18-related proteins were evaluated by RT-qPCR and Western blotting. A dual-luciferase reporter assay in MLE12 cells tested the interaction between miR-197 and the NLRP3 3' UTR. Hyperoxia increased lung wet-to-dry ratios and mean linear intercept, reduced radial alveolar count, and caused progressive alveolar simplification. miR-197 expression decreased, whereas NLRP3 mRNA increased, at all examined time points; NLRP3 protein and inflammasome-related protein changes were most evident from postnatal day 7 onward. The miR-197 mimic reduced luciferase activity in the wild-type but not mutant NLRP3 3' UTR reporter. These findings show that neonatal hyperoxia is associated with reduced miR-197 and increased NLRP3/inflammasome-related signaling and support a sequence-specific interaction between miR-197 and the NLRP3 3' UTR, although causal regulation in vivo requires further investigation.

Animals↗

PubMed2014

MiR-16 targets Bcl-2 in paclitaxel-resistant lung cancer cells and overexpression of miR-16 along with miR-17 causes unprecedented sensitivity by simultaneously modulating autophagy and apoptosis.

Non-small cell lung cancer is one of the most aggressive cancers as per as the mortality and occurrence is concerned. Paclitaxel based chemotherapeutic regimes are now used as an important option for the treatment of lung cancer. However, resistance of lung cancer cells to paclitaxel continues to be a major clinical problem nowadays. Despite impressive initial clinical response, most of the patients eventually develop some degree of paclitaxel resistance in the course of treatment. Previously, utilizing miRNA arrays we reported that downregulation of miR-17 is at least partly involved in the development of paclitaxel resistance in lung cancer cells by modulating Beclin-1 expression [1]. In this study, we showed that miR-16 was also significantly downregulated in paclitaxel resistant lung cancer cells. We demonstrated that anti-apoptotic protein Bcl-2 was directly targeted miR-16 in paclitaxel resistant lung cancer cells. Moreover, in this report we showed that the combined overexpression of miR-16 and miR-17 and subsequent paclitaxel treatment greatly sensitized paclitaxel resistant lung cancer cells to paclitaxel by inducing apoptosis via caspase-3 mediated pathway. Combined overexpression of miR-16 and miR-17 greatly reduced Beclin-1 and Bcl-2 expressions respectively. Our results indicated that though miR-17 and miR-16 had no common target, both miR-16 and miR-17 jointly played roles in the development of paclitaxel resistance in lung cancer. miR-17 overexpression reduced cytoprotective autophagy by targeting Beclin-1, whereas overexpression of miR-16 potentiated paclitaxel induced apoptotic cell death by inhibiting anti-apoptotic protein Bcl-2.

3' Untranslated Regions↗

PubMed2026

Pan-cancer multi-omics machine learning defines a lactylation-associated immune-excluded tumor state with proteomic and experimental corroboration.

BACKGROUND: Histone lactylation links lactate metabolism to chromatin regulation, but whether lactylation-program-associated transcriptional patterns delineate recurrent pan-cancer tumor states remains unclear. METHODS: We integrated mRNA, lncRNA, and miRNA profiles from 9712 TCGA tumors across 33 cancer types with GTEx references, six GEO cohorts, IMvigor210, and an institutional clear-cell renal cell carcinoma (ccRCC) cohort used for exploratory DIA-NN proteomic corroboration. Random-effects co-expression meta-analysis, multi-omics consensus clustering, regulon inference, immune deconvolution, TIDE, oncoPredict, and SHAP-based machine learning were applied. hsa-miR-431-5p was functionally evaluated as a proof-of-concept CS2-associated miRNA in bladder cancer models. RESULTS: LacCoEx-Atlas comprised 398,491 lactylation-related co-expression pairs across 24,667 RNA features under a random-effects framework (median I² = 88.6%). Consensus clustering identified two subtypes: CS2 showed glycolytic-mesenchymal-immune-excluded features, M2 macrophage enrichment, CD8⁺ T-cell depletion, elevated HDAC4/NSD3/KDM6B activity, and worse survival, whereas CS1 showed oxidative, sirtuin-active programs. CS2 had fewer predicted ICI responders (18.3% vs. 52.0%) and a lower observed ORR in IMvigor210 (15.3% vs. 24.0%). oncoPredict identified NU7441 as a hypothesis-generating CS2-associated sensitivity signal (Hedges' g = 1.17). DIA-NN proteomics in 50 ccRCC specimens provided exploratory support for CS2-associated hypoxia, ECM degradation, and metastasis programs. The 10-feature mRNA LARItools model achieved an apparent AUC of 0.9413, while a separate multi-omics model achieved 0.971; neither was independently validated. LARItools reproduced prognostic separation across six GEO cohorts. miR-431-5p promoted malignant phenotypes and EMT in bladder cancer cells, with concordant CMU4h expression findings. CONCLUSIONS: Lactylation-program-associated transcriptional patterns delineate a recurrent immune-excluded pan-cancer tumor state associated with adverse prognosis, reduced predicted immunotherapy responsiveness, exploratory single-cancer protein-level support, and testable DNA damage response-targeting hypotheses. LacCoEx-Atlas and LARItools provide open resources for lactylation-program-associated tumor-state stratification and future translational research.

Humans↗

PubMed2006

Allele-specific RNA interference for neurological disease.

Suppressing the expression of toxic genes through RNAi holds great promise for the treatment of human disease. Allele-specific approaches have now been used to silence dominant toxic genes implicated in several neurological disorders. Here, we review strategies used to achieve allele-specific silencing in light of recent developments in the field of RNAi biology. In particular, new insights into siRNA and miRNA processing may be used to improve efficiency and specificity of RNAi therapy. We further discuss steps that can be taken to maximize the therapeutic benefits of this powerful technology.

Alleles↗

PubMed2026

Multi-omics analysis reveals coordinated epigenetic dysregulation in atrazine-induced dopaminergic neurotoxicity.

Atrazine (ATR), a widely used triazine herbicide, has been linked to neurotoxicity, yet the epigenetic mechanisms underlying its dopaminergic effects remain unclear. This study investigated whether coordinated miRNA dysregulation and DNA methylation alterations contribute to ATR-induced Parkinson's disease (PD)-like neurotoxicity. Male Sprague-Dawley rats were administered ATR (50 mg/kg/day) for 90 days, resulting in motor and cognitive deficits with dopaminergic dysfunction, including increased α-synuclein and reduced tyrosine hydroxylase expression. Small RNA sequencing identified 72 differentially expressed miRNAs in the substantia nigra, enriched in PI3K-Akt, MAPK, and Ras signaling pathways. In a cohort of six PD patients and six matched controls, genome-wide DNA methylation profiling revealed 4694 differentially methylated positions, predominantly hypomethylated, with overlapping enrichment in neuronal signaling pathways. Weighted gene co-expression network analysis identified a PD-associated module strongly correlated with disease status (r = -0.95, P < 0.001). Multi-omics integration identified CASP3 as a central hub gene. External validation supported CASP3 relevance in PD (AUC = 0.833), and molecular docking suggested potential ATR-CASP3 interaction. Further analysis predicted upregulated miR-3552 as a potential upstream regulator of CASP3. These findings indicate that ATR-induced neurotoxicity may be mediated through the miR-3552/CASP3 signaling axis, ultimately regulating apoptosis and contributing to neurodegeneration.

Animals↗

PubMedDate not supplied

HSV LAT and neuronal survival.

Herpes Simplex Virus (HSV) establishes a latent infection within sensory neurons and periodically reactivates in response to stress. HSV's ability to inhabit neurons for the life of the host involves a number of virally encoded functions that tightly regulate the latency-reactivation cycle, preventing uncontrolled spread of reactivating virus and large-scale death of neurons. The HSV latency-associated transcript (LAT) is a complex transcription unit expressed primarily in neurons containing latent genomes. While mutational analyses indicate LAT is nonessential for viral replication, the 5' exon of LAT greatly enhances reactivation. Several studies have also identified LAT mutations that reduce establishment of latency and enhance virulence. Recently, LAT has also been shown to inhibit cell death through by blocking caspase-8 and caspase-9 pathways. While blocking apoptosis is not essential for either establishment of latency or reactivation, it likely augments these processes and may contribute to HSV's long-term persistence and spread.

Animals↗

PubMed2006

Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells.

The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells.

Cell Differentiation↗

PubMed2026

Enhanced exonuclease-Cas9 systems promote multiple nucleotide deletions with higher efficiency and broader targeting scope in plants.

CRISPR-Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease-Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND-Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND-Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND-Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9-NG and SpG variants, generating MND-Cas9-NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND-Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3'UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND-Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease-Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.

CRISPR-Cas Systems↗

PubMed2005

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells.

Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.

Apoptosis↗