SERPINA4 Human

What is SERPINA4 and what alternative nomenclature exists in the literature?

SERPINA4, officially designated as Serpina family A member 4, is also known as kallistatin, KAL, or KST in scientific literature. First identified as a tissue kallikrein-binding protein in the 1900s, it belongs to the serpin (serine protease inhibitor) superfamily . When designing research protocols, investigators should account for these alternative designations when conducting literature searches or database queries to ensure comprehensive data collection.

What are the primary biological functions of SERPINA4 in human physiology?

SERPINA4 functions as a novel anti-angiogenesis agent with multiple regulatory effects on inflammation, angiogenesis, and tumor growth . Mechanistically, SERPINA4 suppresses angiogenesis and inflammation through several pathways:

• Inhibition of TNF-α-induced NF-κB activation

• Blocking of VEGF signaling pathways

• Induction of apoptosis in cultured human endothelial cells

Additionally, SERPINA4 inhibits xenograft tumor growth in experimental models by antagonizing VEGF-related cell proliferation, migration, and invasion of endothelial cells . Beyond its anti-angiogenic properties, SERPINA4 directly inhibits cancer cell proliferation, migration, and invasion by modulating cancer cell signaling in various malignancies including lung, breast, and hepatocellular carcinoma .

How is SERPINA4 typically expressed across normal human tissues versus cancerous tissues?

Research utilizing tissue microarray (TMA) analysis of 327 normal colorectal mucosa specimens revealed a distinctive expression pattern:

This distribution demonstrates that SERPINA4 is significantly downregulated in colorectal cancer tissues compared to matched normal mucosa (P < 0.001) . Quantitative analyses have confirmed this downregulation at both transcriptional (mRNA) and translational (protein) levels .

What experimental techniques are most effective for quantifying SERPINA4 expression in human tissue samples?

Comprehensive SERPINA4 expression analysis typically employs multiple complementary techniques:

• Quantitative real-time PCR (qRT-PCR): For measuring SERPINA4 mRNA expression levels, with results typically normalized using ΔCt values relative to housekeeping genes. In CRC studies, the average SERPINA4 expression (ΔCt value) was 11.03 ± 1.11 in tumor tissue versus 9.73 ± 1.32 in normal tissue (P < 0.001) .

• Western blot analysis: For semi-quantitative assessment of SERPINA4 protein levels. Studies have shown protein expression levels of 0.64 ± 0.04 in tumor tissues versus 1.20 ± 0.30 in paired normal mucosa (P < 0.001) .

• Immunohistochemistry (IHC): For characterizing expression patterns and subcellular localization. SERPINA4 is primarily detected in the membrane and cytoplasm of colorectal epithelium, with staining intensity typically classified as negative, weak, or strong .

• Tissue microarray (TMA): Enables simultaneous evaluation of SERPINA4 expression across multiple specimens, facilitating robust statistical analysis of expression patterns in relation to clinicopathological parameters .

How can researchers effectively establish SERPINA4 overexpression or knockdown models for functional studies?

Several approaches have proven effective for manipulating SERPINA4 expression in experimental systems:

• Plasmid-mediated overexpression: SERPINA4 expression plasmids can be transfected into cancer cell lines (e.g., Caco2 and RKO cells), with successful transfection verified by qRT-PCR and western blot analysis .

• RNA interference: siRNA or shRNA targeting SERPINA4 can be utilized for knockdown experiments to elucidate the consequences of reduced SERPINA4 expression.

• Cell proliferation assays: Following genetic manipulation, Cell Counting Kit-8 (CCK-8) assays can evaluate effects on cell growth at various time points (12, 24, 48, and 72 hours) .

• Colony formation assays: These provide insights into the effects of altered SERPINA4 expression on long-term cell survival and proliferation capacity .

What in vivo models are appropriate for studying SERPINA4's physiological and pathological roles?

Xenograft models have provided valuable insights into SERPINA4's functions:

• Subcutaneous xenograft approach: Cancer cells with manipulated SERPINA4 expression (e.g., RKO-SERPINA4 and RKO-Control cells) are injected subcutaneously into immunocompromised mice .

• Tumor growth assessment: Studies have demonstrated that RKO-SERPINA4 tumors exhibit significantly lower growth indices compared to RKO-Control tumors .

• Histological and immunohistochemical analysis: H&E staining and IHC for proliferation markers (e.g., Ki67) on xenograft sections reveal reduced proliferation in SERPINA4-overexpressing tumors compared to controls .

How does SERPINA4 expression correlate with clinicopathological parameters in human cancers?

SERPINA4 expression demonstrates significant associations with multiple clinicopathological parameters in colorectal cancer:

These correlations indicate that decreased SERPINA4 expression is significantly associated with more advanced disease features, including deeper invasion, increased metastasis, higher AJCC stage, and poorer differentiation .

What is the prognostic significance of SERPINA4 expression in colorectal cancer?

SERPINA4 serves as an independent prognostic indicator in colorectal cancer:

What molecular mechanisms underlie SERPINA4's tumor-suppressive effects?

SERPINA4 exerts tumor-suppressive effects through multiple mechanisms:

• Anti-angiogenic activity: SERPINA4 inhibits angiogenesis by blocking VEGF signaling pathways and inducing apoptosis in endothelial cells .

• Direct inhibition of cancer cell proliferation: In vitro experiments using colorectal cancer cell lines have demonstrated that SERPINA4 overexpression significantly reduces cell growth and colony formation, with observable inhibition of cell growth at 12, 24, 48, and 72 hours after transfection (P < 0.05) .

• Inhibition of migration and invasion: SERPINA4 directly inhibits the migratory and invasive capabilities of cancer cells by modulating related signaling pathways .

• Suppression of inflammation: By inhibiting TNF-α-induced NF-κB activation, SERPINA4 may reduce inflammation-driven tumor progression .

How do researchers address technical challenges in detecting low-abundance SERPINA4 in clinical samples?

Detection of downregulated SERPINA4 in clinical samples presents several technical challenges that researchers address through:

• Sample enrichment techniques: Concentrating protein fractions before analysis to enhance detection of low-abundance SERPINA4.

• Enhanced sensitivity detection methods: Utilizing amplified detection systems for immunohistochemistry or highly sensitive ELISA assays.

• Digital PCR: For more precise quantification of low-abundance mRNA transcripts compared to conventional qRT-PCR.

• Laser capture microdissection: To isolate specific cell populations of interest, minimizing dilution effects from non-target cells within heterogeneous tumor samples.

What are the current limitations in understanding SERPINA4's role across different cancer types?

Despite progress in colorectal cancer research, several limitations persist:

• Tissue-specific differences: The expression and function of SERPINA4 may vary across different tissue types and cancer lineages, requiring tissue-specific investigation protocols.

• Context-dependent effects: SERPINA4's interactions with other molecular players may vary depending on the tumor microenvironment and genetic background.

• Lack of standardization: Variations in detection methods, cutoff values for expression analysis, and scoring systems make cross-study comparisons challenging.

• Mechanistic understanding gaps: While associations between SERPINA4 expression and clinical outcomes are established, the complete mechanistic pathways mediating these effects remain incompletely characterized .

How can researchers identify and validate SERPINA4-interacting proteins and regulatory pathways?

Advanced methodologies for exploring SERPINA4's interactome and regulatory networks include:

• Co-immunoprecipitation followed by mass spectrometry: To identify proteins that physically interact with SERPINA4.

• Chromatin immunoprecipitation (ChIP): To investigate transcription factors regulating SERPINA4 expression.

• Yeast two-hybrid screening: To discover novel protein-protein interactions involving SERPINA4.

• Pathway inhibitor studies: Using specific inhibitors of pathways hypothesized to interact with SERPINA4 (e.g., VEGF, NF-κB) to elucidate functional relationships.

• Bioinformatic analyses: Leveraging public databases to identify potential regulatory elements and interaction networks associated with SERPINA4.

How might SERPINA4's tumor-suppressive properties be leveraged for therapeutic development?

Several therapeutic approaches could potentially exploit SERPINA4's tumor-suppressive properties:

• Recombinant SERPINA4 administration: Given its anti-tumor and anti-angiogenic effects, administration of recombinant SERPINA4 protein could potentially inhibit tumor growth and progression .

• Gene therapy approaches: Strategies to restore SERPINA4 expression in tumors where it is downregulated could have therapeutic benefits.

• Combination therapies: SERPINA4-targeted approaches could potentially enhance the efficacy of existing treatment modalities, including chemotherapy and targeted therapies.

What biomarker potential does SERPINA4 hold for cancer diagnostics and prognostics?

SERPINA4 demonstrates considerable potential as a biomarker:

• Prognostic biomarker: The significant correlation between SERPINA4 expression and patient survival suggests utility as a prognostic biomarker .

• Risk stratification tool: SERPINA4 expression could help stratify patients into risk categories, potentially informing treatment decisions.

• Complementary marker: SERPINA4 could be incorporated into multi-marker panels alongside established biomarkers to improve prognostic accuracy in colorectal cancer.

• Response prediction: Preliminary evidence suggests SERPINA4 status might predict response to specific therapeutic interventions, though further validation is required.