Upregulated lncRNA-UCA1 contributes to metastasis of bile duct carcinoma through regulation of miR-122/ CLIC1 and activation of the ERK/MAPK signaling pathway

Abstract

In the present study, we aimed to identify specific lncRNAs and miRNAs, as well as mRNAs, involved in bile duct carcinoma (BDC) and to further explore the way in which lncRNA UCA1 regulates cell metastasis ability. Differentially expressed RNAs were screened out from the TCGA database. In in vitro experiments, qRT-PCR was used to measure lncRNA UCA1, miR-122 and CLIC1 expression. We performed a dual luciferase assay to validate the target relationships among UCA1, CLIC1 and miR-122. The cell migration ability was measured by a wound healing assay, and Transwell assays were applied to detect cell invasive ability. Western blot analysis was employed to detect the expression of related proteins in the MAPK signaling pathway. According to the bioinformatics analysis, lncRNA UCA1 and CLIC1 were both significantly upregulated in BDC, while the expression of miR-122 declined compared with the normal group. The target relationship among UCA1, CLIC1 and miR-122 was verified. UCA1 promoted BDC cell migration and invasiveness, while miR-122 inhibited their progression. CLIC1 served as the downstream target gene of miR-122 and had opposite effects. The ERK/MAPK signaling pathway was activated after upregulating UCA1. LncRNA-UCA1 promoted the metastasis of BDC cells by regulating the expression of miR-122 and its downstream gene mRNA CLIC1 and promoted the activation of the ERK/MAPK pathway, which expanded the horizons of targeted therapy of cholangiocarcinoma.

Keywords: Bile duct carcinoma; ERK/MAPK signaling pathway; UCA1; miR-122.

Figure 1.

LncRNA UCA1 expression was upregulated in human bile duct carcinoma. (a) Volcano map. x-axis: log2 (fold change); y-axis: log10 (P-value). the vertical blue lines correspond to 2-fold up and down, and the horizontal blue line represents a P value of 0.05. the red and green points in the plot represent differentially expressed lncRNAs with statistical significance (red: up, green: down). (b) Heatmap showing the differential expression and hierarchical clustering of lncRNAs between BDC and adjacent normal tissues. the top 10 low-expressed and high-expressed lncRNAs in tumor tissue were screened and presented. the red and green blocks in the map represent differentially expressed lncRNAs (red: high-expressed, green: low-expressed). (c) Kaplan–Meier plots for overall survival for a discriminatory median UCA1 expression, from TCGA sequencing data to assess prognostic accuracy. P values were calculated using the log-rank test. (d) the relative expression levels of UCA1 in BDC cell lines and normal bile duct epithelial cells were detected by qRT-PCR. **P < 0.01 compared to the HIBEC group. (e) the relative expression levels of UCA1 in 22 pairs of BDC tissues and para-carcinoma tissues were detected by qRT-PCR. **P < 0.01 compared to the para-carcinoma group. (f) qRT-PCR was used to detect the transfection efficiency of UCA1 in BDC cells treated with pcDNA3.1-UCA1 and siUCA1. **P < 0.01 compared to the NC group.

Figure 2.

Effects of UCA1 on cell migration and invasion. (a-b) Cell migration ability was detected using wound healing assay in CCLP1 and QBC939 cells after transfection for 0 h, 24 h and 48 h. (c-d) Transwell assays were performed to investigate changes in cell invasion ability. Scale bars represent 80 μm.

Figure 3.

Effects of UCA1 on activation of ERK/MAPK signaling pathway. (a-b) the relative protein expression levels of the ERK/MAPK signaling pathway in CCLP1 cells and QBC939 cells were detected by western blot. compared to the NC group, overexpressed UCA1 enhanced the phosphorylation of ERK and MAPK proteins while the silencing of UCA1 weakened the phosphorylation level. all tendency of results was consistent in CCLP1 and QBC939 cells. **P < 0.01 compared to the NC group.

Figure 4.

UCA1 is a target of miR-122. (a) Volcano plot. x-axis: log2 (fold change); y-axis: log10 (P-value). The vertical blue lines correspond to 2.0-fold up and down, and the horizontal blue line represents a P-value of 0.05. The red and green points in the plot represent differentially expressed microRNAs with statistical significance (red: up, green: down). (b) Heatmap showing the differential expression and hierarchical clustering of microRNAs between BDC and adjacent normal tissues. The top 10 low-expressed and high-expressed microRNAs in tumor tissue were screened and presented. The red and green blocks in the map represent differentially expressed microRNAs (red: high-expressed, green: low-expressed). (c) Kaplan–Meier plots for overall survival for a discriminatory median miR-122 expression, from TCGA sequencing data to assess prognostic accuracy. P values were calculated using the log-rank test. (d) Base pairing complement suggested the putative miR-122 binding position on UCA1. (e) Luciferase reporter assay of BDC cells cotransfected with UCA1-wt or UCA1-mut and miR-122 mimics or the miR-NC. **P < 0.01 compared to the NC group. (f) The relative expression levels of miR-122 in BDC cell lines and normal bile duct epithelial cells were detected by qRT-PCR. **P < 0.01 compared to the HIBEC group. (g) The relative expression levels of miR-122 in 22 pairs of BDC tissues and para-carcinoma tissues were detected by qRT-PCR. **P < 0.01 compared to the para-carcinoma group. (h) Scatter diagram exhibited a negative correlation of UCA1 and miR-122 in 22 pairs of BDC tissues by qRT-PCR. (i) Effects of lnc-UCA1 on expression levels of miR-122 were detected by qRT-PCR after transfection. **P < 0.01 compared to the NC group.

Figure 5.

Effects of miR-122 on cell migration and invasion. (a-b) BDC cell migration ability was detected using wound healing assays in CCLP1 and QBC939 cells after transfection for 0 h, 24 h and 48 h. (c-d) Transwell assays were performed to investigate changes in cell invasion ability. Scale bars represent 80 μm.

Figure 6.

Effects of miR-122 on activation of ERK/MAPK signaling pathway. (a-b) The relative protein expression levels of the ERK/MAPK signaling pathway in the CCLP1 and QBC939 cells were detected by western blot. Compared with the NC group, the overexpressed miR-122 weakened the phosphorylation of ERK and MAPK proteins while the phosphorylation level in mimics+UCA1 group showed no significant difference. All tendency of results was consistent in CCLP1 and QBC939 cells. **P < 0.01 compared to the NC group.

Figure 7.

CLIC1 is upregulated in human bile duct carcinoma. (a) Volcano plot. x-axis: log2 (fold change); y-axis: log10 (P-value). The vertical blue lines correspond to 2.0-fold up and down, and the horizontal blue line represents a P-value of 0.05. The red and green points in the plot represent differentially expressed mRNAs with statistical significance (red: up, green: down). (b) Heat map showing the differential expression and hierarchical clustering of mRNAs between BDC and adjacent normal tissues. The top 10 low-expressed and high-expressed mRNAs in tumor tissue were screened and presented. The red and green blocks in the map represent differentially expressed mRNAs (red: high-expressed, green: low-expressed). (c) Kaplan–Meier plots for overall survival for a discriminatory median CLIC1 expression, from TCGA sequencing data to assess prognostic accuracy. P values were calculated using the log-rank test. (d) Coexpression network of lncRNA-UCA1 with their associated miRNAs and mRNAs. The network is based on the Pearson correlation coefficient, and orange solid lines mean positive correlations while gray dotted lines mean negative correlations.

Figure 8.

CLIC1 is a target gene of miR-122. (a) Base pairing complement suggested the putative miR-122 binding position in the 3′-UTR of CLIC1. (b) Luciferase reporter assay of BDC cells cotransfected with CLIC1-wt or CLIC1-mut and miR-122 mimics or the miR-NC. **P < 0.01 compared to the NC group. (c) The relative expression levels of CLIC1 in BDC cell lines and normal bile duct epithelial cells were detected by qRT-PCR. **P < 0.01 compared to the HIBEC group. (d) The relative expression levels of CLIC1 in 22 pairs of BDC tissues and para-carcinoma tissues were detected by qRT-PCR. **P < 0.01 compared to the para-carcinoma group. (e) Effects of lnc-UCA1 and miR-122 on expression levels of CLIC1 were detected by qRT-PCR after transfection. **P < 0.01 compared to the NC group. (f) Scatter diagram exhibited a negative correlation of CLIC1 and miR-122 in 22 pairs of BDC tissues by qRT-PCR.

Figure 9.

Effects of CLIC1 on cell migration and invasion in BDC. (a-b) BDC cell migration ability was detected using wound healing assays in CCLP1 and QBC939 cells after transfection for 0 h, 24 h and 48 h. (c-d) Transwell assays were performed to investigate changes in cell invasion ability. Scale bars represent 80 μm. **P < 0.01 compared to the NC group.

Figure 10.

Effects of CLIC1 on activation of ERK/MAPK signaling pathway. (a-b) The relative protein expression levels of the ERK/MAPK signaling pathway in CCLP1 and QBC939 cells were detected by western blotting. Compared with the NC group, the knockdown of CLIC1 weakened the phosphorylation of ERK and MAPK proteins while the overexpression of CLIC1 enhanced the phosphorylation of ERK and MAPK proteins, and the phosphorylation level in mimics+CLIC1 and siUCA1+ CLIC1 groups showed no significant difference. All tendency of results were consistent in the CCLP1 and QBC939 cells. **P < 0.01 compared to the NC group.