Recombinant Human Putative uncharacterized protein encoded by LINC00305 (LINC00305)

What is LINC00305 and how was it initially identified?

LINC00305 is a long non-coding RNA (lncRNA) that spans approximately 69 kb in length and is located in chromosome 18q22.1, downstream of the serpin peptidase inhibitor, clade B (SERPINB) genes. It was initially identified through genome-wide association studies (GWAS) when researchers discovered an atherosclerosis-associated single-nucleotide polymorphism (SNP) rs2850711 and its putative causal variant rs2676671 located in the intron of LINC00305. Analysis using the Coding Potential Assessment Tool (CPAT) confirmed its low coding potential, classifying it as a true long non-coding RNA .

What is the cellular localization of LINC00305 and which cell types express it predominantly?

LINC00305 is primarily localized in the cytoplasm of cells. Expression analysis has shown that LINC00305 is predominantly expressed in monocytes and is enriched in atherosclerotic plaques. RNA-FISH and immunofluorescence assays have demonstrated that LINC00305 co-localizes with LIMR (lipocalin-1 interacting membrane receptor) in THP-1 cells, a human monocytic cell line used to study monocyte/macrophage functions and mechanisms .

How does LINC00305 differ from other inflammation-associated lncRNAs?

Unlike many lncRNAs that function in cis by regulating neighboring genes, LINC00305 operates in trans, affecting genes located on different chromosomes. While many inflammation-associated lncRNAs interact with heterogeneous nuclear ribonucleoproteins (hnRNPs), NF-κB pathway proteins, or the PRC2 complex, LINC00305 distinctively binds to the transmembrane protein LIMR and influences AHRR (aryl-hydrocarbon receptor repressor) localization. This unique interaction mechanism differentiates LINC00305 from other inflammation-associated lncRNAs that directly interact with transcription factors or chromatin-modifying complexes .

What are the recommended protocols for studying LINC00305 expression in clinical samples?

For studying LINC00305 expression in clinical samples, researchers should consider:

• RNA isolation from atherosclerotic plaques or peripheral blood mononuclear cells (PBMCs) using specialized kits designed for lncRNA preservation

• Quantitative real-time PCR (qRT-PCR) with specifically designed primers for LINC00305

• RNA in situ hybridization for spatial localization in tissue sections

• RNA sequencing (RNA-seq) for comprehensive transcriptomic analysis

For more accurate results when working with atherosclerotic samples, it's important to include healthy vascular tissue controls and normalize expression using appropriate reference genes .

How can researchers effectively overexpress or knock down LINC00305 in experimental models?

For LINC00305 manipulation in experimental models:

Overexpression approaches:

• Lentiviral vectors containing the full LINC00305 sequence (as described in the Zhang et al. study)

• Use of different multiplicity of infection (MOI) to achieve concentration-dependent effects

• Verification of overexpression efficiency via qRT-PCR

Knockdown approaches:

• siRNA targeting specific regions of LINC00305

• CRISPR-Cas9 approaches for genomic deletion

• Antisense oligonucleotides (ASOs) for targeted degradation

The study by Zhang et al. successfully used lentiviral overexpression systems in THP-1 cells at two different expression levels to demonstrate concentration-dependent effects of LINC00305 on inflammatory gene expression .

What methods are recommended for studying LINC00305 protein interactions?

To investigate LINC00305 protein interactions, the following methodologies have proven effective:

• RNA Pull-down Assay: Using biotinylated LINC00305 RNA as bait, followed by mass spectrometry analysis to identify interacting proteins. The antisense LINC00305 RNA should be used as a negative control.

• RNA Immunoprecipitation (RIP): Expressing HA-tagged LIMR in cells followed by immunoprecipitation with anti-HA antibody to confirm binding with LINC00305 in vivo.

• RNA-FISH and Immunofluorescence: Dual staining to visualize co-localization of LINC00305 with potential protein partners (as demonstrated with LIMR in THP-1 cells).

• GST Pull-down Assay: For identifying secondary interaction partners (as shown for LIMR-AHRR interaction).

• Immunoprecipitation (IP): To confirm protein-protein interactions in vivo with appropriate tags (such as His-tagged AHRR and LIMR) .

How does LINC00305 activate the NF-κB signaling pathway?

LINC00305 activates the NF-κB pathway through a complex molecular mechanism:

• LINC00305 directly interacts with LIMR (lipocalin-1 interacting membrane receptor) in the cytoplasm

• This interaction enhances the binding between LIMR and AHRR (aryl-hydrocarbon receptor repressor)

• The enhanced LIMR-AHRR interaction promotes AHRR protein expression and nuclear localization

• Nuclear AHRR represses AHR (aryl hydrocarbon receptor) signaling by competing for binding to ARNT (AHR nuclear translocator)

• AHR typically suppresses inflammation, and its inhibition by AHRR promotes NF-κB activation

• Activated NF-κB translocates to the nucleus, where it upregulates pro-inflammatory genes

Importantly, LINC00305 can only activate NF-κB in the presence of both LIMR and AHRR, as demonstrated through reporter assays in 293T cells. Inhibition of NF-κB abolishes LINC00305-mediated activation of cytokine expression, confirming the essential role of this pathway in LINC00305's pro-inflammatory function .

What is the relationship between LINC00305, LIMR, and AHRR in molecular signaling?

The relationship between LINC00305, LIMR, and AHRR represents a novel signaling axis:

• LINC00305 physically binds to LIMR, a 9-pass transmembrane protein known to mediate the endocytosis of lipocalin-1

• This interaction facilitates and strengthens the binding between LIMR and AHRR, as demonstrated by increased co-immunoprecipitation in the presence of LINC00305

• The enhanced LIMR-AHRR interaction leads to increased AHRR protein levels without affecting AHRR mRNA expression, suggesting post-transcriptional regulation

• LINC00305 promotes AHRR nuclear translocation, where it can compete with AHR for binding to ARNT

• This competition inhibits AHR signaling, which normally suppresses inflammation, resulting in enhanced NF-κB activity

This signaling axis is unique as it involves an lncRNA interacting with a transmembrane receptor to affect a nuclear receptor's localization and function, ultimately leading to transcription factor activation .

How does LINC00305 influence gene expression patterns in monocytes?

LINC00305 significantly alters the transcriptional profile of monocytes, particularly enhancing inflammation-associated genes:

• Microarray analysis of THP-1 cells overexpressing LINC00305 revealed upregulation of numerous pro-inflammatory genes

• Gene Ontology (GO) analysis confirmed enrichment of inflammation-associated pathways in LINC00305-upregulated genes

• LINC00305 specifically increases expression of inflammatory cytokines and chemokines in a concentration-dependent manner

• This effect is mediated through NF-κB activation, as NF-κB inhibition abolishes the pro-inflammatory effect

• LINC00305 promotes P65 (RelA) nuclear localization and enhances P65 binding to target gene promoters

• Importantly, LINC00305 functions in trans, as it does not affect expression of neighboring SERPINB genes

The data suggests LINC00305 acts as a master regulator of inflammatory responses in monocytes by orchestrating a comprehensive pro-inflammatory transcriptional program .

What is the role of LINC00305 in atherosclerosis development?

LINC00305 promotes atherosclerosis development through multiple mechanisms:

• Enhanced monocyte inflammation: LINC00305 upregulates pro-inflammatory genes in monocytes, leading to increased cytokine production

• Vascular smooth muscle cell phenotype switching: LINC00305-expressing THP-1 cells induce phenotypic switching in co-cultured human aortic smooth muscle cells (HASMCs) from a contractile to a synthetic phenotype, evidenced by decreased expression of contractile markers

• NF-κB pathway activation: LINC00305 activates NF-κB signaling, a central pathway in atherosclerosis development

• Increased expression in atherosclerotic tissues: LINC00305 expression is significantly enriched in atherosclerotic plaques compared to normal arterial tissues

• Genetic association: An atherosclerosis-associated SNP (rs2850711) is located within the LINC00305 gene, suggesting a genetic predisposition

These mechanisms collectively contribute to the progression of atherosclerotic lesions by promoting inflammation, altering vascular cell phenotypes, and enhancing plaque formation .

How can LINC00305 expression levels be correlated with atherosclerosis progression?

To correlate LINC00305 expression with atherosclerosis progression, researchers should:

• Collect atherosclerotic plaque samples at various stages of progression (classified by established histopathological criteria)

• Isolate RNA with methods optimized for lncRNA preservation

• Perform qRT-PCR to quantify LINC00305 expression levels

• Conduct in situ hybridization to localize LINC00305 within plaque regions

• Correlate expression levels with:

  • Plaque stage and severity

  • Inflammatory cell infiltration

  • HASMC phenotype markers

  • Clinical parameters including lipid profiles and inflammatory markers

  • Patient outcomes such as cardiovascular events

• Use multivariate analysis to control for confounding factors

The study by Zhang et al. observed significantly enhanced LINC00305 expression in atherosclerotic plaques as well as in the PBMCs of atherosclerosis patients, suggesting its potential as a biomarker for disease progression .

Beyond atherosclerosis, what other inflammatory diseases might involve LINC00305 dysregulation?

Given LINC00305's role in promoting inflammation through NF-κB activation, its dysregulation might be involved in other inflammatory diseases including:

• Rheumatoid arthritis: Chronic inflammatory disorder affecting joints where NF-κB plays a crucial role

• Inflammatory bowel diseases: Including Crohn's disease and ulcerative colitis, which involve dysregulated inflammatory responses

• Chronic obstructive pulmonary disease (COPD): Characterized by chronic inflammation of the airways

• Psoriasis: An inflammatory skin condition with NF-κB pathway involvement

• Systemic lupus erythematosus: An autoimmune disease with aberrant inflammatory responses

Researching LINC00305 expression in these conditions would require tissue-specific sampling, careful selection of control samples, and correlation with established disease markers. The research by Zhang et al. suggests LINC00305 functions as a general pro-inflammatory factor, making it a potential contributor to various inflammatory pathologies .

How can LINC00305 SNPs be effectively genotyped and functionally characterized?

For effective genotyping and functional characterization of LINC00305 SNPs:

Genotyping approaches:

• TaqMan SNP genotyping assays for high-throughput analysis of rs2850711 and rs2676671

• Next-generation sequencing for comprehensive variant discovery across the LINC00305 locus

• Digital droplet PCR for accurate allele frequency determination in heterogeneous samples

Functional characterization methods:

• Reporter gene assays with constructs containing different SNP variants to assess their effect on transcriptional activity

• CRISPR-based approaches to introduce specific SNP variants into cellular models

• Chromatin immunoprecipitation (ChIP) to identify transcription factors binding differentially to SNP variants

• Chromosome conformation capture techniques (3C, 4C, Hi-C) to determine long-range chromatin interactions affected by SNPs

• RNA structure analysis to determine if SNPs alter LINC00305 secondary structure and function

As identified in the Zhang et al. study, the putative causal variant rs2676671 in the LINC00305 locus should be prioritized for functional studies to understand how it might affect LINC00305 expression or function .

What advanced bioinformatic approaches would be most valuable for predicting LINC00305 structure and function?

Advanced bioinformatic approaches for LINC00305 analysis should include:

• RNA Secondary Structure Prediction:

  • Use of algorithms like RNAfold, Mfold, or RNAstructure

  • Incorporation of SHAPE-seq or DMS-seq experimental data for structure validation

  • Prediction of structural motifs that might be involved in protein binding

• Subcellular Localization Prediction:

  • Machine learning algorithms trained on lncRNA localization patterns

  • Analysis of sequence motifs associated with nuclear, cytoplasmic, or membrane localization

• Protein Interaction Network Analysis:

  • Text mining of literature for additional LINC00305 interactions

  • Integration of LIMR and AHRR interaction networks to identify potential functional pathways

  • Molecular docking simulations of LINC00305-LIMR interaction

• Evolutionary Conservation Analysis:

  • Comparative genomics to identify conserved LINC00305 regions across species

  • Analysis of SNPs in conserved regions to prioritize functionally important variants

• Transcription Factor Binding Site Prediction:

  • Identification of regulatory elements in the LINC00305 promoter region

  • Analysis of how inflammatory stimuli might regulate LINC00305 expression

These approaches could help predict structural domains critical for LINC00305 function and identify potential regulatory mechanisms governing its expression .

What are the challenges and methodological considerations in developing LINC00305 as a therapeutic target?

Developing LINC00305 as a therapeutic target presents several challenges and methodological considerations:

Challenges:

• Specificity of targeting an lncRNA without affecting other RNA species

• Delivery of therapeutic agents to specific cell types (particularly monocytes)

• Achieving sufficient knockdown in vivo to produce therapeutic effects

• Potential off-target effects on unrelated inflammatory pathways

• Balancing inflammatory inhibition without compromising immune responses

Methodological considerations:

• Target validation:

  • CRISPR-Cas9 knockout models to confirm phenotypes

  • Animal models of atherosclerosis (ApoE-/- or LDLR-/- mice) with LINC00305 manipulation

  • Analysis of effects in multiple cell types beyond monocytes

• Therapeutic approaches:

  • Antisense oligonucleotides (ASOs) specifically designed for LINC00305

  • Small molecule inhibitors targeting LINC00305-LIMR interaction

  • LIMR or AHRR antagonists to disrupt the downstream signaling pathway

  • RNA aptamers to selectively bind and inhibit LINC00305

• Delivery systems:

  • Lipid nanoparticles for RNA-based therapeutics

  • Monocyte-targeting strategies using surface markers

  • Local delivery systems for atherosclerotic plaque targeting

• Safety monitoring:

  • Comprehensive transcriptomic analysis to detect off-target effects

  • Immune function assays to ensure pathogen response is preserved

  • Long-term studies to assess effects on wound healing and tissue repair

Since LINC00305 promotes inflammation primarily through the LIMR-AHRR-NF-κB axis, targeting specific components of this pathway might provide more selective anti-inflammatory effects than global NF-κB inhibition .

What quantitative data exists regarding LINC00305 expression in healthy versus diseased tissues?

While comprehensive quantitative data comparing LINC00305 expression across multiple tissue types is limited in the available search results, the Zhang et al. study provides critical observations:

• LINC00305 expression is significantly enhanced in atherosclerotic plaques compared to healthy arterial tissue

• PBMCs from atherosclerosis patients show elevated LINC00305 expression compared to healthy controls

• Among blood cells, LINC00305 is predominantly expressed in monocytes

For future research, a systematic quantitative analysis across multiple tissue types and disease states would be valuable. This could include:

• RNA-seq data from normal and atherosclerotic vessels at different anatomical sites

• Single-cell RNA-seq to identify cell-specific expression patterns

• Correlation analyses between LINC00305 expression levels and clinical parameters of inflammation

• Longitudinal studies tracking expression changes during disease progression

This type of comprehensive expression data would further strengthen the association between LINC00305 and inflammatory disease states .

What is the molecular interaction map of LINC00305 and its protein partners?

Figure 1: Molecular Interaction Map of LINC00305 Signaling Pathway

Based on the Zhang et al. study, the molecular interaction map of LINC00305 includes:

• Primary interaction: LINC00305 directly binds to LIMR (lipocalin-1 interacting membrane receptor)

• Secondary interaction: LIMR binds to AHRR (aryl-hydrocarbon receptor repressor)

• LINC00305 enhances the LIMR-AHRR interaction

• AHRR protein expression and nuclear localization increases

• Nuclear AHRR competes with AHR for binding to ARNT

• This competition inhibits AHR signaling (which normally suppresses inflammation)

• Inhibition of AHR signaling promotes NF-κB activation

• Activated NF-κB translocates to the nucleus

• Nuclear NF-κB binds to target gene promoters

• This leads to increased expression of pro-inflammatory genes

This interaction map represents a novel signaling pathway connecting a long non-coding RNA to nuclear receptor signaling and ultimately to transcriptional regulation of inflammatory genes. Future research should focus on identifying additional components of this pathway and understanding how they are regulated in different physiological and pathological contexts .

What are the most promising research directions for understanding LINC00305 function in different cell types?

Future research on LINC00305 function across cell types should focus on:

• Single-cell transcriptomics: Analyzing LINC00305 expression and effects at single-cell resolution in atherosclerotic plaques to identify all responsive cell populations beyond monocytes

• Conditional expression models: Developing tissue-specific LINC00305 expression or knockout models to determine cell-autonomous versus non-cell-autonomous effects

• Vascular smooth muscle cell (VSMC) direct effects: Investigating whether LINC00305 can be expressed in or directly affect VSMCs rather than just through monocyte-mediated paracrine effects

• Endothelial cell interactions: Exploring if and how LINC00305 affects endothelial cell function, which is critical in early atherosclerosis

• Macrophage differentiation: Analyzing how LINC00305 influences monocyte-to-macrophage differentiation and polarization toward pro- or anti-inflammatory phenotypes

• Tissue-resident macrophage responses: Comparing effects on circulating monocytes versus tissue-resident macrophages in the arterial wall

• Lipid metabolism interactions: Investigating potential effects of LINC00305 on cellular lipid handling and foam cell formation

This multi-cell type analysis would provide a comprehensive understanding of LINC00305's role in complex inflammatory tissues .

How might genetic variation in LINC00305 contribute to individual differences in inflammatory responses?

Genetic variation in LINC00305 could influence inflammatory responses through several mechanisms:

• Expression level variation: SNPs in regulatory regions could alter basal or inducible LINC00305 expression levels, affecting inflammatory sensitivity

• Structural effects: Variants within the LINC00305 sequence might alter its secondary structure, potentially affecting LIMR binding affinity

• Splicing alterations: Some variants could influence splicing patterns, potentially generating isoforms with different functional properties

• Stability differences: SNPs might affect LINC00305 stability or half-life, influencing the duration of inflammatory signaling

• Cell-type specific effects: Certain variants might alter expression patterns across different cell types, changing the inflammatory landscape

Research approaches should include:

• Genotype-phenotype correlation studies in diverse populations

• Functional characterization of identified variants using CRISPR-based approaches

• eQTL (expression quantitative trait loci) analysis for LINC00305 in relevant tissues

• Allele-specific expression analysis in heterozygous individuals

The atherosclerosis-associated SNP (rs2850711) and its putative causal variant (rs2676671) identified in the LINC00305 locus provide starting points for understanding genetic contributions to inflammatory diversity .

What technologies and methodological advances could accelerate research on LINC00305 and similar lncRNAs?

Several emerging technologies and methodological advances could significantly accelerate LINC00305 research:

• CRISPR-based techniques:

  • CRISPRi and CRISPRa for precise manipulation of LINC00305 expression

  • CRISPR tiling screens to identify functional domains within LINC00305

  • CRISPR-Cas13 for RNA-targeting approaches

• RNA structure determination:

  • SHAPE-seq and DMS-seq for in vivo structure determination

  • Cryo-EM techniques adapted for lncRNA-protein complexes

  • PARIS (Psoralen Analysis of RNA Interactions and Structures) to map intramolecular interactions

• Spatial transcriptomics:

  • Methods like MERFISH or Visium spatial transcriptomics to map LINC00305 expression within atherosclerotic plaques with spatial resolution

• Proteomics approaches:

  • RNA-protein interaction mapping using CHART-MS or RAP-MS

  • Proximity labeling techniques (BioID, APEX) to identify LINC00305-proximal proteins

• Single-molecule imaging:

  • RNA tracking in living cells to monitor LINC00305 dynamics

  • Super-resolution microscopy to visualize LINC00305-protein interactions

• Organoid and microphysiological systems:

  • Vascular organoids to study LINC00305 in a more physiologically relevant context

  • Organ-on-chip approaches modeling atherosclerotic vessels

• Computational approaches:

  • Machine learning algorithms for lncRNA function prediction

  • Network analysis tools integrating multi-omics data

These technological advances would help overcome current limitations in studying lncRNA biology and accelerate understanding of LINC00305's complex regulatory mechanisms .

How might LINC00305 serve as a biomarker for inflammatory diseases or atherosclerosis?

LINC00305 shows considerable potential as a biomarker for inflammatory diseases, particularly atherosclerosis:

Biomarker applications:

• Diagnostic biomarker: Elevated LINC00305 expression in PBMCs could help identify patients with active atherosclerotic disease

• Risk stratification: LINC00305 levels might correlate with plaque vulnerability or progression risk

• Treatment response monitoring: Changes in LINC00305 expression could indicate effectiveness of anti-inflammatory therapies

• Genetic risk assessment: Genotyping LINC00305 SNPs could contribute to cardiovascular risk prediction

Methodological considerations for biomarker development:

• Standardization of LINC00305 detection methods in clinical samples

• Establishment of reference ranges in diverse populations

• Correlation with existing inflammatory markers and clinical outcomes

• Longitudinal studies to determine predictive value

• Integration into multi-marker panels for improved specificity and sensitivity

The finding that LINC00305 expression is enriched in atherosclerotic plaques and monocytes from patients with atherosclerosis provides preliminary validation of its potential as a disease biomarker. Further clinical studies are needed to establish its utility in various clinical scenarios .

What therapeutic strategies could target LINC00305 or its signaling pathway to modulate inflammation?

Several therapeutic strategies could be developed to target LINC00305 or its signaling pathway:

• Direct LINC00305 targeting:

  • Antisense oligonucleotides (ASOs) designed to bind and neutralize LINC00305

  • Small interfering RNAs (siRNAs) for selective knockdown

  • CRISPR-Cas13 RNA-targeting approaches for specific degradation

• LIMR-LINC00305 interaction inhibition:

  • Small molecule inhibitors designed to disrupt the binding interface

  • Peptide mimetics competing for the binding site

  • RNA aptamers targeting the interaction domain

• LIMR-AHRR pathway modulation:

  • LIMR antagonists to prevent downstream signaling

  • AHRR inhibitors to prevent competition with AHR

  • AHR agonists to counteract AHRR-mediated suppression

• Downstream pathway targeting:

  • Selective NF-κB inhibitors with improved safety profiles

  • Cell-specific delivery of pathway inhibitors to monocytes/macrophages

• Genetic approaches:

  • CRISPR-based therapeutic strategies to modify the LINC00305 locus

  • Gene therapy delivering LINC00305 antagonist constructs

The effectiveness of these approaches would depend on delivery methods, cell-type specificity, and timing of intervention in the disease process. Since LINC00305 appears to function through a well-defined pathway involving LIMR, AHRR, and NF-κB, multiple points of intervention could be explored .

What ethical and methodological considerations should guide translational research on LINC00305?

Translational research on LINC00305 should be guided by several ethical and methodological considerations:

Ethical considerations:

• Ensuring diverse participant representation in genetic and expression studies to avoid population biases

• Transparent communication of potential therapeutic applications and limitations

• Careful evaluation of risk-benefit ratios for any LINC00305-targeting therapies

• Consideration of potential long-term effects of immunomodulation

• Responsible data sharing to advance scientific knowledge while protecting privacy

Methodological considerations:

• Standardization and reproducibility:

  • Development of validated assays for LINC00305 quantification

  • Transparent reporting of experimental conditions and limitations

  • Use of appropriate cell lines and animal models

• Translational pathway planning:

  • Clearly defined preclinical milestones before human studies

  • Early consideration of delivery methods for RNA-based therapeutics

  • Identification of appropriate patient populations for clinical trials

• Comprehensive safety assessment:

  • Investigation of LINC00305 functions beyond inflammation

  • Evaluation of effects on normal immune responses to pathogens

  • Assessment of potential compensatory mechanisms after inhibition

• Regulatory considerations:

  • Early engagement with regulatory agencies regarding novel RNA therapeutics

  • Development of appropriate biomarkers for clinical trials

  • Consideration of companion diagnostics for patient selection

These considerations would help ensure that translational research on LINC00305 proceeds responsibly and effectively toward meaningful clinical applications .