NREP Human

What is NREP and where is it expressed in human tissues?

NREP is a conserved RNA-binding intracellular protein that was first identified by Studler and colleagues. It is relatively conserved across many species and is expressed in multiple human tissues including the brain, smooth muscles, regenerated tissue, and malignant glioblastomas . The protein plays essential roles in promoting wound closure and tissue regeneration.

Methodological Answer: To accurately characterize NREP expression patterns in human tissues, researchers should employ a multi-platform approach:

• Utilize immunohistochemistry (IHC) with validated NREP-specific antibodies on tissue microarrays containing multiple organ samples

• Confirm protein expression with western blotting of tissue lysates

• Quantify mRNA expression through RT-qPCR and RNA-sequencing

• For subcellular localization, implement confocal microscopy with fluorescently-tagged antibodies

• Validate findings using public transcriptomic databases (e.g., GTEx, Human Protein Atlas)

How is NREP gene expression regulated under normal and hypoxic conditions?

NREP expression is significantly regulated by hypoxia through the direct action of HIF-1α (Hypoxia-Inducible Factor 1-alpha). Under hypoxic conditions, NREP is upregulated in cells, and this promotion is reversed when HIF-1α is suppressed .

Methodological Answer: To investigate NREP regulation, researchers should:

• Expose cells to controlled hypoxic conditions (1-2% O₂) in a hypoxia chamber

• Measure NREP mRNA and protein expression changes at various time points

• Implement luciferase reporter assays with NREP promoter constructs to assess transcriptional activity

• Perform chromatin immunoprecipitation (ChIP) assays to confirm direct binding of HIF-1α to the NREP promoter

• Conduct promoter deletion experiments to identify critical regulatory regions (particularly -735 to -383 bp and -12 to +193 bp regions which show significant HIF-1α binding)

• Use siRNA or shRNA to knockdown HIF-1α and observe effects on NREP expression

What functional roles has NREP been demonstrated to have in normal human physiology?

NREP plays several important roles in normal physiological processes:

Methodological Answer: To characterize NREP's physiological functions, researchers should:

• Implement CRISPR-Cas9 gene editing to create NREP knockout cell lines

• Use RNAi approaches (siRNA/shRNA) for transient and stable knockdown experiments

• Perform wound healing assays to assess effects on cellular migration and wound closure

• Measure fibroblast proliferation using BrdU incorporation or EdU labeling assays

• Analyze cellular migration through Transwell and scratch assays

• Employ rescue experiments by reintroducing wild-type or mutant NREP to knockdown cells

• Develop tissue-specific conditional knockout mouse models for in vivo functional studies

How does NREP contribute to breast cancer progression and metastasis?

Bioinformatics analysis shows NREP is highly expressed in breast cancer and closely correlated with poor survival outcomes. Research demonstrates NREP plays multiple roles in promoting breast cancer progression:

Methodological Answer: To thoroughly investigate NREP's role in breast cancer, implement the following comprehensive approaches:

• Generate stable NREP knockdown and overexpression breast cancer cell lines

• Assess cell proliferation using MTT assays, colony formation assays, and EdU incorporation tests

• Analyze cell cycle progression through flow cytometry with propidium iodide staining

• Measure apoptosis using Annexin V/PI staining and TUNEL assays

• Quantify caspase-3 activity with specific activity assays

• Evaluate angiogenic potential through tube formation assays with HUVECs

• Analyze migration and invasion using Transwell chambers with and without Matrigel coating

• Assess EMT markers (E-cadherin, N-cadherin) via western blotting and immunofluorescence

• Measure metabolic changes through glucose consumption, ATP levels, and lactate production assays

• Confirm in vivo effects using xenograft models, analyzing tumor growth, microvessel density, and metastatic potential

What molecular mechanisms underlie NREP's effects on cellular metabolism in cancer cells?

NREP significantly influences cancer cell metabolism, particularly glycolysis. Research shows NREP promotes cellular glycolysis and enhances glucose consumption, ATP production, lactate production, and glucose transporter expression .

Methodological Answer: To elucidate NREP's metabolic effects, researchers should:

• Measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using Seahorse XF Analyzer

• Quantify glycolytic intermediates through targeted metabolomics

• Analyze expression of key glycolytic enzymes (HK2, PKM2, LDHA) via western blotting and qPCR

• Implement ¹³C-glucose isotope tracing combined with mass spectrometry to track carbon flux

• Assess glucose uptake using fluorescent glucose analogs (2-NBDG)

• Measure mitochondrial membrane potential with JC-1 or TMRM dyes

• Evaluate the impact of glycolysis inhibitors (2-DG, 3-BP) on NREP-mediated phenotypes

• Perform metabolic rescue experiments by modulating specific metabolic pathways in NREP-manipulated cells

What is the relationship between NREP and the EMT process in human cancers?

NREP appears to be a significant promoter of epithelial-mesenchymal transition (EMT) in cancer cells.

Methodological Answer: To comprehensively characterize NREP's role in EMT:

• Analyze expression of epithelial markers (E-cadherin, claudins, occludin) and mesenchymal markers (N-cadherin, vimentin, fibronectin) at mRNA and protein levels

• Implement immunofluorescence microscopy to visualize changes in cell morphology and marker localization

• Perform invasion assays using 3D matrices that better recapitulate in vivo conditions

• Analyze expression of EMT-inducing transcription factors (SNAIL, SLUG, TWIST, ZEB1/2) following NREP manipulation

• Use chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify NREP-regulated EMT-related genes

• Establish cell lines with inducible NREP expression to monitor temporal changes in EMT

• Assess cell-cell adhesion properties using aggregation assays

• Evaluate clinical correlations between NREP expression and EMT marker status in patient samples

What are the optimal methods for manipulating NREP expression in experimental systems?

Methodological Answer: For effective NREP manipulation, researchers should consider:

Knockdown approaches:

• siRNA transfection for short-term suppression (48-72 hours)

• shRNA delivered via lentiviral vectors for stable long-term knockdown

• CRISPR-Cas9 gene editing for complete knockout

• Validate knockdown efficiency using both qRT-PCR and western blotting

• Include rescue experiments with wildtype NREP to confirm specificity

Overexpression approaches:

• Clone full-length NREP cDNA into expression vectors with strong promoters

• Use lentiviral or retroviral systems for stable integration

• Consider inducible expression systems (Tet-On/Off) for controlled expression

• Add epitope tags (HA, Flag, GFP) for easy detection and immunoprecipitation

• Verify overexpression at both mRNA and protein levels

For both approaches, include appropriate controls (scrambled siRNA, empty vector) and multiple clones to account for clonal variation .

How should researchers design animal models to study NREP function in cancer progression?

Methodological Answer: For robust in vivo NREP studies:

• Xenograft models:

  • Inject NREP-manipulated human cancer cells subcutaneously into immunodeficient mice

  • Monitor tumor growth by caliper measurements or bioluminescence imaging

  • Analyze tumors for proliferation (Ki67), apoptosis (TUNEL), angiogenesis (CD31), and EMT markers

• Orthotopic models:

  • Implant cells directly into the tissue of origin (e.g., mammary fat pad for breast cancer)

  • Provides more physiologically relevant microenvironment

  • Better recapitulates metastatic potential

• Metastasis models:

  • Utilize tail vein or intracardiac injection to study later stages of metastasis

  • Monitor by whole-body imaging if cells express luciferase

  • Analyze metastatic burden in organs by histopathology and PCR

• Transgenic models:

  • Develop conditional NREP knockout mice using Cre-loxP system

  • Cross with tumor-prone mouse models (e.g., MMTV-PyMT for breast cancer)

  • Analyze tumor initiation, progression, and metastasis

• Patient-derived xenografts:

  • Implant human tumor fragments maintaining original NREP expression

  • Evaluate therapeutic responses in the context of NREP status

What bioinformatic approaches are most effective for analyzing NREP expression in cancer datasets?

Methodological Answer: For comprehensive bioinformatic analysis:

How should researchers interpret contradictory findings regarding NREP function across different cancer types?

Methodological Answer: To resolve contradictions in NREP research:

• Systematic literature review:

  • Conduct meta-analysis of published studies

  • Compare methodologies, cell lines, and experimental conditions

  • Identify potential sources of variability

• Context-dependent analysis:

  • Investigate NREP function in multiple cell lines from the same cancer type

  • Compare effects in different cancer subtypes (e.g., breast cancer molecular subtypes)

  • Assess impact of tumor microenvironment factors on NREP function

• Pathway analysis:

  • Determine if NREP interacts with different signaling networks in different cancers

  • Use RNA-seq and pathway enrichment analysis to identify cancer-specific mechanisms

  • Validate key pathways using specific inhibitors

• Protein interaction studies:

  • Perform immunoprecipitation followed by mass spectrometry in different cell types

  • Identify cell-type specific binding partners that may explain divergent functions

  • Validate key interactions using co-IP and proximity ligation assays

• Genetic background considerations:

  • Analyze influence of common genetic alterations (p53, PTEN, RAS) on NREP function

  • Use isogenic cell lines differing only in specific mutations to isolate effects

What are the best approaches to validate NREP as a potential therapeutic target in cancer?

Methodological Answer: For therapeutic target validation:

• Essential role confirmation:

  • Demonstrate that NREP inhibition reduces cancer cell viability in multiple models

  • Show selective toxicity in cancer vs. normal cells

  • Verify effects persist in 3D culture systems and in vivo models

• Target validation techniques:

  • Implement inducible knockdown systems to model therapeutic inhibition

  • Use CRISPR interference (CRISPRi) for dose-dependent suppression

  • Evaluate combination approaches with standard therapies

• Resistance mechanisms:

  • Develop resistant cell lines through long-term NREP suppression

  • Identify compensatory pathways activated upon NREP inhibition

  • Test combination strategies to overcome potential resistance

• Biomarker development:

  • Identify patient subgroups most likely to benefit from NREP targeting

  • Develop assays to measure NREP activity in patient samples

  • Correlate NREP status with response to other therapies

• Therapeutic approaches:

  • Design small molecule inhibitors targeting NREP-protein interactions

  • Develop antisense oligonucleotides or siRNA-based approaches

  • Consider antibody-based strategies if NREP has accessible domains

Table 1: NREP Expression Analysis Across Multiple Breast Cancer Databases

Table 2: Effects of NREP Manipulation on Breast Cancer Cell Functions

Table 3: HIF-1α Binding Sites in the NREP Promoter Region