Investigating novel biomarkers in uterine corpus endometrial carcinoma: in silico analysis and clinical specimens validation via RT-qPCR and immunohistochemistry

Abstract

The rising incidence and mortality rate of Uterine Corpus Endometrial Carcinoma (UCEC) pose significant health concerns. CC and CXC chemokines have been linked to tumorigenesis and cancer progression. Recognizing the growing significance of CC and CXC chemokines' diagnostic and prognostic significance in diverse cancer types, our objective was to comprehensively analyze the diagnostic and prognostic values of hub genes from the CC and CXC chemokines in UCEC, utilizing both in silico and clinical samples and cell lines-based approaches. In silico analyses include STRING, Cytoscape, Cytohubba, The Cancer Genome Atlas (TCGA) datasets analysis via the UALCAN, GEPIA, OncoDB, and MuTarget, SurvivalGenie, MEXPRESS, cBioPoratal, TIMER, ENCORI, and DrugBank. Meanwhile, clinical samples and cell lines based analyses include Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), targeted bisulfite sequencing (bisulfite-seq) analysis, and immunohistochemistry (IHC). Through present study, we identified CCL25 (CC motif chemokine ligand 25), CXCL10 (C-X-C motif chemokine ligand 10), CXCL12 (C-X-C motif chemokine ligand 12), and CXCL16 (C-X-C motif chemokine ligand 16) as crucial hub genes among the CC and CXC chemokines. Analyzing the expression data from TCGA, we observed a significant up-regulation of CCL25, CXCL10, and CXCL16 in UCEC samples compared to controls. In contrast, we noted a significant down-regulation of CXCL12 expression in UCEC samples. On clinical UCEC samples and cell lines analysis, the significant higher expression of CCL25, CXCL10, and CXCL16 and significant lower expression of CXCL12 were also denoted in UCEC samples than the controls via RT-qPCR and IHC analyses. Moreover, in silico analysis also confirmed the abnormal promoter methylation levels of the hub genes in TCGA UCEC samples, which was later validated by the clinical samples using targeted based bisulfite-seq analysis. In addition, various additional aspects of the CCL25, CXCL10, CXCL12, and CXCL16 have also been uncovered in UCEC during the present study. Our findings offer novel insights that contribute to the clinical utility of CCL25, CXCL10, CXCL12, and CXCL16 chemokines as potential diagnostic and prognostic biomarkers in UCEC.

Keywords: CC and CXC chemokines; UCEC; biomarkers.

Figure 1

A PPI network of the CC and CXC Families member genes and identified hub genes. (A, B) PPI networks of the CC and CXC Families member genes and (C) A PPI network of identified four hub genes. PPI, Protein-protein interaction.

Figure 2

Expression profiling of the CCL25, CXCL10, CXCL12, and CXCL16 in UCEC samples paired with controls via UALCAN. (A) A heatmap of CCL25, CXCL10, CXCL12, and CXCL16 hub genes in UCEC sample group and normal control group and (B) Box plot presentation of CCL25, CXCL10, CXCL12, and CXCL16 hub genes expression in UCEC sample group and normal control group. A p-value less than 0.05 was considered as significant. UCEC, Uterine Corpus Endometrial Carcinoma.

Figure 3

Expression profiling of CCL25, CXCL10, CXCL12, and CXCL16 in UCEC samples of different clinical variables relative to controls via UALCAN. (A) Expression profiling of CCL25 in UCEC samples of different clinical variables, (B) Expression profiling of CXCL10 in UCEC samples of different clinical variables, (C) Expression profiling of CXCL12 in UCEC samples of different clinical variables, and (D) Expression profiling of CXCL16 in UCEC samples of different clinical variables. A p-value less than 0.05 was considered as significant. UCEC, Uterine Corpus Endometrial Carcinoma.

Figure 4

Expression validation of CCL25, CXCL10, CXCL12, and CXCL16 using additional TCGA datasets. (A) Expression validation of CCL25, CXCL10, CXCL12, and CXCL16 in UCEC and normal samples via GEPIA database, (B) Expression validation of CCL25, CXCL10, CXCL12, and CXCL16 in UCEC and normal samples via OncoDB database, (C) Expression validation of CCL25, CXCL10, CXCL12, and CXCL16 in UCEC and normal samples via MuTarget database, and (D) Expression heatmap of CCL25, CXCL10, CXCL12, and CXCL16 in UCEC and normal samples via MuTarget database. A p-value less than 0.05 was considered as significant. UCEC, Uterine Corpus Endometrial Carcinoma.

Figure 5

Survival analysis of CCL25, CXCL10, CXCL12, and CXCL16 in UCEC patients using SurvivalGenie database. A p-value less than 0.05 was considered as significant. UCEC, Uterine Corpus Endometrial Carcinoma.

Figure 6

Methylation status exploration of CCL25, CXCL10, CXCL12, and CXCL16 via MEXPRESS in UCEC and normal samples. (A) CCL25, (B) CXCL10, (C) CXCL12, and (D) CXCL16. A p-value less than 0.05 was considered as significant. UCEC, Uterine Corpus Endometrial Carcinoma.

Figure 7

Exploration of genetic alteration frequencies and co-express genes with hub gens in UCEC samples via cBioPortal. (A) Frequencies, and location of the genetic alterations in CCL25, CXCL10, CXCL12, and CXCL16, and (B) Co-express genes with hub genes in UCEC groups. UCEC, Uterine Corpus Endometrial Carcinoma.

Figure 8

Gene enrichment analysis of CCL25, CXCL10, CXCL12, and CXCL16. (A) CCL25, CXCL10, CXCL12, and CXCL16 associated CC terms, (B) CCL25, CXCL10, CXCL12, and CXCL16 associated MF terms, (C) CCL25, CXCL10, CXCL12, and CXCL16 associated BP terms, and (D) CCL25, CXCL10, CXCL12, and CXCL16 associated KEGG terms. A p-value less than 0.05 was considered as significant. CC, Cellular components; MF, Molecular functions; BP, Biological process; KEGG, Encyclopedia of Genes and Genomes.

Figure 9

Correlation analysis of CCL25, CXCL10, CXCL12, and CXCL16 hub genes expression with different immune cells (CD8+ T, CD4+ T, and macrophages) infiltration level. (A) CCL25, (B) CXCL10, (C) CXCL12, and (D) CXCL16. A p-value less than 0.05 was considered as significant.

Figure 10

miRNA-mRNA co-regulatory network of CCL25, CXCL10, CXCL12, and CXCL16 hub genes. (A) A PPI of miRNAs targeting hub genes, (B) A PPI highlighting most important miRNA (hsa-mir-744-5p) targeting all hub genes, and (C) RT-qPCR based expression profiling of has-miR-27a-5p. A p-value less than 0.05 was considered as significant. miRNA, MicroRNA; mRNA, Messenger RNA; RT-qPCR, Reverse transcription-quantitative polymerase chain reaction.

Figure 11

Validating CCL25, CXCL10, CXCL12, and CXCL16 expressions and promoter methylation levels using UCEC tissue samples and cell lines paired with controls via RT-qPCR and targeted bisulfite-seq analyses. (A) Relative expression profile of CCL25, CXCL10, CXCL12, and CXCL16 across UCEC tissue samples paired with controls via RT-qPCR, (B) Relative expression profile of CCL25, CXCL10, CXCL12, and CXCL16 using UCEC cell lines paired with control via RT-qPCR, (C) Beta values based promoter methylation based validation of CCL25, CXCL10, CXCL12, and CXCL16 across UCEC tissue samples paired with controls, and (D) IHC-based differential expression of CCL25, CXCL10, CXCL12, and CXCL16 in UCEC and normal tissues. A p-value less than 0.05 was considered as significant. UCEC, Uterine Corpus Endometrial Carcinoma; RT-qPCR, Reverse transcription-quantitative polymerase chain reaction.