CeRNA regulatory network-based analysis to study the roles of noncoding RNAs in the pathogenesis of intrahepatic cholangiocellular carcinoma

Abstract

To explore and understand the competitive mechanism of ceRNAs in intrahepatic cholangiocarcinoma (ICC), we used bioinformatics analysis methods to construct an ICC-related ceRNA regulatory network (ceRNET), which contained 340 lncRNA-miRNA-mRNA regulatory relationships based on the RNA expression datasets in the NCBI GEO database. We identified the core regulatory pathway RP11-328K4.1-hsa-miR-27a-3p-PROS1, which is related to ICC, for further validation by molecular biology assays. GO analysis of 44 differentially expressed mRNAs in ceRNET revealed that they were mainly enriched in biological processes including "negative regulation of epithelial cell proliferation" and "positive regulation of activated T lymphocyte proliferation." KEGG analysis showed that they were mainly enriched in the "complement and coagulation cascade" pathway. The molecular biology assay showed that lncRNA RP11-328K4.1 expression was significantly lower in the cancerous tissues and peripheral plasma of ICC patients than in normal controls (p<0.05). In addition, hsa-miR-27a-3p was found to be significantly upregulated in the cancer tissues and peripheral plasma of ICC patients (p<0.05). Compared to normal controls, the expression of PROS1 mRNA was significantly downregulated in ICC patient cancer tissues (p<0.05) but not in peripheral plasma (p>0.05). Furthermore, ROC analysis revealed that RP11-328K4.1, hsa-miR-27a-3p, and PROS1 had significant diagnostic value in ICC. We concluded that the upregulation of lncRNA RP11-328K4.1, which might act as a miRNA sponge, exerts an antitumor effect in ICC by eliminating the inhibition of PROS1 mRNA expression by oncogenic miRNA hsa-miR-27a.

Keywords: biomarkers; ceRNA; ceRNA regulatory network; intrahepatic cholangiocarcinoma; prognosis.

Figure 1

Heat map (A) and volcano map (B) of differentially expressed mRNAs in the GSE61850 dataset.

Figure 2

Heat map (A) and volcano map (B) of differentially expressed lncRNAs in the GSE61850 dataset.

Figure 3

Heat map (A) and volcano map (B) of differentially expressed mRNAs in the GSE103909 dataset.

Figure 4

Heat map (A) and volcano map (B) of differentially expressed lncRNAs in the GSE103909 dataset.

Figure 5

Heat map (A) and volcano map (B) of differentially expressed mRNAs in the GSE57555 dataset.

Figure 6

Heat map (A) and volcano map (B) of differentially expressed miRNAs in the GSE57555 dataset.

Figure 7

Heat map (A) and volcano map (B) of differentially expressed miRNAs in the GSE53992 dataset

Figure 8

Heat map (A) and volcano map (B) of differentially expressed miRNAs in the GSE53870 dataset

Figure 9

Venn diagram of differentially expressed mRNAs, lncRNAs, miRNAs in all datasets (from A to B to C: mRNA, lncRNA, miRNA).

Figure 10

The ICC-related ceRNA network map (green circles indicate the downregulated mRNAs, red circles indicate the upregulated mRNA, dark blue diamonds indicate the downregulated lncRNA, light purple diamonds indicate the upregulated lncRNA, pink triangles indicate the upregulated miRNA, brown triangles indicate the downregulated miRNA, and gray triangles indicate that upregulation or downregulation of miRNAs cannot be determined. The gray arrow indicates the regulatory relationship between miRNA and lncRNA, the yellow arrow indicates the regulatory relationship between miRNA and mRNA, and the green dotted line indicates the synergistic expression relationship between lncRNA and mRNA).

Figure 11

GO terms (including biological process (BP), cellular component (CC) and molecular function (MF)) and KEGG pathways involved in the construction of ICC-related ceRNETs of 44 DE mRNAs.

Figure 12

qRT-PCR analysis showed the difference in lncRNA RP11-328K4.1 expression between the experimental group and control group in ICC fresh tissue samples after normalization to internal controls. RP11-328K4.1 was normalized to β-actin.

Figure 13

qRT-PCR analysis showed the difference in lncRNA RP11-328K4.1 expression between the experimental group and control group in peripheral plasma samples after normalization to internal controls. RP11-328K4.1 was normalized to β-actin.

Figure 14

(A–C) ROC analysis of RP11-328K4.1, hsa-miR-27a-3p, and PROS1 in ICC tumor tissues and matched adjacent nontumor tissues. (D) ROC analysis of RP11-328K4.1, hsa-miR-27a-3p, and PROS1 in early ICC and advanced ICC.

Figure 15

The flowchart of the study.