Biomarker cystatin B expression correlates with pathogenesis in cervical cancer

Abstract

Objective: Cervical cancer (CC) is one of the most common gynecologic malignancies worldwide. Although rapid improvements have been made regarding its prevention and treatment, little is known about disease pathogenesis and the clinical relevance of reliable biomarkers. The present study evaluated the expression of cystatin B (CSTB) as a potential biomarker of CC.

Methods: Tissue microarray analysis and immunohistochemical staining were performed to detect CSTB expression, while CSTB mRNA and protein expression levels of freshly isolated CC tissue were measured by quantitative real-time PCR and western blot, respectively. Bioinformatics were used to analyze the CSTB co-expression network and functional enrichments.

Results: We observed high CSTB mRNA and protein expression levels in CC tissues, which was confirmed by tissue microarray in a comparison with paired adjacent non-cancerous cervical tissue samples. CSTB gene enrichments and associations with co-expressed genes were also observed. Further analysis showed that elevated CSTB expression was associated with pathological progress in CC.

Conclusion: Our data demonstrate that CSTB has the potential to be used as a tissue biomarker with clinical value in patients with CC, which may aid the development of intervention strategies.

Keywords: Cystatin B; cervical cancer; lesion; pathological progress; tissue microarray; tumor.

Figure 1.

Expression of CSTB in human cervical tissue. (a) Comparison of CSTB transcripts between healthy cervical tissues and primary tumor tissues of cervical squamous cell carcinoma from the cancer data analysis portal of the University of Alabama at Birmingham using TCGA samples. Analysis was performed using the Student’s t-test, and data are presented as the mean ± SD. ****, P < 0.0001. (b) CSTB mRNA detection by qRT-PCR. One-way ANOVA followed by Tukey’s multiple comparison test was used. Data are presented as the mean ± SD. (c) Representative image of western blot for the CSTB protein. (d) Semi-quantitative analysis of (c). One-way ANOVA followed by Tukey’s multiple comparisons test was used. Data are presented as the mean ± SD. CSTB, cystatin B; N, normal; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; CC, cervical cancer; TCGA, the cancer genome atlas; qRT-PCR, quantitative real-time PCR; ANOVA, analysis of variance.

Figure 2.

Detection of CSTB protein expression in tissue microarray. (a) Detection of CSTB protein expression in non-tumorous cervical tissues (Normal) and primary cervical cancer tissues (cancer) by immunohistochemistry staining. Representative images are shown. Original, magnification ×200; Amplified, magnification ×400. (b) Comparison of CSTB protein expression between 70 paired normal and cancer tissues after immunohistochemistry staining. The number of cases in each score group is indicated. (c) Analysis of the IHC score for different tumor stages. Data are presented as the mean ± SD. ****, P < 0.001 tumor (T) vs. normal (N); T1-4, tumor stage 1-4. (d) Analysis of the overall survival (OS) of patients with CC. (e) Analysis of the time to progression (TTP) of patients with CC. CSTB, cystatin B; IHC, immunohistochemistry; TNM, tumor, node and metastasis; I–IV, TNM stages I–IV; CC, cervical cancer.

Figure 3.

Association of CSTB mRNA with clinical features in patients with CC. (a) Comparison of CSTB mRNA expression between patients aged ≤65 and >65 years old using the Student’s t-test. (b) Comparison of CSTB mRNA expression between CC tumor stages from T1 to T4 using one-way ANOVA followed by Tukey’s multiple comparisons test. (c) Comparison of CSTB mRNA expression between the pathological grades of CC from G1 to G3 using one-way ANOVA followed by Tukey’s multiple comparisons test. Data are presented as the mean ± SD. CSTB, cystatin B; ANOVA, analysis of variance; CC, cervical cancer.

Figure 4.

Association of CSTB expression with that of other genes. (a) Heatmap representing genes positively and negatively correlated with high and low expression of CSTB. (b) GSEA analysis showed functional enrichment of CSTB expression in CC cohorts from the TCGA database. (c) Gene–gene functional correlation analysis visualized the association of the top 20 positively related genes with CSTB expression. CSTB, cystatin B; GSEA, gene set enrichment analysis; TCGA, the cancer genome atlas; CC, cervical cancer.

Figure 5.

Functional enrichment analyses of intersecting genes correlated with CSTB. (a) Five pathways identified from KEGG enrichment analysis. (b) GO term analysis. The bubble plot shows the top seven elements significantly enriched in GO categories BP, CC, and MF. GeneRatio refers to the ratio of the number of genes enriched in the term/pathway to the total number of genes in the terms/pathways. CSTB, cystatin B; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene ontology; BP, biological process; CC, cellular component; MF, molecular function.