Computational Insights into CRISP3 Downregulation in Cervical Cancer and Its Cervical Lineages Pattern

Cervical Cancer (CC) is the fourth most common malignant tumor among women worldwide, particularly in low- and middle-income countries, where its incidence and mortality rates remain high. According to statistics, there were over 600,000 new cases and more than 340,000 deaths globally in 2020. The primary cause of cervical cancer is the persistent infection of high-risk Human Papillomavirus (HPV), especially HPV16 and HPV18. Although the promotion of HPV vaccines has reduced the incidence of cervical cancer to some extent, due to uneven vaccine coverage and the treatment needs of already infected populations, the prevention and control of cervical cancer still face significant challenges.

In this context, the search for new biomarkers and therapeutic targets has become an important direction in cervical cancer research. CRISP3 (Cysteine-Rich Secretory Protein 3) has garnered attention as a potential biomarker in various cancers in recent years. Initially isolated from human neutrophil secretions in 1996, its role in immune responses and inflammatory processes has gradually been uncovered. However, the expression pattern and clinical significance of CRISP3 in cervical cancer remain unclear. This study aims to explore the expression characteristics of CRISP3 in cervical cancer and its potential as a prognostic marker through computational biology and experimental validation.

Source of the Paper

This paper was co-authored by Ricardo Cesar Cintra, Andrés Galindo Céspedes, Mércia Patrícia Ferreira Conceição, and others, with the research team coming from multiple institutions including Universidade de São Paulo, Almanzor Aguinaga Asenjo National Hospital, and Universidad Privada del Este. The paper was published on July 24, 2024, in the journal Precision Clinical Medicine, titled Computational Insights into CRISP3 Downregulation in Cervical Cancer and Its Cervical Lineages Pattern.

Research Process and Results

1. Data Source and Differential Gene Expression Analysis

The study first screened datasets related to cervical cancer from the Gene Expression Omnibus (GEO) database, ultimately selecting the GSE63514 dataset for analysis. Using the GEO2R tool, the researchers identified differentially expressed genes (DEGs) and constructed a Protein-Protein Interaction (PPI) network. CRISP3 was selected as the focus of subsequent analysis due to its significant downregulation in tumor tissues.

Results:

In the GSE63514 dataset, CRISP3 expression was significantly lower in cervical cancer tissues compared to normal tissues. Additionally, as the grade of Cervical Intraepithelial Neoplasia (CIN) increased, CRISP3 expression showed a gradual decline, particularly in CIN3 and cervical cancer tissues, where this downregulation was more pronounced.

2. Protein-Protein Interaction Network and Pathway Enrichment Analysis

The researchers used the STRING database to construct a CRISP3-related PPI network and performed pathway enrichment analysis. The results showed that CRISP3 interacts closely with several classic markers (such as estrogen receptor ESR1 and androgen receptor AR) and inflammation-related genes (such as IL-6 and IL-8).

Results:

CRISP3 was highly correlated with genes such as CRISP2 (a protein from the same family) and TCN1 (a vitamin B12-binding protein). Pathway enrichment analysis indicated that CRISP3 may be involved in multiple biological processes, including “epidermal development,” “TGF-β signaling pathway,” “cell cycle regulation,” and “inflammatory response.”

3. TCGA and GENT2 Data Analysis

The researchers further utilized The Cancer Genome Atlas (TCGA) and GENT2 databases to analyze the expression characteristics of CRISP3 in cervical cancer patients and its relationship with clinicopathological parameters. Using RNA sequencing data, patients were divided into high and low CRISP3 expression groups, and correlations with age, HPV type, histological subtype, and other parameters were analyzed.

Results:

CRISP3 expression was significantly lower in Squamous Cell Carcinoma (SCC) and HPV16-positive patients, and low expression was associated with poorer Overall Survival (OS). Additionally, CRISP3 expression was negatively correlated with patient age, and HPV18-positive patients showed significantly higher CRISP3 expression compared to other HPV types.

4. Cell Culture and Epigenetic Drug Treatment

The researchers selected five cervical cancer cell lines (Siha, Sw756, C33A, HeLa, and Caski) and treated them with the epigenetic drugs Trichostatin A (TSA) and 5-Aza-2’-Deoxycytidine (5-Aza). CRISP3 expression changes were detected via RT-qPCR.

Results:

In all cell lines, TSA treatment significantly increased CRISP3 transcription levels, while 5-Aza treatment also upregulated CRISP3 in most cell lines. Particularly in Siha and Sw756 cells, the combined use of TSA and 5-Aza showed a synergistic effect, further enhancing CRISP3 expression.

5. miRNA Prediction and Survival Analysis

The researchers predicted miRNAs potentially regulating CRISP3 using the miRTarBase and miRWalk databases and analyzed the prognostic value of these miRNAs using the TCGA database.

Results:

miR-1229-3p was identified as a potential regulator of CRISP3, and its high expression was associated with poorer OS and Relapse-Free Survival (RFS). Additionally, high expression of miR-3614-5p was also associated with poorer OS.

Conclusions and Significance

This study, through computational biology and experimental validation, systematically explored the expression characteristics and clinical significance of CRISP3 in cervical cancer for the first time. The findings revealed that CRISP3 is significantly downregulated in cervical cancer tissues, particularly in SCC and HPV16-positive patients, where low expression is associated with poorer prognosis. Additionally, the epigenetic drugs TSA and 5-Aza significantly upregulated CRISP3 expression, suggesting that CRISP3 transcription may be regulated through epigenetic mechanisms.

Scientific Value:

1. Biomarker Potential: CRISP3, as a potential prognostic marker, may provide new references for personalized treatment in cervical cancer patients.
2. Epigenetic Regulatory Mechanisms: The study revealed the potential role of TSA and 5-Aza in regulating CRISP3 expression through epigenetic mechanisms, offering new directions for cervical cancer treatment.
3. HPV Type Correlation: The study found that CRISP3 expression is closely related to HPV types, indicating its unique role in HPV-associated cervical cancer.

Application Value:

1. Prognostic Assessment: CRISP3 expression levels may become an important indicator for prognostic assessment in cervical cancer patients.
2. Therapeutic Target: The upregulation of CRISP3 by epigenetic drugs may provide new targets for cervical cancer treatment.

Research Highlights

1. Multidimensional Analysis: The study combined computational biology, database analysis, and experimental validation to comprehensively explore the role of CRISP3 in cervical cancer.
2. Epigenetic Drug Effects: For the first time, the study revealed the regulatory effects of TSA and 5-Aza on CRISP3 expression, providing new research directions for cervical cancer treatment.
3. HPV Type Specificity: The study found that CRISP3 expression is closely related to HPV types, highlighting its unique role in HPV-associated cervical cancer.

Other Valuable Information

1. miRNA Regulatory Network: The study revealed the potential regulatory roles of miR-1229-3p and miR-3614-5p on CRISP3, providing new clues for molecular mechanism research in cervical cancer.
2. Cell Line Models: The study used multiple cervical cancer cell lines, covering different HPV types and histological subtypes, enhancing the generalizability of the results.

Through this study, the downregulation of CRISP3 in cervical cancer and its clinical significance have been systematically revealed, offering new perspectives and potential targets for the prognosis and treatment of cervical cancer.