Recombinant Mouse Suppressor of tumorigenicity 7 protein-like (St7l)

What is Recombinant Mouse Suppressor of Tumorigenicity 7 Protein-like (St7l)?

St7l (also referred to as ST7R or ST7-like) is a novel gene homologous to the tumor suppressor gene ST7. It encodes a 575-amino-acid polypeptide characterized by a leucine zipper domain and three tyrosine-phosphorylation sites. The protein demonstrates significant homology to human ST7 (72.1% total-amino-acid identity) and Drosophila CG3634 (56.8% total-amino-acid identity) . Recombinant forms of this protein are produced for research applications to investigate its functional properties and potential therapeutic applications in cancer biology.

The St7l gene consists of at least 15 exons, with four St7l isoforms transcribed through alternative splicing mechanisms. Structurally, the protein contains three conserved domains (S7H1, S7H2, and S7H3) that are shared among ST7R, ST7, and CG3634, underscoring evolutionary conservation of these functional regions .

What genomic characteristics define St7l?

The St7l gene is located on human chromosome 1p13, a region frequently associated with allelic loss or rearrangements in various cancers including breast cancer, germ cell tumors, squamous cell carcinoma of head and neck, non-small cell lung cancer, and several others . A distinctive genomic feature is that St7l and WNT2B genes are clustered in a tail-to-tail manner with an interval of less than 5.0-kb. This arrangement mirrors the clustering of ST7-WNT2, suggesting that these gene clusters might have originated from the duplication of an ancestral gene cluster .

The leucine zipper domain is unique to St7l compared to its homologs, while Tyr 268 and Tyr 441 are conserved across St7l, ST7, and CG3634, suggesting functional importance of these residues in signaling pathways .

What cell culture systems are most effective for studying St7l function?

For studying St7l function, researchers should consider several cell line models based on experimental objectives:

• HCT-116 human colorectal carcinoma cells: These cells have demonstrated sensitivity to recombinant mouse PDGFRL Fc Chimera (a protein with related research applications), with an ED50 of 1-6 μg/mL in proliferation inhibition assays . While this specifically relates to PDGFRL rather than St7l, the methodology and response evaluation are applicable to St7l research.

• Pancreatic cancer cell lines: Given St7l's established role in pancreatic cancer progression and drug resistance, pancreatic cancer cell lines provide valuable models. When establishing such experiments, researchers should implement both wild-type and drug-resistant variants to assess St7l's contribution to chemoresistance phenotypes .

The experimental approach should include:

• Stable overexpression and knockdown of St7l using lentiviral or CRISPR-Cas9 systems

• Comparative analysis of proliferation, migration, invasion, and drug response

• Analysis of downstream signaling pathway components, particularly β-catenin activation markers

How should researchers design experiments to investigate St7l's role in drug resistance?

When investigating St7l's role in drug resistance, researchers should implement a multifaceted experimental approach:

• Establish baseline expression: Determine St7l expression levels in sensitive and resistant cell populations using qRT-PCR and Western blot analysis.

• Modulate expression: Create cell lines with St7l overexpression or knockdown using appropriate vector systems.

• Drug sensitivity testing: Subject modified cells to dose-response curves with relevant chemotherapeutic agents (e.g., gemcitabine for pancreatic cancer models) .

• Pathway analysis: Examine changes in Wnt/β-catenin signaling components, as St7l downregulation has been shown to inhibit β-catenin activation .

• miRNA regulation: Investigate the regulatory relationship between St7l and miR-331-3p, which has been shown to target St7l in pancreatic cancer .

Data collection should include:

• IC50 values before and after St7l modulation

• Apoptosis markers (caspase activation, PARP cleavage)

• EMT markers (E-cadherin, vimentin, N-cadherin)

• Cell cycle analysis

How does St7l interact with the Wnt/β-catenin signaling pathway?

The relationship between St7l and Wnt/β-catenin signaling represents a critical aspect of its tumor suppressor function. Studies have demonstrated that St7l downregulation inhibits the activation of β-catenin . This suggests that St7l may function as a negative regulator of this pathway under normal conditions.

The methodological approach to investigating this interaction should include:

• Co-immunoprecipitation assays: To identify direct protein-protein interactions between St7l and components of the Wnt/β-catenin destruction complex.

• TOPFlash/FOPFlash reporter assays: To quantitatively measure β-catenin-mediated transcriptional activity following St7l modulation.

• Subcellular fractionation: To determine whether St7l expression affects nuclear translocation of β-catenin.

• Chromatin immunoprecipitation (ChIP): To assess whether St7l influences binding of β-catenin to target gene promoters.

The genomic arrangement where St7l and WNT2B genes are clustered in a tail-to-tail manner with an interval of less than 5.0-kb provides additional evidence for a potential functional relationship between St7l and Wnt signaling .

What is the relationship between St7l and miR-331-3p in cancer progression?

The interaction between St7l and miR-331-3p represents a significant regulatory mechanism in cancer biology. Research has demonstrated that miR-331-3p is upregulated in pancreatic cancer and promotes cell proliferation and EMT-mediated metastasis by targeting St7l . This suggests a tumor suppressor role for St7l that is counteracted by miR-331-3p overexpression.

Methodological approaches to explore this relationship include:

• Luciferase reporter assays: Using constructs containing the St7l 3'UTR with wild-type or mutated miR-331-3p binding sites to confirm direct targeting.

• Expression correlation analysis: Examining the inverse relationship between miR-331-3p and St7l levels in patient samples and cell lines.

• Rescue experiments: Determining whether St7l overexpression can reverse the oncogenic effects of miR-331-3p.

• Drug resistance implications: Investigating whether modulation of the miR-331-3p/St7l axis affects sensitivity to chemotherapeutic agents .

What are the optimal conditions for recombinant St7l protein production and storage?

Based on related recombinant protein methodologies, researchers should consider the following guidelines for St7l:

• Expression system selection: Mammalian expression systems (such as HEK293 or CHO cells) are recommended for producing recombinant St7l with proper post-translational modifications.

• Purification strategy: Implement a two-step purification process, typically involving affinity chromatography followed by size exclusion chromatography.

• Formulation and storage:

  • Lyophilize from a 0.2 μm filtered solution in PBS

  • Store lyophilized protein at -20°C to -80°C

  • Reconstitute at approximately 500 μg/mL in PBS

  • Avoid repeated freeze-thaw cycles by aliquoting before freezing

• Stability assessment: Implement periodic activity testing to confirm protein stability over time.

How can researchers validate St7l functionality in experimental systems?

Validating St7l functionality requires multiple complementary approaches:

• Proliferation inhibition assays: Similar to those performed with related proteins where ED50 values can be determined (typically in the range of 1-6 μg/mL for similar tumor suppressor proteins) .

• Wnt/β-catenin signaling assessment: Measure downstream targets of the pathway, as St7l downregulation has been shown to inhibit β-catenin activation .

• Binding assays: Implement surface plasmon resonance or similar techniques to confirm interaction with predicted binding partners.

• Functional rescue experiments: Determine whether recombinant St7l can restore normal phenotypes in St7l-deficient systems.

How might St7l be exploited as a therapeutic target in cancer treatment?

Given St7l's potential tumor suppressor functions, several therapeutic strategies warrant investigation:

• miRNA inhibition: Developing miR-331-3p inhibitors to restore St7l expression in cancers where this regulatory mechanism is active .

• Recombinant protein administration: Evaluating whether recombinant St7l or engineered variants can directly suppress tumor growth.

• Synthetic lethality: Identifying genes that, when inhibited, cause selective death of St7l-deficient cancer cells.

• Drug resistance modulation: Exploring St7l restoration as a strategy to overcome chemoresistance in pancreatic cancer and other malignancies .

Methodological considerations include:

• Selection of appropriate delivery systems for RNA therapeutics

• Development of stabilized recombinant protein formulations

• High-throughput screening for synthetic lethal interactions

• Combination therapy approaches with standard chemotherapeutics

What computational approaches can enhance St7l research?

Modern computational methods offer powerful tools for advancing St7l research:

• Silencer element prediction: Implementing simple subtractive analysis (SSA) approaches similar to those used in genome-wide silencer element identification . This can help identify regulatory elements controlling St7l expression.

• Support Vector Machine (SVM) modeling: Applying gapped k-mer SVM (gkmSVM) algorithms to predict functional elements relevant to St7l regulation, with potential model performance achieving area under the curve (AUC) values of approximately 0.79-0.80 for both ROC and precision-recall curves .

• ChIP-seq data integration: Analyzing datasets for known repressors to validate predictions about St7l regulation.

• Disease variant association: Examining the enrichment of disease-associated variants in predicted regulatory regions of St7l in relevant cell types or lineages .

How can researchers address antibody specificity issues in St7l studies?

Antibody specificity remains a critical concern in St7l research. Researchers should implement a comprehensive validation approach:

• Multiple antibody validation: Test at least two antibodies targeting different epitopes of St7l.

• Positive and negative controls: Include overexpression and knockdown systems as controls.

• Cross-reactivity assessment: Test antibodies in tissues known to lack St7l expression.

• Peptide competition assays: Confirm binding specificity through pre-incubation with immunizing peptides.

• Western blot validation: Verify specific band detection at the expected molecular weight (approximately 65-70 kDa for full-length St7l).

The implementation of these validation steps is essential for generating reliable data, particularly when investigating the four St7l isoforms produced through alternative splicing .