HMGB3 Human

What is HMGB3 and what is its molecular structure?

HMGB3 (also known as HMG-4 and HMG-2a) is a 23 kDa nuclear protein and member of the HMGB family. Human HMGB3 is synthesized as a 200 amino acid (aa) precursor, which is demethylated to produce the 199 aa mature chain. The protein contains two HMG box DNA-binding domains (amino acids 9-79 and 93-161) and an acidic Asp/Glu-rich region (amino acids 181-200). Human HMGB3 shares 98% amino acid sequence identity with mouse and bovine HMGB3 . The protein is encoded by an X-linked gene and is classified with HMGB1 and HMGB2 in the HMGB subfamily .

What is the normal expression pattern of HMGB3 in human tissues?

HMGB3 expression is primarily restricted to embryogenesis and is normally diminished or silent in adult tissues . It is predominantly expressed in bone marrow hematopoietic stem cells and embryonic cells, while being absent or barely expressed in other normal tissues . Studies investigating HMGB3 expression demonstrate its importance in early developmental processes, with expression becoming progressively limited as tissues differentiate and mature.

What are the primary biological functions of HMGB3?

HMGB3 plays important roles in DNA recombination, repair, replication, and transcription as part of the HMGB subfamily . It binds preferentially to single-stranded DNA and possesses the ability to unwind double-stranded DNA . In hematopoietic stem cells, HMGB3 helps regulate the balance between self-renewal and differentiation into progenitor cells . Its restricted expression pattern in embryonic and stem cells suggests a critical role in development and cellular differentiation processes.

How is HMGB3 expression altered in human cancers?

Overexpression of HMGB3 has been documented in numerous human cancers. Pan-cancer investigation using GEPIA and UALCAN databases revealed high levels of HMGB3 expression across different malignancies . These include breast cancer, pheochromocytoma, paraganglioma, cervical squamous cell carcinoma, bladder urothelial carcinoma, cholangiocarcinoma, colon adenocarcinoma, head and neck squamous cell carcinoma, kidney cancers, esophageal carcinoma, lung cancers, liver hepatocellular carcinoma, prostate adenocarcinoma, uterine corpus endometrial carcinoma, and stomach adenocarcinoma . This widespread upregulation in diverse cancer types suggests a fundamental role for HMGB3 in oncogenic processes.

What cellular processes does HMGB3 affect in gastric cancer?

Research on gastric cancer cells demonstrates that HMGB3 significantly impacts multiple cellular processes. In gastric cancer cell lines MGC803 and BGC823, knockdown of HMGB3 expression leads to:

• Inhibition of cell proliferation, with significantly reduced proliferation rates at 24, 48, and 72 hours after silencing

• Cell cycle arrest at the G0/G1 phase

• Reduced cell migration and invasion capabilities

• Increased sensitivity to chemotherapeutic agents including oxaliplatin, cisplatin, and paclitaxel

• Molecular changes including upregulation of p21, p53, and Bax proteins and downregulation of Bcl-2 protein levels

These findings indicate that HMGB3 promotes gastric cancer progression through multiple mechanisms involving cell cycle regulation, apoptotic resistance, and cellular motility.

How does HMGB3 expression correlate with clinical features in breast cancer?

In breast cancer, HMGB3 expression correlates with numerous clinical and molecular features. High HMGB3 expression shows positive correlations with:

• Human epidermal growth factor receptor 2 (HER2) status

• Nottingham Prognostic Index (NPI)

• Basal-like status

• Nodal status (N+)

• Triple-negative status

• Scarff-Bloom-Richardson (SBR) grade

What are effective methods for detecting HMGB3 expression in human samples and cell lines?

Multiple techniques have been validated for detecting HMGB3 expression:

Western Blot Analysis:
Western blot has proven effective for detecting HMGB3 protein in various cell lines including HeLa (cervical epithelial carcinoma), 293T (embryonic kidney), and Jurkat (acute T cell leukemia) cells. Using Human/Mouse HMGB3 Antigen Affinity-purified Polyclonal Antibody, HMGB3 appears as a specific band at approximately 29 kDa under reducing conditions .

Immunohistochemistry (IHC):
IHC scoring systems have been established for evaluating HMGB3 expression in tissue samples. One validated approach uses a composite score based on staining intensity (0-3) and percentage of positive cells (0-3), with final scores ranging from 0 to 6. Scores can be categorized as negative ("-", score 0-2), weakly positive ("+", score 3), moderately positive ("++", score 4), or strongly positive ("+++", score ≥5) .

Gene Expression Analysis:
Transcriptomic analysis using public databases such as GEPIA and UALCAN can provide insights into HMGB3 mRNA expression across different tissues and cancer types .

What are effective approaches for modulating HMGB3 expression in experimental models?

RNA interference using small interfering RNA (siRNA) has been successfully implemented to knockdown HMGB3 expression in cancer cell lines. In the gastric cancer study, GC MGC803 and BGC823 cells were transfected with siRNA targeting the HMGB3 gene, which effectively reduced HMGB3 protein expression as confirmed by Western blot assays . This approach enables researchers to investigate the functional consequences of HMGB3 downregulation on various cellular processes.

For genetic models, Hmgb3-deficient mice (Hmgb3−/Y) have been developed to study the role of HMGB3 in vivo, particularly in hematopoietic stem cell regulation . These models allow for investigation of HMGB3 function at the organismal level and under various physiological and stress conditions.

How can researchers evaluate the functional effects of HMGB3 knockdown in cancer cells?

Based on published methodologies, the following assays can effectively evaluate HMGB3's functional roles:

Cell Proliferation:

• MTT assay at 24, 48, and 72 hours after transfection to assess viable cell count

Cell Cycle Analysis:

• Flow cytometry to determine cell distribution across G0/G1, S, and G2/M phases

Migration Capacity:

• Wound scratch assay to measure cell motility and wound closure rates

Invasion Potential:

• Transwell assay to quantify invasive capabilities

Chemosensitivity Assessment:

• Treating cells with varying concentrations of chemotherapeutic agents (e.g., oxaliplatin, cisplatin, paclitaxel) for 24 hours followed by MTT assay to determine cell viability

Protein Expression Analysis:

• Western blot to detect changes in related proteins including p21, p53, Bax, and Bcl-2

How does HMGB3 regulate hematopoietic stem cell function?

HMGB3 plays a critical role in maintaining the balance between hematopoietic stem cell (HSC) self-renewal and differentiation. Studies with Hmgb3-deficient mice (Hmgb3−/Y) revealed that while these mice contain normal numbers of HSCs capable of self-renewal and hematopoietic repopulation, they have fewer common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) .

Hmgb3−/Y HSCs exhibit constitutive activation of the canonical Wnt signaling pathway, which regulates stem cell self-renewal. This increased Wnt signaling corresponds to elevated expression of Dvl1, a positive regulator of the canonical Wnt pathway . These alterations appear to bias HSCs toward self-renewal at the expense of progenitor production, suggesting HMGB3 normally helps maintain the proper balance between these processes.

How does HMGB3 influence hematopoietic recovery after stress?

When Hmgb3−/Y mice were subjected to hematopoietic stress through 5-fluorouracil treatment, they exhibited a faster recovery of functional HSCs compared to wild-type mice. This accelerated recovery may be attributed to the increased Wnt signaling observed in Hmgb3-deficient HSCs . Importantly, the recovery of HSC numbers in Hmgb3−/Y mice occurred more rapidly than the recovery of CLP and CMP populations, further supporting the model where HMGB3 deficiency biases toward HSC self-renewal rather than differentiation into progenitor cells .

What are the molecular mechanisms through which HMGB3 regulates cancer cell chemoresistance?

While current research has demonstrated that HMGB3 knockdown enhances chemosensitivity in cancer cells, the precise molecular mechanisms remain incompletely defined. Investigating the following pathways would advance understanding:

• Apoptotic Regulation: Further characterize how HMGB3 modulates the balance between pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins beyond correlation studies .

• DNA Damage Response: Given HMGB3's role in DNA processes, examine how it might affect DNA damage recognition and repair mechanisms that influence chemotherapy efficacy.

• Drug Efflux Mechanisms: Investigate whether HMGB3 regulates expression or function of multidrug resistance transporters that affect cellular accumulation of chemotherapeutic agents.

What is the relationship between HMGB3 and immune infiltration in the tumor microenvironment?

Analysis of breast cancer samples has shown that the amount of immunological infiltration is substantially linked with high expression of HMGB3 . This correlation raises important questions about the potential immunomodulatory roles of HMGB3. Future research should investigate:

• How HMGB3 expression levels affect specific immune cell populations within tumors

• Whether HMGB3 influences immune checkpoint molecules and pathways

• If targeting HMGB3 might enhance immunotherapy responses

What are the key pathways and protein interactions mediating HMGB3's oncogenic functions?

Enrichment analysis has indicated that HMGB3 is involved in the chromosome centromeric region, ATPase activity, and the cell cycle . To better understand how HMGB3 promotes cancer progression, researchers should:

• Perform comprehensive interactome studies to identify HMGB3 binding partners in cancer cells

• Conduct ChIP-seq experiments to map HMGB3 binding across the genome in different cancer types

• Develop systems biology approaches to model how HMGB3 interacts with other oncogenic and tumor suppressor pathways