SPINT2 Human

What is SPINT2 and what are its primary biological functions?

SPINT2, also known as hepatocyte growth factor activator inhibitor type-2, belongs to the Kunitz family of serine protease inhibitors. Its primary function is to bind to and inactivate HGFA, thereby impairing the conversion of inactive pro-HGF/SF (hepatocyte growth factor/scatter factor) into bioactive HGF/SF . This regulatory mechanism is significant because HGF can improve cell viability and invasiveness, stimulate angiogenesis, and function as a tumor progression factor .

To study SPINT2 function, researchers should employ gene expression analysis (RT-qPCR, RNA-seq), protein-protein interaction studies, and functional assays examining cell proliferation, migration, and invasion in various cell types with modulated SPINT2 expression. Western blotting combined with immunofluorescence provides valuable insights into protein expression levels and cellular localization patterns.

What experimental methods are most effective for detecting and measuring SPINT2 expression?

Multiple complementary approaches should be employed for comprehensive SPINT2 detection:

• RNA-level detection:

  • RT-qPCR: Essential for quantitative measurement of SPINT2 mRNA expression

  • RNA-sequencing: Provides transcriptome-wide context for SPINT2 expression

• Protein-level detection:

  • Western blotting: Quantifies SPINT2 protein levels in tissue lysates

  • Immunohistochemistry: Visualizes tissue distribution of SPINT2

  • Immunofluorescence: Enables co-localization studies (e.g., co-staining SPINT2 with α-SMA)

• Bioinformatic approaches:

  • Mining public databases (GEO, TCGA) for expression patterns across tissues and diseases

  • Using tools like GEO2R for differential expression analysis

  • Correlation analysis with clinical features and survival outcomes

For optimal results, researchers should validate findings using multiple techniques, including appropriate positive and negative controls, and standardize protocols across experimental conditions.

How does SPINT2 interact with the HGF/Met signaling pathway?

SPINT2 serves as a critical upstream regulator of the HGF/Met signaling pathway through the following mechanisms:

• Direct inhibition of HGFA: SPINT2 binds to and inactivates HGFA, which is responsible for converting pro-HGF to active HGF . This inhibitory action creates a rate-limiting step in the activation of HGF/SF.

• Regulation of downstream signaling: By controlling HGF activation, SPINT2 indirectly modulates Met receptor signaling and subsequent cellular responses including proliferation, migration, survival, and angiogenesis .

• Impact on tissue-specific functions: In prostate cancer cells, SPINT2 overexpression suppresses pro-HGF activation, extracellular matrix degradation, and cancer cell invasion . In smooth muscle cells, SPINT2 modulates PDGF receptor β expression, affecting phenotypic switching .

To experimentally investigate this interaction, researchers should employ:

• Protein-protein interaction assays to detect SPINT2-HGFA binding

• HGF activation assays measuring pro-HGF to HGF conversion

• Phosphorylation analysis of Met and downstream signaling components

• Functional readouts including migration, invasion, and proliferation assays

What pathways and biological processes are enriched in SPINT2 co-expressed genes?

Gene Ontology (GO) and KEGG pathway analyses of SPINT2 co-expressed genes reveal enrichment in several key biological processes:

• Cell adhesion and migration: SPINT2 co-expressed genes participate in regulating cell adhesion and migration pathways, suggesting a role in tissue architecture and cellular motility .

• Immune response: Functional enrichment analysis demonstrates significant involvement in immune-related processes, supporting SPINT2's role in modulating the tumor immune microenvironment .

• Embryonic development: SPINT2 and its co-expressed genes are enriched in developmental processes, consistent with its known functions in embryogenesis .

• Cancer-related pathways: KEGG analysis shows enrichment in pathways associated with cancer occurrence and metastasis .

To conduct similar analyses, researchers should:

• Identify SPINT2 co-expressed genes using correlation analysis in large datasets

• Employ tools like clusterProfiler for GO and KEGG enrichment analyses

• Visualize results using chord plots and other graphical representations

• Validate key pathway components through focused experimental studies

What factors influence SPINT2 expression in different human tissues?

SPINT2 expression exhibits considerable tissue-specific and context-dependent regulation:

• Age-related factors: In ovarian cancer, SPINT2 expression is significantly higher in patients above 40 years old compared to younger patients .

• Tumor grade: Higher SPINT2 expression is observed in ovarian tumors above grade 2 compared to grade 1 tumors .

• Tissue type: According to The Human Protein Atlas data, most cancer tissues display weak to moderate SPINT2 expression, while upregulated expression is observed in ovarian, prostate, pancreatic, breast, colorectal, and testis cancers .

• Pathological conditions: In thoracic aortic dissection (TAD), SPINT2 is expressed at significantly lower levels compared to normal aortic tissues .

• Growth factor stimulation: PDGF-BB treatment can modulate SPINT2 expression in smooth muscle cells, with concentration-dependent effects .

To study these influences, researchers should employ:

• Stratified expression analysis across patient demographics and clinical features

• In vitro stimulation with different growth factors and cytokines

• Epigenetic profiling to identify potential regulatory mechanisms

• Promoter analysis to identify tissue-specific transcription factor binding sites

How does SPINT2 influence immune cell infiltration in cancer microenvironments?

SPINT2 significantly impacts immune cell infiltration in cancer, particularly affecting macrophages through multiple mechanisms:

• Correlation with immune infiltration: TIMER analysis reveals SPINT2 expression positively correlates with macrophage infiltration in ovarian cancer (correlation coefficient r = 0.219, p = 1.29 × 10^-6) and neutrophil infiltration (r = 0.137, p = 2.57 × 10^-3) .

• M2 macrophage recruitment: SPINT2 mediates the migration of M2 macrophages, potentially through chemotactic mechanisms involving cytokine or chemokine secretion .

• Macrophage polarization: SPINT2 promotes polarization from M0 to M2 macrophages, as evidenced by increased expression of M2 markers (CD163 and CD206) and enhanced IL-10 release .

To experimentally investigate these mechanisms, researchers should:

• Use THP-1 differentiation models with PMA stimulation to generate macrophage-like cells

• Conduct migration assays to assess SPINT2's chemotactic effects

• Measure M2 markers by western blotting and flow cytometry

• Quantify cytokine production (especially IL-10) using ELISA

• Perform co-culture experiments with SPINT2-overexpressing cancer cells and monocytes

These findings explain how SPINT2 may contribute to creating an immunosuppressive tumor microenvironment, potentially contributing to its association with poor prognosis in certain cancers.

What are the contradictory roles of SPINT2 in cancer progression?

SPINT2 exhibits context-dependent roles in cancer, with evidence supporting both tumor-suppressive and oncogenic functions:

Tumor-suppressive evidence:

• In glioma, SPINT2 decreases cell migration and invasion via downregulation of MMP-2 expression and activity .

• In endometrial cancer, SPINT2 reduces vimentin levels, potentially inhibiting epithelial-to-mesenchymal transition .

• In prostate cancer, SPINT2 overexpression suppresses pro-HGF activation, ECM degradation, and cancer cell invasion .

Oncogenic evidence:

To address these contradictions, researchers should:

• Conduct comprehensive expression profiling across multiple cancer types

• Perform functional studies in diverse cancer cell lines

• Develop in vivo models to evaluate SPINT2's net impact on tumor growth

• Investigate isoform-specific effects and post-translational modifications

• Employ single-cell analysis to understand heterogeneous expression patterns

These contradictions likely reflect SPINT2's multifunctional nature and context-dependent interactions, emphasizing the need for cancer-specific therapeutic approaches.

What experimental approaches are most effective for studying SPINT2's role in cell migration and invasion?

To comprehensively investigate SPINT2's role in cell migration and invasion, researchers should employ multiple complementary methods:

• Transwell assays:

  • Migration assay: Cells are placed in the upper chamber of a transwell insert to assess migration toward chemoattractants .

  • Invasion assay: Similar to migration assay but with membrane coated with ECM components (e.g., Matrigel) .

• Gene expression modulation:

  • Adenoviral vectors for SPINT2 overexpression, as described in the research where cells were infected at MOI of 100 for 24h .

  • RNA interference (siRNA/shRNA) or CRISPR/Cas9 for knockdown/knockout studies.

• Molecular pathway analysis:

  • Western blotting to assess migration/invasion markers (MMPs, vimentin, E-cadherin) .

  • Immunofluorescence to visualize cytoskeletal changes and cell morphology .

  • Gelatin zymography to measure MMP activity.

• HGF/Met pathway-specific assays:

  • Measurement of pro-HGF processing

  • Met phosphorylation analysis

  • Quantification of downstream signaling components

• Advanced techniques:

  • 3D cell culture models (spheroids, organoids)

  • Live-cell imaging for real-time visualization

  • Co-culture systems to study effects on other cell types, such as immune cells

For optimal results, researchers should validate findings using multiple approaches and include appropriate controls for each method.

How can researchers effectively modulate SPINT2 expression in experimental models?

Several strategies can be employed to modulate SPINT2 expression in cell culture models, each with specific advantages:

• Adenoviral vector systems:

  • Methodology: SPINT2 sequence is synthesized, ligated to a vector (e.g., RedTrack-CMV), packaged in 293A cells, and used to infect target cells at optimized MOI (e.g., 100 for 24h) .

  • Advantages: High transduction efficiency in both dividing and non-dividing cells, temporal control of expression.

  • Validation: RT-qPCR and western blotting should confirm SPINT2 overexpression at both mRNA and protein levels .

• CRISPR/Cas9 gene editing:

  • For creating stable knockout or knock-in cell lines.

  • Protocol involves designing guide RNAs targeting the SPINT2 locus, transfecting cells with Cas9 and guide RNA constructs, and screening for desired modifications.

  • Advantages: Permanent genetic modification, allows for complete knockout.

• RNA interference:

  • siRNA for transient knockdown or shRNA for stable knockdown.

  • Include appropriate negative controls (scrambled sequences).

  • Validate knockdown efficiency at both mRNA and protein levels.

• Growth factor modulation:

  • PDGF-BB treatment (20 ng/ml for 24h) can modulate endogenous SPINT2 expression in some cell types, particularly smooth muscle cells .

  • Measure expression changes by RT-qPCR and western blotting.

• Inducible expression systems:

  • Tet-On/Tet-Off systems allow for controlled temporal expression.

  • Advantages: Adjustable expression levels by varying inducer concentration.

How does SPINT2 expression correlate with clinical outcomes in different cancer types?

SPINT2 expression shows varied correlations with clinical outcomes across cancer types:

To study these correlations, researchers should:

• Conduct Kaplan-Meier survival analysis stratified by SPINT2 expression levels

• Mine public repositories (GEO, TCGA, UALCAN) for correlations with clinical features

• Perform multivariate analysis to determine independent prognostic value

• Validate findings in independent patient cohorts using tissue microarrays

These findings highlight the importance of tumor-specific analysis when evaluating SPINT2 as a prognostic biomarker or therapeutic target.