CENPU Human

What is CENPU and what is its primary function in human cells?

CENPU is a centromere component crucial for mitosis with a vital role in orchestrating kinetochore-microtubule attachment . It functions primarily as a centromere-binding protein required for cellular mitosis and has been identified as a putative transcriptional repressor . The protein is localized to human chromosome 4q35.1 and is expressed in the nuclei and cytoplasm of cells across various tissues, particularly in fetal liver, bone marrow, thymus, and testicular tissue .

In normal cellular function, CENPU plays a critical role in the G1/S transition of the cell cycle, and its deregulation can contribute to uncontrolled cell proliferation characteristic of cancer cells .

How is CENPU expression detected in clinical research settings?

CENPU expression can be evaluated through several methodological approaches:

• RNA level detection: Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used to measure CENPU mRNA expression levels in tissue samples or cell lines .

• Protein level detection: Western blotting using polyclonal rabbit anti-human CENPU antibodies (such as cat. no. ab117078, Abcam) is employed to quantify protein expression . Standard protocols involve:

  • Harvesting and lysing cells in SDS buffer

  • Protein quantification using BCA Protein assay

  • Separation of equal amounts of protein (typically 20 μg) using 10% SDS-PAGE

  • Electrotransfer onto polyvinylidene difluoride membranes

  • Blocking with 5% dry skimmed milk in TBST (0.1%)

  • Incubation with primary antibody (1:500 dilution) at 4°C overnight

• Tissue analysis: Immunohistochemical staining on formalin-fixed, paraffin-embedded tissue sections (4-μm thickness) after antigen retrieval in EGTA buffer (pH 9.0) for 25 minutes, followed by incubation with anti-CENPU antibody (1:50 dilution) .

What criteria are used to classify CENPU expression as "high" versus "low" in research studies?

Studies typically employ statistical methods to categorize CENPU expression levels. For example, in nasopharyngeal carcinoma research, patients are stratified into high and low CENPU expression groups based on statistical analysis of immunohistochemical staining or RT-qPCR results . The classification criteria often involve:

• Scoring systems based on staining intensity and percentage of positive cells

• Median or mean expression values as cutoff points

• Statistical analyses using SPSS and GraphPad Prism software

The classification is clinically relevant as demonstrated in the following patient distribution table from a nasopharyngeal carcinoma study:

*Statistically significant (P<0.05)

Which cancer types demonstrate significant CENPU upregulation?

Research has documented CENPU upregulation in multiple cancer types:

• Nasopharyngeal carcinoma (NPC): CENPU is significantly upregulated in NPC tissues compared to normal nasopharyngeal epithelium and correlates with advanced clinical stage (P=0.03) .

• Hepatocellular carcinoma (HCC): High CENPU expression in HCC tissue correlates positively with poor prognosis in patients .

• Breast cancer: CENPU expression is significantly upregulated in human breast cancer tissues compared to matched adjacent normal breast tissue, as confirmed by TCGA database analysis (n=106; P<0.001) .

• Bladder cancer (BCa): qPCR analysis reveals higher CENPU gene expression in BCa tissues compared to cancer-adjacent normal tissues, with high expression strongly correlated with tumor size and TNM stage .

• Other documented cancers: Triple-negative breast cancer, lung adenocarcinoma, prostate cancer, cervical cancer, and liver cancer .

How does CENPU expression correlate with patient survival and prognosis?

Multiple studies have established a clear correlation between CENPU expression and patient outcomes:

• Nasopharyngeal carcinoma: Overexpression of CENPU is associated with poorer survival in NPC patients .

• Hepatocellular carcinoma: High CENPU expression correlates positively with poor prognosis in HCC patients .

• Bladder cancer: Kaplan-Meier survival analysis indicates that high CENPU levels are associated with reduced survival rates .

The consistent association between elevated CENPU expression and poor prognosis across multiple cancer types suggests its potential utility as a prognostic biomarker in clinical oncology.

What cellular processes are affected by CENPU alteration in cancer cells?

CENPU affects several key cellular processes relevant to cancer development:

• Cell proliferation: CENPU knockdown significantly suppresses proliferation activity in various cancer cell lines, including breast cancer MDA-MB-231 cells and bladder cancer T24 cells .

• Cell cycle progression: CENPU silencing leads to G1 phase cell cycle arrest in multiple cancer cell types . In HCC cells, CENPU knockdown inhibits the G1/S transition both in vivo and in vitro .

• Apoptosis: Flow cytometry analysis demonstrates that apoptosis is significantly increased in CENPU-silenced cells compared to control cells .

• Colony formation: Giemsa staining shows that CENPU-silenced cells display significantly lower numbers of cell colonies compared to control cells, indicating reduced clonogenic capacity .

• Metastasis: CENPU has been implicated in cancer metastasis in various cancer types, with knockdown studies showing reduced metastatic potential .

Which signaling pathways interact with CENPU in cancer development?

Several key signaling pathways have been identified in CENPU-mediated cancer development:

• ERK1/2 and p38 pathways: In nasopharyngeal carcinoma, CENPU promotes growth and metastasis by activating the ERK1/2 and p38 pathways. Gene chip and ingenuity pathway analysis (IPA) revealed that p38/MAPK and ERK1/2 were strongly activated when CENPU was knocked down .

• E2F1-mediated signaling: In hepatocellular carcinoma, CENPU physically interacts with E2F6 and promotes its ubiquitin-mediated degradation, affecting the transcription level of E2F1 and accelerating G1/S transition .

• HMGB1 signaling pathway: Network analysis by Ingenuity Pathway Analysis (IPA) in bladder cancer revealed that CENPU is associated with the HMGB1 signaling pathway. CENPU knockdown downregulated expression levels of ILB, CXCL8, RAC1, and IL1A within this pathway .

• DUSP6 regulation: CENPU promotes the development of nasopharyngeal carcinoma by negatively regulating DUSP6 expression, with coimmunoprecipitation analysis revealing a physical interaction between CENPU and DUSP6 .

What protein interactions have been experimentally verified for CENPU?

Several critical protein interactions have been experimentally confirmed:

• DUSP6 interaction: Co-IP and reciprocal western blotting analysis revealed that CENPU was coimmunoprecipitated with DUSP6 and, conversely, DUSP6 was coimmunoprecipitated with CENPU in CNE-2 cells .

• E2F6 interaction: In HCC cells, CENPU physically interacts with E2F6 and promotes its ubiquitin-mediated degradation, thus affecting E2F1 transcription .

• Feedback loop mechanisms: A positive feedback loop of CENPU/E2F6/E2F1 has been identified in HCC, where E2F1 directly binds to the CENPU promoter and increases CENPU transcription, forming a positive regulatory loop .

These protein interactions provide mechanistic insights into how CENPU contributes to cancer progression and may offer potential targets for therapeutic intervention.

How does CENPU influence gene expression in cancer cells?

Genome-wide effects of CENPU manipulation have been studied through various high-throughput approaches:

• Differential gene expression: In bladder cancer research, CENPU knockdown resulted in 1,274 differentially expressed genes, including 809 downregulated genes and 465 upregulated genes .

• Network analysis: IPA analysis identified 25 distinct signaling pathways affected by CENPU knockdown, with the top-ranked network being "Cellular compromise, organismal injury and abnormalities, skeletal and muscular disorders" .

• Gene chip analysis: In NPC research, hierarchical clustering of differentially expressed genes between shCENPU and shCtrl groups showed high similarity within groups and low similarity between groups. Gene chip analysis found 172 upregulated genes and 397 downregulated genes following CENPU knockdown .

![Gene expression pattern example: Hierarchical clustering would show distinct patterns between CENPU knockdown and control groups]

What are the established protocols for CENPU gene silencing in experimental models?

Lentiviral-mediated RNA interference is the predominant method for CENPU silencing in cancer research:

• Vector construction: Short hairpin RNAs (shRNAs) targeting CENPU are designed and cloned into lentiviral vectors. For example:

  • Target sequence for CENPU: 5'-GCTGAAGTGCTAGAAAGGAAA-3'

  • Control sequence: 5'-TTCTCCGAACGTGTCACGT-3'

• Lentivirus production:

  • Recombinant lentiviral vectors carrying CENPU shRNA and non-silencing RNA are packaged by co-transfecting with Helper 1.0 and Helper 2.0 plasmids in 293(T) cells

  • Viral particles are harvested by centrifugation (4°C, 10 min, 4,000 × g) and purification after 48-hour cell culture

• Cell transfection:

  • Target cells in logarithmic phase are treated with trypsin (0.25%, pH=8.0) and re-suspended in appropriate media

  • Cell suspension (3–5×10^4 cells) is seeded onto six-well plates and incubated until reaching 30% confluence

  • Lentivirus (2×10^6 TU/ml) is added according to predetermined multiplicity of infection

  • Transfection efficiency is measured by green fluorescent protein (GFP) fluorescence

  • CENPU gene expression is evaluated using RT-qPCR and western blotting 3 days post-transfection

What cell-based assays are appropriate for assessing CENPU function?

Several validated assays are employed to evaluate the functional consequences of CENPU manipulation:

• Cell proliferation assays:

  • Celigo Imaging Cytometer: Transfected cells are plated in 96-well plates at equal densities (2,000 cells/100 μl), cultured for 24 hours, and then analyzed for GFP expression over a 5-day period

  • BrdU incorporation assay: Measures DNA synthesis rate as an indicator of proliferation

  • MTT assay: Evaluates cell viability and proliferation through metabolic activity

• Cell cycle analysis:

  • Flow cytometry: Cells are harvested, fixed, stained with propidium iodide, and analyzed for cell cycle distribution (G0/G1, S, and G2/M phases)

• Apoptosis detection:

  • Flow cytometry with Annexin V-APC staining: Quantifies early and late apoptotic cell populations

• Colony formation assay:

  • Cells are seeded in 6-well plates at appropriate densities and cultured for 10-14 days

  • Colonies are fixed with methanol, stained with Giemsa, and counted

What animal models are appropriate for studying CENPU function in vivo?

In vivo models provide critical insights into CENPU's role in tumor development:

• Xenograft tumor models:

  • CENPU-knockdown or overexpressing cancer cells are injected subcutaneously into immunodeficient mice (typically BALB/c nude mice)

  • Tumor growth is monitored by measuring tumor dimensions periodically

  • Tumor volume is calculated using the formula V = (length × width²)/2

  • At study endpoint, tumors are excised, weighed, and processed for histological and molecular analyses

• Metastasis models:

  • Cancer cells with manipulated CENPU expression are injected into the tail vein or other appropriate sites to model metastatic spread

  • Metastatic burden is assessed through techniques such as bioluminescence imaging, histological examination, or PCR-based detection of human-specific sequences

All animal experiments should be conducted in accordance with institutional guidelines and reporting checklists such as ARRIVE and MDAR .

How might CENPU serve as a therapeutic target in cancer treatment?

CENPU represents a promising therapeutic target based on several lines of evidence:

• Consistent oncogenic role: CENPU acts as a cancer-promoting gene across multiple cancer types, with overexpression consistently associated with poor survival .

• Critical cellular functions: CENPU knockdown inhibits proliferation, induces apoptosis, and arrests cell cycle progression in various cancer cell lines, suggesting that therapeutic targeting could effectively impair cancer growth .

• Pathway intervention: Targeting CENPU could disrupt multiple cancer-related signaling pathways, including ERK1/2, p38/MAPK, and HMGB1 pathways .

• Potential approaches:

  • RNA interference therapeutics targeting CENPU mRNA

  • Small molecule inhibitors disrupting CENPU protein interactions

  • PROTAC (Proteolysis Targeting Chimera) technology to induce CENPU degradation

The specific inhibition of CENPU may represent a novel strategy for cancer therapy, particularly in tumors with confirmed CENPU overexpression.

What are the challenges in developing CENPU-targeted therapies?

Several research challenges must be addressed:

• Delivery methods: Efficient delivery of RNA interference agents or other CENPU-targeting molecules to tumor cells remains challenging.

• Off-target effects: CENPU's role in normal cell division suggests potential toxicity in rapidly dividing normal tissues.

• Resistance mechanisms: Alternative pathways might compensate for CENPU inhibition, necessitating combination approaches.

• Biomarker development: Identification of patient populations most likely to benefit from CENPU-targeted therapy requires robust biomarker development.

How can CENPU expression be integrated into cancer prognostic models?

CENPU expression has demonstrated prognostic value across multiple cancer types:

• Multivariate analysis: The Cox proportional hazards regression model can be used to estimate the hazard ratio of CENPU expression while controlling for other clinical variables .

• Combined biomarker panels: Integration of CENPU with other molecular markers may enhance prognostic accuracy.

• Statistical methods:

  • Kaplan-Meier method for estimating survival probability

  • Log-rank test for evaluating differences between high and low CENPU expression groups

  • Spearman's correlation analysis for evaluating relationships between CENPU and other molecular markers

CENPU expression analysis could potentially be incorporated into clinical decision-making to identify high-risk patients who might benefit from more aggressive treatment approaches.

What are the knowledge gaps in understanding CENPU's role in cancer?

Despite significant advances, several aspects of CENPU biology remain unexplored:

• Tissue specificity: The mechanisms underlying CENPU's differential effects across cancer types require further investigation.

• Post-translational modifications: Limited information exists regarding how phosphorylation, ubiquitination, or other modifications regulate CENPU function.

• Epigenetic regulation: How CENPU expression is controlled at the epigenetic level remains largely unknown.

• Immune interactions: CENPU's potential role in modulating tumor-immune interactions has not been thoroughly explored.

What emerging technologies might advance CENPU research?

Several cutting-edge approaches could enhance our understanding of CENPU:

• CRISPR-Cas9 genome editing: Precise modification of CENPU or its regulatory elements could provide new insights into its function.

• Single-cell analysis: Examination of CENPU expression at the single-cell level could reveal heterogeneity within tumors.

• Proteomics approaches: Advanced mass spectrometry techniques could identify novel CENPU-interacting proteins.

• Structural biology: Determination of CENPU's three-dimensional structure could facilitate rational drug design.

• Patient-derived organoids: Testing CENPU manipulation in patient-derived 3D culture models could better recapitulate in vivo responses.