Recombinant Human Vacuolar fusion protein MON1 homolog B (MON1B)

What is MON1B and what are its primary cellular functions?

MON1B (Vacuolar fusion protein MON1 homolog B) is a mammalian homolog of the yeast MON1 protein that functions as an essential docking factor for vesicular fusion. In mammalian cells, MON1B plays a critical role in the membrane recruitment necessary for vesicular fusion of early endosomes . The protein contains NF-κB binding sites and is involved in fundamental cellular processes related to endosomal trafficking and fusion. MON1B is also critical for apoptotic cell clearance, similar to its yeast counterpart, indicating conservation of function across species . Research techniques for studying MON1B's cellular functions typically involve fluorescence microscopy to track vesicular movement, co-immunoprecipitation to identify interaction partners, and siRNA-mediated knockdown to observe functional consequences.

How is MON1B expression detected in tissue and cell samples?

MON1B expression can be reliably detected using several complementary techniques. RT-qPCR is commonly employed to quantify mRNA expression levels, using specific primer sequences for MON1B (as shown in research protocols) . For protein detection, Western blot analysis is the standard approach, utilizing rabbit anti-MON1B antibodies (such as Novus, NBP1-92131, at 1:2000 dilution) . For experimental validation, researchers typically use β-actin as a housekeeping control gene. When designing experiments to detect MON1B, it's important to include appropriate positive controls (such as colon cancer cell lines with known high MON1B expression) and negative controls (normal epithelial cells) to establish a comparative baseline .

What cell lines are appropriate models for studying MON1B function?

According to research data, several human colon cancer cell lines demonstrate elevated MON1B expression compared to normal colon mucosal epithelial cells. The LoVo cell line particularly shows significantly higher MON1B expression, making it an excellent model for studying MON1B knockdown effects . Other suitable cell lines include HT-29, SW480, and COLO205, all of which display increased MON1B levels compared to the normal colon mucosal epithelial NCM460 cells . When selecting cell models, researchers should consider the specific research question, as different cell lines may exhibit varying responses to MON1B manipulation depending on their genetic background and molecular characteristics.

What is the relationship between MON1B expression and colon cancer progression?

MON1B expression demonstrates a significant relationship with colon cancer progression across multiple parameters. Research has established that MON1B expression levels correlate with several clinical features:

• MON1B expression is significantly elevated in colon cancer tissues compared to adjacent normal tissues at both mRNA and protein levels .

• Higher MON1B expression correlates with advanced pathological grades. Specifically, patients with pathological grades III-IV show significantly higher MON1B expression compared to those with grades I-II (p=0.009) .

• MON1B expression is significantly associated with metastasis status (p=0.026), with higher expression in patients with metastatic disease .

• Poor differentiation in tumors correlates with higher MON1B expression (p=0.016) .

• Survival analysis indicates that patients with lower MON1B levels have longer survival times compared to those with higher expression levels .

These correlations suggest MON1B may serve as both a prognostic biomarker and potential therapeutic target in colon cancer. Methodologically, researchers investigating these relationships should employ multivariate analysis to control for confounding factors when assessing MON1B's independent prognostic value.

What molecular mechanisms underlie MON1B's effects on cancer cell invasion and migration?

MON1B influences cancer cell invasion and migration through several interconnected molecular pathways. When MON1B is silenced in colon cancer cells:

• Matrix metalloproteinases MMP-2 and MMP-9, which degrade extracellular matrix components facilitating tumor invasion, show decreased expression at both mRNA and protein levels .

• Tissue inhibitor of metalloproteinase (TIMP-2), which counteracts MMP activity, displays increased expression .

• Metastasis-associated gene MTA-1 expression is reduced, limiting cellular migration potential .

• NF-κB expression decreases while IκB levels increase, suggesting MON1B regulates the NF-κB signaling pathway, which is crucial for cancer progression .

• Chemokine receptor CXCR-4, important for directed migration, shows reduced expression .

Methodologically, researchers investigating these mechanisms should employ multiple complementary approaches including gene expression analysis, protein interaction studies, and functional assays. Pathway inhibition experiments would further validate the specific contributions of each molecular component to the observed phenotypes.

How can MON1B knockdown be effectively achieved in experimental settings?

Efficient MON1B knockdown can be achieved through RNA interference techniques. The research data indicates successful implementation of siRNA-mediated MON1B knockdown in LoVo colon cancer cells . The experimental methodology involves:

• Selection of appropriate siRNA sequences targeting MON1B (commercially available from sources like Thermo Fisher) .

• Transfection of target cells (e.g., LoVo cells) at approximately 70% confluence using Lipofectamine 3000 according to manufacturer's protocols .

• Optimized transfection with 30 pmol siRNA per reaction .

• Inclusion of both untransfected control and negative control siRNA groups to distinguish specific from non-specific effects .

• Verification of knockdown efficiency through both RT-qPCR and Western blot analysis, with successful knockdown showing significant reductions in both mRNA and protein levels .

For researchers attempting MON1B knockdown, optimization of transfection conditions for specific cell lines is critical, as transfection efficiency varies between cell types. Alternative approaches such as stable knockdown using shRNA or CRISPR-Cas9 gene editing might provide more sustained MON1B suppression for long-term studies.

What assays are most appropriate for evaluating MON1B's effects on cancer cell properties?

Based on established research protocols, several complementary functional assays are recommended for comprehensive assessment of MON1B's effects on cancer cell properties:

• For cell proliferation: Cell Counting Kit-8 (CCK-8) assay provides time-dependent (12, 24, and 48 hours) quantitative assessment of cell viability following MON1B manipulation . This colorimetric assay offers advantages in sensitivity and reproducibility over traditional MTT assays.

• For cell migration: Wound healing assay (scratch test) with time-point observations at 12 and 24 hours post-wounding allows for quantification of migratory capacity . Analysis should include calculation of wound closure percentages and appropriate statistical comparison between experimental groups.

• For cell invasion: Transwell chambers (8-μm pore filters) coated with Matrigel GFR provide three-dimensional assessment of invasive capacity . The experimental setup should include:

  • Seeding 5×10⁴ cells in upper chambers with serum-free media

  • Adding 10% FBS-containing media to lower chambers as chemoattractant

  • 48-hour incubation period

  • Fixation with 4% paraformaldehyde

  • Staining with 0.1% crystal violet

  • Quantification of invaded cells under optical microscopy

When implementing these assays, researchers should ensure appropriate controls and technical replicates (minimum triplicate) to ensure statistical validity of results.

How can researchers properly design RT-qPCR experiments to study MON1B and related genes?

Robust RT-qPCR experimental design for studying MON1B and related genes requires careful attention to several methodological considerations:

• RNA extraction protocol: Total RNA should be extracted using TRIzol Reagent or equivalent methods that preserve RNA integrity .

• Quality control: RNA quantity and quality should be assessed via spectrophotometry (A260/A280 ratio) and gel electrophoresis or Bioanalyzer analysis.

• Reverse transcription: Standardized amounts of RNA (e.g., 1 μg) should be converted to cDNA using validated reverse transcription kits such as EN-QuantiTect Reverse Transcription kit .

• PCR amplification conditions: Fast SYBR Green Master Mix with appropriate thermal cycling parameters (e.g., 25s at 95°C for initial denaturation, 40 cycles of 20s at 95°C, 20s at 57°C, 30s at 72°C, and 10 min at 72°C for final extension) .

• Primer design: Specific primers for MON1B and related genes (MMP-2, MMP-9, TIMP-2, MTA-1, NF-κB, IκB, CXCR-4) with appropriate annealing temperatures and amplicon sizes .

• Reference gene selection: β-actin has been validated as an appropriate housekeeping gene for normalization in this research context .

• Technical and biological replicates: Minimum triplicate technical replicates and appropriate biological replicates to account for variability.

• Data analysis: Relative quantification using the 2^-ΔΔCt method with appropriate statistical analysis.

This methodological approach ensures reliable quantification of gene expression changes in response to experimental manipulation of MON1B.

How does MON1B expression correlate with clinical outcomes in cancer patients?

Clinical evidence demonstrates significant correlations between MON1B expression and patient outcomes. In colon cancer, MON1B expression levels are strongly associated with:

• Survival rates: Patients with lower MON1B expression demonstrate longer survival times compared to those with higher expression levels over a 2-year follow-up period .

• Tumor characteristics: Higher MON1B levels correlate with advanced pathological grades (III-IV), presence of metastasis, and poor differentiation status .

The following table summarizes clinical correlations with MON1B expression:

For researchers investigating MON1B as a prognostic marker, multivariate analysis is essential to determine its independent predictive value when accounting for other established prognostic factors. Additionally, larger cohort studies with extended follow-up periods would strengthen these clinical correlations.

What are the methodological considerations for analyzing MON1B in patient tissue samples?

Analysis of MON1B in patient tissue samples requires careful attention to several methodological considerations:

• Tissue collection and processing:

  • Paired sampling of tumor and adjacent normal tissues provides optimal comparative analysis

  • Rapid freezing in liquid nitrogen for molecular studies or formalin fixation for histological studies

  • Consistent processing protocols to minimize variability

• Expression analysis techniques:

  • RT-qPCR for mRNA quantification using validated primers

  • Western blot for protein detection using specific antibodies (e.g., rabbit anti-MON1B, Novus, NBP1-92131, 1:2000)

  • Immunohistochemistry for spatial distribution analysis within tissue architecture

• Scoring and classification:

  • Establishment of standardized thresholds for categorizing "high" versus "low" MON1B expression

  • Use of digital pathology and image analysis software for objective quantification

  • Blinded assessment by multiple pathologists to reduce subjective bias

• Clinical correlation:

  • Comprehensive collection of patient clinical data including follow-up information

  • Statistical approaches that account for potential confounding factors

  • Survival analysis using Kaplan-Meier curves and Cox regression models

These methodological considerations ensure reliable and reproducible assessment of MON1B in patient samples for translational research applications.

What novel therapeutic approaches might target MON1B-dependent pathways?

Based on current understanding of MON1B's role in cancer progression, several therapeutic approaches warrant investigation:

• Direct MON1B inhibition strategies:

  • Development of small molecule inhibitors targeting MON1B protein interactions

  • Antisense oligonucleotides or siRNA-based therapies for specific MON1B knockdown in tumors

  • Peptide-based inhibitors targeting functional domains of MON1B

• Targeting downstream effectors in MON1B pathways:

  • MMP inhibitors to counteract the elevated MMP-2/MMP-9 activity associated with high MON1B expression

  • NF-κB pathway modulators, given MON1B's regulatory effect on this signaling cascade

  • CXCR-4 antagonists to address the chemokine signaling influenced by MON1B

• Combination approaches:

  • MON1B inhibition combined with conventional chemotherapy

  • Dual targeting of MON1B and related endosomal trafficking proteins

  • Integration with immunotherapy approaches, particularly if MON1B influences immune cell recognition

Researchers exploring these therapeutic directions should employ both in vitro and in vivo models, with particular attention to potential off-target effects given MON1B's role in fundamental cellular processes.

How might MON1B function differ across various cancer types?

While current research has established MON1B's significance in colon cancer , its role likely extends to other malignancies with potential tissue-specific variations. Researchers investigating MON1B across cancer types should consider:

• Comparative expression analysis:

  • Systematic assessment of MON1B expression across cancer types using publicly available databases (TCGA, GEO)

  • Validation in tissue microarrays representing multiple cancer types

  • Correlation with cancer-specific molecular subtypes and genetic alterations

• Functional heterogeneity:

  • Comparison of phenotypic effects following MON1B manipulation in cell lines representing different cancer types

  • Investigation of tissue-specific interaction partners that may modify MON1B function

  • Analysis of differential pathway activation downstream of MON1B across cancer types

• Clinical correlation variations:

  • Assessment of whether MON1B's prognostic significance is consistent across malignancies

  • Investigation of cancer-specific correlations with treatment response

Previous research has indicated correlations between MON1B and prostate cancer progression , suggesting its oncogenic function may extend beyond colon cancer. This comparative approach would establish whether MON1B represents a broadly applicable therapeutic target or if intervention strategies need to be tailored to specific cancer types.