MYEOV Antibody

What is MYEOV and why is it significant in cancer research?

MYEOV (Myeloma-overexpressed gene, also known as OCIM) is a candidate oncogene initially identified in multiple myeloma. It is located on chromosome 11q13.3, a region frequently amplified in various cancers . MYEOV has gained research significance due to its overexpression in multiple cancer types including myeloma, breast, lung, pancreatic, and esophageal cancers, where it often correlates with poor prognosis . Recent studies have revealed that MYEOV may have originated as an enhancer element that acquired protein-coding potential in primates while maintaining regulatory functions, potentially affecting the expression of nearby genes like CCND1 (Cyclin D1) . This dual nature makes MYEOV particularly interesting in understanding cancer mechanisms and as a potential prognostic biomarker.

What types of MYEOV antibodies are currently available for research?

Several types of MYEOV antibodies are available for different research applications:

• Rabbit Polyclonal antibodies such as Abcam's ab121387, validated for immunohistochemistry on paraffin-embedded tissues (IHC-P) and immunocytochemistry/immunofluorescence (ICC/IF) with human samples

• Rabbit Polyclonal antibodies like Proteintech's 11151-1-AP, suitable for Western blotting and IHC-P applications

• Antibodies used in quantitative sandwich enzyme immunoassay kits (ELISA) for detection of MYEOV in human serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids

When selecting antibodies, researchers should consider their specific application needs and the validation data available for each product.

What are the optimal protocols for detecting MYEOV in tissue samples using immunohistochemistry?

For immunohistochemical detection of MYEOV in tissues, the following optimized protocol has been successfully employed:

• Section paraffin-embedded tissues at 4-μm thickness and mount on polylysine-coated glass slides

• Incubate slides at 62°C for 2 hours, followed by deparaffinization and rehydration

• Perform heat-mediated antigen retrieval in 10 mmol/L Tris-citrate buffer (pH 7.0) in a pressure cooker, or alternatively use Tris-EDTA buffer (pH 9.0)

• Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes at room temperature

• Block nonspecific binding with 5% normal goat serum in PBS containing 0.1% Tween 20 for 1 hour

• Incubate with primary MYEOV antibody overnight at 4°C:

  • For ab121387: Use at 1:400 dilution

  • For 11151-1-AP: Use at 1:200 dilution

• Apply appropriate secondary antibody and develop signal according to standard protocols

• Counterstain, dehydrate, and mount

For scoring MYEOV expression in tissue samples, a standard approach combines intensity and distribution:

• Intensity scoring: 0 (negative), 1 (weak), 2 (moderate), or 3 (strong)

• Distribution scoring: 0 (negative) for no staining, 1 (focal) for <50% positive staining, 2 (diffuse) for 51-80% positive staining, or 3 for 81-100% positive staining

• Final score = Intensity score × Distribution score (0 = "negative", 1-3 = "weak", 4-6 = "moderate", ≥7 = "strong positive")

This scoring system has been successfully used to demonstrate correlations between MYEOV expression and clinical outcomes in cancer studies .

What challenges might researchers encounter when detecting MYEOV protein versus mRNA?

Researchers face several specific challenges when detecting MYEOV protein compared to its mRNA:

• Functional protein uncertainty: Despite the presence of an ORF, evidence for a functional MYEOV protein remains limited, making protein detection potentially more challenging than mRNA detection .

• Dual functional nature: Recent studies indicate MYEOV may function both as a protein-coding gene and as an enhancer element, complicating the interpretation of protein detection results .

• Antibody validation concerns: Due to the limited characterization of the MYEOV protein, antibody specificity requires rigorous validation through appropriate controls including knockdown experiments.

• Expression level discrepancies: While MYEOV mRNA is overexpressed in various cancers, protein levels may not directly correlate with mRNA levels due to potential post-transcriptional regulation .

• Evolutionary considerations: Since the MYEOV ORF appears to be primate-specific, translation may be subject to evolutionary-specific regulatory mechanisms not present for more conserved genes .

To address these challenges, a multi-modal approach combining mRNA detection (qRT-PCR, RNA-seq), protein detection (Western blot, IHC, ICC), and functional studies is recommended to gain a comprehensive understanding of MYEOV's role in experimental models.

How can researchers quantitatively measure MYEOV in biological samples?

For quantitative measurement of MYEOV in biological samples, researchers can employ several approaches:

• ELISA-based detection:

  • Quantitative sandwich enzyme immunoassay techniques are available for measuring MYEOV in human serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids

  • These assays employ antibodies specific for human MYEOV pre-coated onto microplates

  • The detection process typically involves:

    • Adding samples and standards to wells where MYEOV binds to immobilized antibody

    • Washing away unbound substances

    • Adding detection antibody specific for MYEOV

    • Adding enzyme conjugate and substrate solution

    • Measuring color development proportional to MYEOV concentration

• Western blot quantification:

  • Extract proteins using standard lysis buffers

  • Separate by SDS-PAGE and transfer to membranes

  • Probe with MYEOV antibodies (e.g., 11151-1-AP at 1:600 dilution)

  • Use appropriate secondary antibodies and chemiluminescence detection

  • Quantify band intensity relative to loading controls

• qRT-PCR for mRNA quantification:

  • Extract total RNA from samples

  • Perform reverse transcription

  • Quantify MYEOV mRNA levels using specific primers

  • Normalize to appropriate housekeeping genes

  • This approach has been successfully used to demonstrate MYEOV overexpression in multiple cancer types

• Immunohistochemistry scoring:

  • Use the standardized scoring system described in section 2.1

  • For digital quantification, employ image analysis software to measure staining intensity and distribution

  • Correlate scores with clinical parameters for prognostic studies

How does MYEOV expression correlate with prognosis across different cancer types?

MYEOV expression has been studied as a prognostic marker in several cancer types:

These findings collectively suggest that MYEOV may serve as a valuable prognostic biomarker across multiple cancer types, with high expression generally indicating poorer clinical outcomes.

What is known about MYEOV's potential role as a competing endogenous RNA (ceRNA) in cancer?

Emerging evidence suggests MYEOV may function as a competing endogenous RNA (ceRNA) in certain cancer types:

• Mechanism of Action:

  • As a ceRNA, MYEOV transcript could compete for microRNA binding, thereby affecting the regulation of other cancer-related genes

  • This represents a non-protein-coding function that could contribute to cancer progression

• Pancreatic Cancer:

  • Studies have identified MYEOV as potentially forming an interaction network with other pancreatic cancer-related genes through a ceRNA mechanism

  • This network may influence multiple cancer-associated pathways, affecting proliferation, invasion, and metastasis

• Non-Small Cell Lung Cancer:

  • Research suggests MYEOV transcript functions as a ceRNA in NSCLC

  • This mechanism may play a critical role in the invasion and metastasis of NSCLC cells

  • MYEOV expression correlates with immune cell infiltration patterns in NSCLC, suggesting potential immunomodulatory effects that might be mediated through ceRNA functions

• Experimental Evidence:

  • The ceRNA function has been supported by expression correlation analyses and functional studies

  • Knockdown experiments using siRNAs targeting MYEOV have demonstrated effects on cancer phenotypes that may be mediated through this mechanism

This ceRNA function represents an important area for further investigation, as it suggests MYEOV may influence cancer progression even in the absence of a functional protein product.

How can MYEOV antibodies help investigate its relationship with CCND1 and enhancer function?

MYEOV antibodies can be valuable tools for investigating its relationship with CCND1 and potential enhancer function through several approaches:

• Co-expression Analysis:

  • Use MYEOV antibodies in combination with CCND1 antibodies for dual immunofluorescence staining

  • Analyze correlation of expression patterns in tissue samples

  • Identify cell populations where both proteins are co-expressed

• Chromatin Studies:

  • Investigate the enhancer activity of the MYEOV locus using ChIP assays with antibodies against enhancer-associated histone marks (H3K4me1, H3K27ac)

  • Recent studies have shown these enhancer marks overlapping MYEOV in multiple tissues, including B cells, liver, and lung tissue

  • Analysis of 3D genome datasets has revealed chromatin interactions between the MYEOV-3'-enhancer and CCND1

• Evolutionary Conservation:

  • The enhancer element within MYEOV is highly conserved across species, including those lacking the MYEOV ORF

  • A 273 bp conserved region within MYEOV is found in all mammals and even chickens

  • This enhancer consistently appears near CCND1 orthologues across diverse species

  • Similar interactions between the enhancer region and CCND1/Ccnd1 have been observed in both humans and mice

• Cancer Context:

  • MYEOV and CCND1 are often co-amplified in multiple cancers, particularly in multiple myeloma with 11q13 duplication

  • In esophageal squamous cell carcinomas, co-amplification of both genes can lead to epigenetic silencing of MYEOV

  • In primary plasma cell leukemia, chromosomal rearrangements can juxtapose super-enhancers next to both MYEOV and CCND1, leading to overexpression of both genes

These approaches can help elucidate whether MYEOV primarily functions through its protein product, as an enhancer element regulating CCND1, or through both mechanisms in different contexts.

How do current research findings reconcile MYEOV's potential dual function as a coding gene and an enhancer element?

Current research suggests a fascinating evolutionary scenario for MYEOV that helps explain its dual nature:

• Evolutionary Origin:

  • MYEOV appears to be a primate-specific gene with a de novo open reading frame (ORF) that originated within an evolutionarily older enhancer region

  • The enhancer element is deeply conserved across species from amphibians/amniotes divergence

  • This suggests that the protein-coding function is a relatively recent evolutionary innovation, while the enhancer function is ancient

• Functional Evidence of Enhancer Activity:

  • MYEOV's 3' UTR region shows enhancer activity in B cells based on ATAC-STARR-seq data from GM12878 lymphoblastoid B cell line

  • This region displays characteristic enhancer histone marks (H3K4me1, H3K27ac) in multiple healthy human tissues

  • 3D genome analysis reveals chromatin interactions between this enhancer region and CCND1

• Conservation Across Species:

  • Similar enhancer activity and chromatin interactions with Ccnd1 are observed in mice, despite mice lacking the MYEOV ORF

  • The interaction pattern coincides with CTCF binding sites in both humans and mice

  • The enhancer state is conserved in non-human primates, dogs, rats, and mice

• Implications for Cancer:

  • In cancer contexts, MYEOV overexpression might affect both its potential protein function and enhancer activity

  • This could explain why MYEOV and CCND1 are often co-dysregulated in cancers

  • Cancer-associated genomic rearrangements might disrupt the ancient regulatory relationship between these loci

This dual nature makes MYEOV particularly interesting for studying evolutionary processes where regulatory elements acquire protein-coding potential while maintaining their original function. It also suggests that therapeutic strategies might need to target both aspects of MYEOV function.

What experimental approaches can determine if MYEOV's oncogenic effects are mediated through RNA, protein, or enhancer mechanisms?

Distinguishing between MYEOV's potential RNA, protein, or enhancer functions requires sophisticated experimental approaches:

• RNA-Specific Manipulations:

  • Use antisense oligonucleotides to target MYEOV transcript without affecting DNA

  • Employ RNA interference (siRNA) techniques - previous studies have used sequences such as:

    • si-1: 5ʹ-UCA ACG CCC ACU CUA AAG GCU UCU C-3ʹ

    • si-2: 5ʹ-GGA UGU AAG UUA UCA ACU A-3ʹ

  • Analyze effects on cancer phenotypes and CCND1 expression

  • If effects are mediated through RNA mechanisms, these approaches should disrupt oncogenic functions

• Protein Detection and Manipulation:

  • Use Western blotting with validated antibodies (e.g., 11151-1-AP) to detect the ~55 kDa MYEOV protein

  • Perform immunoprecipitation followed by mass spectrometry to confirm protein existence

  • Create mutant constructs that maintain RNA sequence but disrupt protein translation

  • If protein function is critical, these mutations should abrogate oncogenic effects

• Enhancer Function Analysis:

  • Use CRISPR-based approaches to specifically modify the enhancer region without affecting the coding sequence

  • Perform chromatin conformation capture (3C, 4C, Hi-C) to analyze interactions with CCND1

  • Compare enhancer activity (using H3K4me1 and H3K27ac ChIP-seq) in normal vs. cancer cells

  • If enhancer function is primary, these modifications should affect CCND1 expression and oncogenic phenotypes

• Comparative Studies Across Species:

  • Compare MYEOV function in primate cells (with ORF) vs. non-primate cells (without ORF)

  • Analyze whether the conserved enhancer region in mice affects Ccnd1 expression similarly to humans

  • Introduce human MYEOV ORF into mouse cells to determine if protein function adds oncogenic properties

  • These approaches can help distinguish ancestral enhancer functions from more recently evolved protein functions

• Cellular Localization Studies:

  • Use immunofluorescence with MYEOV antibodies to determine protein localization

  • Employ RNA FISH to track MYEOV transcript localization

  • Compare with enhancer-associated markers and CCND1 expression

  • Different functional mechanisms would likely show distinct localization patterns

These complementary approaches can help determine which aspect of MYEOV's complex biology primarily drives its oncogenic effects in different cancer contexts.

How does MYEOV expression correlate with immune infiltration in the tumor microenvironment?

Recent studies have revealed important correlations between MYEOV expression and immune cell infiltration in cancer:

• Non-Small Cell Lung Cancer (NSCLC):

  • MYEOV expression shows distinct correlations with immune cell infiltration depending on NSCLC subtype

  • In lung adenocarcinoma (LUAD): MYEOV expression negatively correlates with tumor purity and B cell infiltration

  • In lung squamous cell carcinoma (LUSC): MYEOV expression negatively correlates with tumor purity but positively associates with CD8+ T cells, CD4+ T cells, dendritic cells, and neutrophils

  • These findings suggest MYEOV may influence the immune microenvironment differently across cancer subtypes

• Potential Mechanisms:

  • MYEOV may function as a competing endogenous RNA (ceRNA) affecting expression of genes involved in immune regulation

  • The enhancer function of MYEOV might influence expression of nearby genes involved in immune response

  • MYEOV-expressing cancer cells might secrete factors that attract or repel specific immune cell populations

• Immune Cell Profiling Techniques:

  • Studies have employed computational methods like the ImmuCellAI algorithm to analyze relationships between MYEOV expression and 24 immune cell types

  • These analyses reveal complex patterns of immune cell infiltration associated with MYEOV expression

• Clinical Implications:

  • The correlation between MYEOV expression and immune infiltration suggests potential implications for immunotherapy response

  • High MYEOV expression might identify tumors with particular immune microenvironment profiles

  • This could inform stratification for immunotherapy approaches

Understanding these relationships could help develop more effective immunotherapeutic strategies for cancers with high MYEOV expression, potentially overcoming resistance mechanisms or enhancing response rates.

What validation methods should be employed to ensure specificity of MYEOV antibodies?

Ensuring MYEOV antibody specificity requires rigorous validation approaches:

Thorough validation is particularly important given the complex nature of MYEOV as both a potential protein-coding gene and an enhancer element.

How should researchers interpret discrepancies between MYEOV mRNA and protein detection results?

Discrepancies between MYEOV mRNA and protein detection are common and can provide important biological insights:

• Potential Biological Explanations:

  • Post-transcriptional regulation: MYEOV mRNA might be subject to microRNA-mediated suppression or other regulatory mechanisms

  • Protein stability issues: MYEOV protein might have a short half-life or undergo rapid degradation

  • Translational efficiency: The primate-specific ORF might be inefficiently translated

  • Dual function: MYEOV might primarily function through its RNA (e.g., as a ceRNA) or enhancer activity rather than as a protein in some contexts

• Technical Considerations:

  • Antibody sensitivity: Protein detection methods might be less sensitive than PCR-based mRNA detection

  • Epitope accessibility: Post-translational modifications or protein interactions might mask antibody epitopes

  • Sample preparation differences: RNA and protein extraction methods affect different subcellular compartments

• Interpretative Framework:

  • Context-dependent translation: Consider whether specific cellular conditions might activate or suppress MYEOV translation

  • Cancer-specific alterations: Genomic or epigenomic changes in cancer might affect the relationship between mRNA and protein levels

  • Evolutionary considerations: The relatively recent emergence of the MYEOV ORF might result in inefficient translation machinery

• Recommended Approach:

  • Always analyze both mRNA and protein in the same samples

  • Consider subcellular localization of both mRNA and protein

  • Correlate with functional readouts (e.g., effects on CCND1 expression or cancer phenotypes)

  • Use multiple antibodies targeting different epitopes

  • Include appropriate positive and negative controls

These discrepancies, rather than being merely technical issues, may reflect the complex biology and evolutionary history of MYEOV as a gene that potentially functions through multiple mechanisms.

What are the key technical considerations when designing experiments to study MYEOV's enhancer function?

Investigating MYEOV's enhancer function requires specific technical considerations:

• Chromatin State Analysis:

  • ChIP-seq for enhancer-associated histone marks (H3K4me1, H3K27ac)

  • ATAC-seq to assess chromatin accessibility

  • DNase-seq to identify DNase I hypersensitivity sites

  • Previous studies have identified enhancer marks overlapping the 3' UTR and flanking region of MYEOV in multiple cell types

• 3D Genome Architecture:

  • Chromosome Conformation Capture (3C, 4C, Hi-C) to map interactions

  • Studies have shown interactions between the MYEOV locus and CCND1 in both humans and mice

  • Pay particular attention to CTCF binding sites, which appear to be conserved across species

• Functional Validation Methods:

  • ATAC-STARR-seq has been used to confirm enhancer activity of the MYEOV 3' region in GM12878 cells

  • Reporter assays with the MYEOV enhancer region driving expression of reporter genes

  • CRISPR/Cas9-mediated deletion or mutation of specific enhancer elements

• Evolutionary Comparisons:

  • Compare enhancer function across species with and without the MYEOV ORF

  • A 273 bp conserved region within MYEOV is found across mammals and even in chickens

  • The enhancer state is conserved in non-human primates, dogs, rats, and mice

• Cell Type Considerations:

  • Enhancer activity may be cell type-specific

  • Enhancer marks have been observed in multiple tissues including B cells, liver, and lung

  • Choose experimental models relevant to cancers where MYEOV is implicated

• Target Gene Analysis:

  • Monitor expression of potential target genes, particularly CCND1 and LTO1 (previously ORAOV1)

  • PCHi-C data from B cells has shown interactions between the MYEOV locus and the promoters of these genes

  • Validate with qRT-PCR and protein detection methods

These technical considerations should guide experimental design to effectively study the enhancer function of MYEOV independently from its potential protein-coding function.