Recombinant Human Putative uncharacterized protein encoded by LINC00301 (LINC00301)

What is LINC00301 and how is it classified in genomic databases?

LINC00301 (Long Intergenic Non-protein Coding RNA 301) is a non-coding RNA gene also known as C11orf64 or NCRNA00301. It is classified as a long non-coding RNA located on chromosome 11q12.2 . The gene structure consists of 7 exons spanning positions 60615751 to 60687149 on chromosome 11 (according to reference assembly NC_000011.10) . Despite being primarily a non-coding RNA, there is evidence of a putative uncharacterized protein product consisting of 95 amino acids that can be recombinantly expressed .

What expression patterns of LINC00301 have been observed in normal and pathological tissues?

LINC00301 shows differential expression patterns between normal and cancerous tissues. It is significantly upregulated in non-small cell lung cancer (NSCLC) compared to adjacent normal tissues, with approximately 5.29-fold higher expression in lung cancer samples . The expression profile of LINC00301 across various cancer cell lines can be accessed through the Cancer Cell Line Encyclopedia (CCLE) . High expression of LINC00301 in NSCLC correlates with poorer prognosis, suggesting its potential value as a prognostic biomarker .

How does LINC00301 contribute to cancer progression mechanisms?

LINC00301 exhibits multiple oncogenic functions in non-small cell lung cancer through several mechanisms:

• Cellular proliferation: LINC00301 facilitates cell proliferation by modulating cell cycle regulation .

• Apoptosis inhibition: It suppresses programmed cell death pathways in cancer cells .

• Metastatic potential: LINC00301 promotes cell migration and invasion capabilities .

• Immune modulation: It increases regulatory T cell (Treg) population while decreasing CD8+ T cell population in tumors through targeting TGF-β pathways, creating an immunosuppressive microenvironment .

These multifaceted roles make LINC00301 a significant contributor to cancer progression and potential therapeutic target.

What are the key signaling pathways modulated by LINC00301 in cancer cells?

LINC00301 interacts with several critical oncogenic pathways:

• FOXC1/LINC00301/EZH2/EAF2/pVHL/HIF1α pathway: LINC00301, mediated by transcription factor FOXC1, binds to EZH2 in the nucleus to promote H3K27me3 at the EAF2 promoter. Downregulation of EAF2 leads to decreased pVHL stability, resulting in increased HIF1α expression, which promotes tumorigenesis .

• FOXC1/LINC00301/miR-1276/HIF1α pathway: In the cytoplasm, LINC00301 functions as a competing endogenous RNA (ceRNA) by sponging miR-1276, thereby indirectly upregulating HIF1α expression .

These dual nuclear and cytoplasmic functions highlight the versatility of LINC00301 in promoting oncogenic processes.

What is the mechanism of FOXC1-mediated regulation of LINC00301 expression?

The transcription factor FOXC1 directly regulates LINC00301 expression by binding to its promoter region. This has been validated through chromatin immunoprecipitation (ChIP) assays that confirmed the binding activity between FOXC1 and LINC00301 promoters . The regulatory relationship suggests a feed-forward oncogenic loop, where FOXC1 activation leads to increased LINC00301 expression, which further promotes cancer progression. Bioinformatic analysis also indicates potential CpG islands in the LINC00301 promoter region that may contribute to its regulation through epigenetic mechanisms .

How does the interaction between LINC00301 and EZH2 influence epigenetic regulation?

LINC00301 directly interacts with EZH2 (enhancer of zeste homolog 2), a histone methyltransferase and core component of Polycomb Repressive Complex 2 (PRC2). Specifically:

• The nucleotides 83-123 of LINC00301 bind to amino acids 612-727 of EZH2 protein .

• This interaction has been confirmed through RNA immunoprecipitation (RIP), RNA pull-down, protein domain mapping, and RNA electrophoresis mobility shift assay (EMSA) .

• The LINC00301-EZH2 complex promotes H3K27 trimethylation (H3K27me3) at the EAF2 promoter, leading to transcriptional repression of EAF2 .

• Quantitative assessment of this interaction has been performed using Alpha assay technology .

This epigenetic regulation mechanism explains how LINC00301 can influence gene expression patterns in cancer cells without directly encoding functional proteins.

What is the role of LINC00301 in immune modulation within the tumor microenvironment?

LINC00301 significantly influences the tumor immune microenvironment by:

• Increasing regulatory T cell (Treg) population while decreasing CD8+ T cell infiltration in tumors .

• Modulating TGF-β signaling pathways, which are critical for immunosuppression .

• Creating conditions favorable for immune evasion, thereby promoting tumor progression.

This immunomodulatory function adds another dimension to LINC00301's oncogenic potential beyond direct effects on cancer cell biology. Understanding these mechanisms could inform immunotherapy approaches targeting LINC00301-associated pathways.

What are the optimal methods for detecting and quantifying LINC00301 expression?

Several complementary techniques are recommended for accurate detection and quantification of LINC00301:

• Quantitative PCR (qPCR): The gold standard for measuring LINC00301 expression in clinical specimens and cell lines . Proper primer design is critical for specificity.

• Fluorescence in situ hybridization (FISH): Optimal for determining the subcellular localization of LINC00301 . Research has shown LINC00301 is predominantly nuclear with some cytoplasmic presence .

• RNA-Seq: Provides comprehensive transcriptome-wide analysis of LINC00301 expression in relation to other genes.

• Northern blotting: Useful for confirming the size and integrity of LINC00301 transcripts.

When designing experiments, researchers should consider the following methodological considerations:

• Include appropriate housekeeping genes for normalization

• Validate expression patterns across multiple techniques

• Account for potential isoforms of LINC00301

What techniques are recommended for studying LINC00301 interactions with proteins such as EZH2?

Several specialized techniques have been successfully employed to study LINC00301-protein interactions:

• RNA Immunoprecipitation (RIP): Effective for exploring the binding activity between LINC00301 and proteins like EZH2 .

• RNA pull-down assays: Used in conjunction with dot-blot analysis to identify specific binding regions between LINC00301 and proteins .

• Protein domain mapping: Helps determine which specific domains of proteins (e.g., EZH2) interact with LINC00301 .

• RNA Electrophoresis Mobility Shift Assay (EMSA): Valuable for confirming direct RNA-protein interactions in vitro .

• Alpha assay technology: Provides quantitative assessment of the interaction between LINC00301 and EZH2 .

• Bioinformatic prediction tools: Resources such as catRAPID (http://s.tartaglialab.com/page/catrapid_group) and RPISeq (http://pridb.gdcb.iastate.edu/RPISeq/) can predict binding potential between LINC00301 and proteins .

What are the considerations for recombinant expression and purification of LINC00301-encoded protein?

For researchers working with the putative protein encoded by LINC00301, consider these methodological approaches:

• Expression system: E. coli has been successfully used for recombinant expression of the full-length human putative protein encoded by LINC00301 .

• Protein tagging: His-tagging has proven effective for purification purposes (His-tagged recombinant protein consisting of amino acids 1-95) .

• Purification quality: Aim for >90% purity as determined by SDS-PAGE for downstream applications .

• Storage and stability:

  • Store at -20°C/-80°C upon receipt

  • Aliquot to avoid repeated freeze-thaw cycles

  • For working solutions, store aliquots at 4°C for up to one week

  • Use Tris/PBS-based buffer with 6% Trehalose, pH 8.0

• Reconstitution protocol:

  • Briefly centrifuge vials before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

How might targeting LINC00301 be developed as a therapeutic strategy in cancers?

Developing LINC00301-targeted therapies presents several promising approaches:

• Antisense oligonucleotides (ASOs): Design specific ASOs targeting LINC00301 to reduce its expression, potentially reversing its oncogenic effects.

• CRISPR-Cas9 gene editing: Targeted disruption of LINC00301 genomic loci or its regulatory elements could provide therapeutic benefit.

• Small molecule inhibitors: Developing compounds that disrupt the LINC00301-EZH2 interaction could prevent downstream epigenetic modifications.

• Immunotherapy combinations: Since LINC00301 modulates the immune microenvironment, combining LINC00301 inhibition with immune checkpoint blockade therapies might enhance treatment efficacy .

• Targeting downstream pathways: Inhibiting the HIF1α pathway components activated by LINC00301 could represent an indirect approach to mitigate its effects .

Challenges to consider include delivery systems for RNA-targeted therapies, potential off-target effects, and identifying appropriate patient populations for clinical trials based on LINC00301 expression profiles.

What are the potential roles of LINC00301 isoforms or mutations in disease heterogeneity?

Further research is needed to explore:

• Alternative splicing variants: Characterizing different LINC00301 isoforms and their specific functions in normal and pathological contexts.

• Single nucleotide polymorphisms (SNPs): Investigating whether genetic variations in LINC00301 correlate with disease susceptibility or treatment outcomes.

• Structural variations: Determining if the functional domains of LINC00301, particularly the EZH2-binding region (nucleotides 83-123), exhibit mutations that affect its oncogenic properties .

• Expression heterogeneity: Exploring whether LINC00301 expression varies across different cancer subtypes or within different regions of the same tumor.

• Epigenetic regulation: Investigating how DNA methylation or histone modifications of the LINC00301 locus might contribute to its expression patterns.

This research direction could provide insights into personalized medicine approaches based on LINC00301 status.

How does LINC00301 interact with other non-coding RNAs in regulatory networks?

LINC00301 functions within complex regulatory networks involving other non-coding RNAs:

• miRNA interactions: Beyond miR-1276, which other miRNAs might interact with LINC00301, and what are their downstream effects?

• Competing endogenous RNA (ceRNA) networks: Comprehensive mapping of the ceRNA network involving LINC00301 could reveal additional indirect regulatory mechanisms .

• Coordinated regulation with other lncRNAs: Investigating whether LINC00301 acts in concert with other lncRNAs in cancer progression.

• Feedback loops: Exploring potential feedback mechanisms where LINC00301 and its target genes reciprocally regulate each other.

• Integration with transcription factor networks: Further investigation of how transcription factors beyond FOXC1 might regulate LINC00301 expression .

Understanding these complex interactions could provide a more complete picture of LINC00301's role in cellular regulatory networks and identify additional intervention points for therapeutic development.