NELFE Human

What is NELFE and what is its role in human cellular processes?

NELFE is one of the subunits of the Negative Elongation Factor (NELF) complex, which cooperates with DRB sensitivity-inducing factor (DSIF) to inhibit RNA Polymerase II elongation . Beyond its canonical role in transcriptional regulation, NELFE functions as an RNA-binding protein (RBP) that can regulate target RNAs at multiple levels, including RNA stabilization, translation, localization, and degradation . The NELF complex contains several subunits that work together to control gene expression, particularly at the promoter-proximal pausing stage .

As an RBP, NELFE can directly bind to specific RNA sequences and alter their fate within the cell. This function is particularly significant as nearly 2,000 RBPs have been identified, though the mechanisms by which many of them influence cancer progression remain unclear . NELFE represents an important example of how RBPs can acquire oncogenic properties through their RNA regulatory abilities.

How is NELFE expression detected and measured in human tissues?

Researchers employ multiple complementary techniques to detect and measure NELFE expression in human tissues:

Reverse Transcription-Quantitative PCR (RT-qPCR): This method is commonly used to quantify NELFE mRNA expression levels in both fresh tissues and cell lines . RNA is extracted from tissues, reverse-transcribed to cDNA, and then amplified using NELFE-specific primers. The amplification is monitored in real-time and compared to housekeeping genes for normalization.

Immunohistochemistry (IHC): For protein-level detection in clinical samples, paraffin-embedded tissue sections are stained with anti-NELFE antibodies (typically 1:100 dilution, such as with Proteintech 10705-1-AP) . This allows visualization of NELFE expression patterns and subcellular localization within tissue architecture. Pathologists evaluate these stained sections using microscopy to score expression levels.

Western Blotting: For protein quantification in fresh tissues or cell lines, proteins are separated by SDS-PAGE, transferred to membranes, and probed with NELFE-specific antibodies (typically at 1:1000 dilution) . This provides information about protein size and relative abundance across samples.

RNA Sequencing: For comprehensive transcriptome analysis, RNA-seq data (such as from TCGA database) can be analyzed to compare NELFE expression between normal and tumor tissues across large patient cohorts .

These methods are often used in combination to provide robust validation of NELFE expression patterns in clinical samples and experimental models.

What experimental models are available for studying NELFE function?

Several experimental models have been established to investigate NELFE function:

Cell Line Models: Human cancer cell lines such as BGC-823 and AGS (gastric cancer), Huh-1 and Hep3B (liver cancer), and various pancreatic cancer cell lines have been used to study NELFE function . These models allow for manipulation of NELFE expression through knockdown or overexpression approaches.

Knockdown Systems: Short hairpin RNA (shRNA) constructs targeting NELFE have been effectively used to reduce NELFE expression in cell lines . This approach allows researchers to observe phenotypic changes associated with NELFE depletion, including effects on cell viability, proliferation, and metastatic potential.

Mouse Xenograft Models: To study NELFE function in vivo, human cancer cells with manipulated NELFE expression can be implanted into immunodeficient mice. This allows for assessment of tumor growth and metastasis in a more physiologically relevant context . For example, NELFE knockdown in gastric cancer cells significantly inhibited tumor formation ability in nude mice.

Clinical Tissue Samples: Paired tumor and adjacent non-tumor tissues from cancer patients provide crucial validation for findings from experimental models . These samples allow researchers to correlate NELFE expression with clinical parameters and patient outcomes.

Structural Analysis Systems: For studying NELFE's molecular architecture, X-ray crystallography of purified protein components has been employed, enabling insights into its RNA-binding domains and interaction surfaces .

How does NELFE contribute to cancer progression at the molecular level?

NELFE contributes to cancer progression through multiple molecular mechanisms:

Regulation of Oncogenic Transcription Factors: In gastric cancer, NELFE regulates E2F2 expression through direct binding to its 3'UTR, stabilizing the mRNA and increasing E2F2 protein levels . Similarly, in hepatocellular carcinoma, NELFE regulates MYC signaling and defines a subset of MYC target genes known as NELFE-dependent MYC targets (NDMTs) .

Promotion of EMT: In pancreatic cancer, NELFE promotes epithelial-to-mesenchymal transition (EMT), a critical process for cancer cell invasion and metastasis . This involves regulation of EMT markers such as E-cadherin, N-cadherin, and Vimentin, facilitating cancer cell plasticity and motility.

Post-transcriptional Regulation: As an RNA-binding protein, NELFE affects mRNA stability of target genes. This was demonstrated in gastric cancer cells where NELFE knockdown accelerated the decay of E2F2 mRNA . The mRNA decay assay revealed that NELFE binding to the 3'UTR of E2F2 significantly extended its half-life.

Modulation of Tumor Microenvironment: NELFE expression correlates with the proportion of tumor-infiltrating immune cells (TICs) in gastric cancer, suggesting a role in modulating the immune microenvironment . Specific immune cell populations including macrophages and regulatory T cells show altered frequencies in tumors with high NELFE expression.

Cell Cycle Regulation: NELFE affects expression of cell cycle regulators including cyclin D1 and survivin, promoting cancer cell proliferation . This contributes to accelerated tumor growth in various cancer types.

These molecular functions collectively enable NELFE to drive cancer progression across multiple tumor types, influencing core hallmarks of cancer including sustained proliferation, invasion, and metastasis.

What methodologies are used to identify and validate RNA targets of NELFE?

Researchers employ several complementary approaches to identify and validate RNA targets of NELFE:

RNA Immunoprecipitation (RIP): This technique involves immunoprecipitating NELFE-RNA complexes from cell lysates using NELFE-specific antibodies, followed by identification of bound RNAs through RT-qPCR or sequencing . This method was used to demonstrate direct binding between NELFE and E2F2 mRNA in gastric cancer cells.

mRNA Decay Assay: To assess NELFE's effect on RNA stability, researchers treat cells with transcriptional inhibitors (like actinomycin D) and measure the decay rate of potential target mRNAs in NELFE-knockdown versus control cells over time . This approach revealed that NELFE knockdown accelerated E2F2 mRNA decay in gastric cancer cells.

Biotin RNA Pull-down Assay: Biotinylated RNA segments (like 5'UTR, coding sequence, or 3'UTR of potential targets) are synthesized and incubated with cell lysates, followed by pull-down with streptavidin beads and western blotting for NELFE . This technique helps identify which specific regions of target RNAs interact with NELFE. For example, NELFE was shown to bind preferentially to the 3'UTR of E2F2 mRNA.

Luciferase Reporter Assay: Reporter constructs containing the 3'UTR of potential target mRNAs are transfected into cells with or without NELFE knockdown/overexpression . Changes in luciferase activity indicate NELFE-mediated post-transcriptional regulation. This approach validated NELFE's regulation of E2F2 through its 3'UTR in gastric cancer cells.

RNA-seq after NELFE Manipulation: Transcriptome-wide changes after NELFE knockdown or overexpression help identify potential targets on a global scale . In gastric cancer cells, RNA-seq after NELFE knockdown identified 23 downregulated genes, suggesting they might be directly stabilized by NELFE.

Cross-linking Immunoprecipitation (CLIP): This technique involves UV cross-linking of RNA-protein complexes in living cells, followed by immunoprecipitation and sequencing of bound RNAs . This method can identify direct binding sites with nucleotide resolution.

These methodologies provide multiple lines of evidence for direct NELFE-RNA interactions and their functional consequences in cancer contexts.

What is the association between NELFE expression and clinical outcomes in cancer patients?

NELFE expression shows significant associations with clinical parameters and patient outcomes across multiple cancer types:

Expression Patterns:
In pancreatic cancer, NELFE showed significantly higher expression in tumor tissues compared to adjacent non-tumor tissues . Similarly, gastric cancer tissues exhibited elevated NELFE expression compared to normal gastric mucosa .

Correlation with Clinicopathological Features:
In pancreatic cancer, high NELFE expression significantly correlated with:

• Larger tumor size (p=0.04)

• Lymph node metastasis (p=0.001)

• Advanced TNM stage (p=0.003)

This clinical correlation is summarized in the following table from pancreatic cancer studies:

Survival Impact:
In gastric cancer, patients with high NELFE expression showed:

These clinical correlations suggest that NELFE may serve as a prognostic biomarker in multiple cancer types, with high expression generally associated with more aggressive disease features and poorer patient outcomes.

How can researchers effectively manipulate NELFE expression in experimental models?

Researchers have employed several strategies to manipulate NELFE expression for functional studies:

RNA Interference Approaches:

• Short hairpin RNA (shRNA): Stable knockdown of NELFE has been achieved using lentiviral or retroviral vectors expressing shRNAs targeting different regions of NELFE mRNA . This approach provides long-term suppression suitable for both in vitro and in vivo experiments.

• Small interfering RNA (siRNA): For transient knockdown experiments, synthetic siRNAs targeting NELFE can be transfected into cells. This approach is useful for short-term studies and can help validate findings from shRNA experiments.

Protocol Example for shRNA-mediated NELFE Knockdown:

• Design at least two independent shRNA sequences targeting different regions of NELFE mRNA

• Clone these sequences into lentiviral vectors with appropriate selection markers

• Package lentiviral particles in HEK293T cells using standard transfection protocols

• Transduce target cancer cells with viral particles at MOI of 10-20

• Select stable transductants using appropriate antibiotics (e.g., puromycin)

• Validate knockdown efficiency at both mRNA (RT-qPCR) and protein (western blot) levels

Overexpression Systems:

• For gain-of-function studies, NELFE cDNA can be cloned into expression vectors with strong promoters (e.g., CMV) and transfected into cells

• To study protein domains, researchers can express truncated or mutated versions of NELFE

CRISPR-Cas9 Gene Editing:

• For complete knockout studies, CRISPR-Cas9 targeting exons of NELFE can generate cell lines with permanent loss of NELFE expression

• CRISPR interference (CRISPRi) can be used for targeted repression of NELFE transcription

Inducible Expression Systems:

• Tetracycline-inducible (Tet-On/Tet-Off) systems allow for temporal control of NELFE expression

• This approach is particularly useful for studying immediate versus long-term effects of NELFE manipulation

Validation of successful manipulation should include both RNA and protein level assessments, ideally using both RT-qPCR and western blotting to confirm changes in NELFE expression .

What is known about the structure-function relationship of NELFE protein?

The structure-function relationship of NELFE has been partly elucidated through crystallography and biochemical studies:

Structural Architecture:
NELFE is part of the larger NELF complex, which includes NELF-A, NELF-B, NELF-C/D, and NELF-E subunits . X-ray crystallography has revealed that NELF-A and NELF-C form a conserved core subcomplex with a novel fold . This structural information provides insights into how NELF subunits interact and bind to RNA.

RNA Binding Domains:
NELFE contains specific domains that mediate its RNA-binding function:

• One side of the NELF-AC subcomplex exhibits a conserved binding face for single-stranded nucleic acids

• NELFE can bind directly to specific regions of target mRNAs, with a preference for 3'UTR sequences as demonstrated in studies of E2F2 regulation in gastric cancer

Functional Interactions:
The interaction between NELFE and target RNAs has been characterized using various methods:

• Biotin pull-down assays have identified specific RNA segments that interact with NELFE

• mRNP immunoprecipitation assays have confirmed enrichment of target mRNAs such as E2F2 in NELFE complexes

• Luciferase reporter assays have demonstrated functional consequences of NELFE binding to 3'UTR regions

Mutational Analysis:
Mutational studies have begun to identify critical residues for NELFE's RNA binding functions:

• Mutations in the RNA-binding domain affect NELFE's ability to stabilize target mRNAs

• These mutational analyses help distinguish RNA binding from other functions of NELFE

Understanding the structure-function relationships of NELFE is critical for developing potential therapeutic approaches that might disrupt its oncogenic functions in cancer cells.

How does NELFE influence the tumor immune microenvironment?

NELFE expression significantly impacts the tumor immune microenvironment, particularly the composition and distribution of tumor-infiltrating immune cells (TICs):

Impact on Immune Cell Composition:
Computational analysis of TCGA data using CIBERSORTx revealed that NELFE expression levels correlate with the proportion of specific immune cell populations in gastric cancer . Analysis identified five types of TICs significantly associated with NELFE expression:

• Resting memory CD4+ T cells

• Activated memory CD4+ T cells

• Follicular helper T cells

• Regulatory T cells (Tregs)

• M0 macrophages

Methodology for Immune Cell Analysis:
Researchers employ several approaches to characterize immune cell populations in relation to NELFE expression:

• Computational deconvolution of bulk RNA-seq data using algorithms like CIBERSORTx to estimate immune cell proportions

• Correlation analysis between NELFE expression and immune cell signatures using Pearson coefficient and Wilcoxon rank sum tests

• Validation of computational findings using immunohistochemical staining for specific immune cell markers in patient samples

• Functional studies in co-culture systems to directly assess NELFE's impact on immune cell recruitment and activation

Potential Mechanisms:
NELFE may influence the tumor immune microenvironment through several mechanisms:

• Regulation of chemokine/cytokine production by cancer cells

• Alteration of adhesion molecule expression affecting immune cell recruitment

• Modulation of inflammatory signaling pathways

• Indirect effects mediated through NELFE-regulated target genes like E2F2

Understanding NELFE's impact on the tumor immune microenvironment may reveal opportunities for combining NELFE-targeted therapies with immunotherapeutic approaches in cancer treatment.

What is the potential of NELFE as a therapeutic target in cancer?

Based on its widespread oncogenic functions, NELFE represents a promising therapeutic target in multiple cancer types:

Rationale for Targeting NELFE:

• Consistent overexpression in multiple cancer types including pancreatic, gastric, and hepatocellular carcinoma

• Significant association with poor clinical outcomes and advanced disease features

• Involvement in multiple cancer hallmarks including proliferation, metastasis, and EMT

• Unique RNA-binding properties that could be specifically targeted

Potential Therapeutic Strategies:

• RNA-based approaches:

  • Antisense oligonucleotides (ASOs) designed to reduce NELFE expression

  • siRNA/shRNA delivery systems for targeted knockdown in cancer cells

• Small molecule inhibitors:

  • Compounds targeting the RNA-binding domains of NELFE

  • Disruptors of protein-protein interactions between NELFE and other NELF complex components

• Combination approaches:

  • NELFE inhibition combined with standard chemotherapies

  • Synergistic targeting of NELFE alongside its downstream effectors (e.g., E2F2, MYC pathways)

Methodological Considerations for Target Validation:
Researchers should employ multiple approaches to validate NELFE as a therapeutic target:

• Generate genetic models with inducible NELFE knockdown/knockout in established tumors

• Assess effects on tumor regression, metastatic burden, and survival

• Evaluate potential toxicities in normal tissues

• Identify biomarkers that predict response to NELFE-targeted therapies

Challenges and Future Directions:

• Developing delivery systems that can effectively reach cancer cells in vivo

• Identifying cancer subtypes most likely to respond to NELFE inhibition

• Understanding potential resistance mechanisms to NELFE-targeted therapies

• Designing highly specific inhibitors that don't affect other RNA-binding proteins

The multifaceted role of NELFE in cancer biology suggests that its targeting could provide therapeutic benefits across multiple tumor types, particularly in cancers with high NELFE expression.