Identification and validation of hub microRNAs dysregulated in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide, and its morbidity is exacerbated by the lack of early symptoms. Bioinformatics analyses enable discovery of differentially expressed genes and non-protein-coding RNAs of potential prognostic and/or therapeutic relevance in ESCC and other cancers. Using bioinformatics tools, we searched for dysregulated miRNAs in two ESCC microarray datasets from the Gene Expression Omnibus (GEO) database. After identification of three upregulated and five downregulated miRNAs shared between databases, protein-protein interaction (PPI) network analysis was used to identify the top 10 hub-gene targets. Thereafter, a miRNA-gene interaction network predicted that most hub genes are regulated by miR-196a-5p and miR-1-3p, which are respectively upregulated and downregulated in ESCC. Functional enrichment analyses in the GO and KEGG databases indicated the potential involvement of these miRNAs in tumorigenesis-related processes and pathways, while both differential expression and correlation with T stage were demonstrated for each miRNA in a cohort of ESCC patients. Overexpression showed that miR-196a-5p increased, whereas miR-1-3p attenuated, proliferation and invasion in human ESCC cell lines grown in vitro. These findings suggest miR-196a-5p and miR-1-3p jointly contribute to ESCC tumorigenesis and are potential targets for diagnosis and treatment.

Keywords: bioinformatics; esophageal squamous cell carcinoma; hub miRNAs; tumorigenesis.

Figure 1

Identification of differently expressed miRNAs (DEMs) in esophageal squamous cell carcinoma (ESCC). (A, B) GSE114110 data before and after normalization. (C, D) GSE43732 data before and after normalization. (E, F) Volcano plots of DEMs in GSE114110 and GSE43732, respectively. Black dots represent genes equally represented between ESCC and normal samples. Red and green dots represent upregulated and downregulated miRNAs, respectively. Volcano plots showing all DEMs. |log2FC| ≥ 1 and P < 0.05 were set as cut-off criteria. (G) Venn diagram analysis showing the top 10 upregulated and downregulated miRNAs in the two GEO datasets. (H) Identification of three upregulated and five downregulated miRNAs overlapping between both GEO datasets.

Figure 2

GO and KEGG analysis of genes targeted by DEMs commonly shared between the two GEO datasets. (A–C) Top 10 GO biological process (BP), cellular component (CC), and molecular function (MF) terms enriched in target genes of the three upregulated miRNAs. (D) Top 10 KEGG pathways enriched in target genes of the three upregulated miRNAs. (E–G) Top 10 GO BP, CC, and MF terms enriched in target genes of the five downregulated miRNAs. (H) Top 10 KEGG pathways enriched in target genes of the five downregulated miRNAs.

Figure 3

GO and KEGG analysis of hub-gene targets. (A) Top 10 hub-gene targets for the three upregulated miRNAs. (B) Top 10 hub-gene targets for the five downregulated miRNAs. (C–E) Top 10 GO BP, CC, and MF terms enriched in the top 20 hub-gene targets. (F) Top 10 KEGG pathways enriched in the 20 hub-gene targets.

Figure 4

Regulatory networks of miRNAs-hub-gene targets. (A) Interaction network of the three upregulated miRNAs and their hub-gene targets. (B) Regulatory network of the five downregulated miRNAs and their hub-gene targets. (C–D) Regulatory networks of miR-196a-5p and miR-1-3p and associated signaling pathways.

Figure 5

Relative expression of hub-gene targets of miR-196a-5p and miR-1-3p. (A–E) Relative expression of MYC, CCND1, PTEN, MAPK1, and CASP3 mRNAs in esophageal carcinoma, compared to normal esophageal tissue samples. (F–J) Relative expression of ACTB, EGFR, CDC42, IL6, and FN1 mRNAs in esophageal carcinoma, compared to normal esophageal tissue samples. Analysis of esophageal carcinoma RNA-seq datasets in TCGA was performed on the UALCAN database.

Figure 6

Expression analysis and prognostic value of miR-196-5p and miR-1-3p in ESCC. (A) Analysis of miR-196a-5p expression by qPCR in ESCC and normal esophageal epithelial (Het1A) cells. (B) Expression of miR-196a-5p in ESCC and normal esophagus samples from TCGA. (C) Expression of miR-196a-5p in ESCC and paired normal tissue samples (n = 32) collected at our institution. (D) Expression of miR-1-3p in ESCC and Het1A cells. (E) Expression of miR-1-3p in ESCC and normal esophagus samples from TCGA. (F) Expression of miR-1-3p in ESCC samples and paired normal tissue samples (n = 32) collected at our institution. (G and H) Kaplan-Meier survival analysis based on miR-196a-5p and miR-1-3p expression in esophageal carcinoma samples from TCGA. *P < 0.05, **P < 0.01. ***P < 0.001, and ****P < 0.0001.

Figure 7

Opposing effects of miR-196a-5p and miR-1-3p on proliferation and migration in cultured ESCC cells. (A–C) CCK-8 cell proliferation assay results from KYSE150, KYSE30, and KYSE410 cells transfected with mimics of miR-196a-5p, miR-1-3p, or negative control (NC). (D, F) EdU cell proliferation assay results from KYSE150, KYSE30, and KYSE410 cells transfected with mimics of miR-196a-5p, miR-1-3p, or NC. (E, G) Transwell migration assay results from KYSE150, KYSE30, and KYSE410 cells transfected with miRNA mimics. *P < 0.05, **P < 0.01, and ***P < 0.001.