Long noncoding RNA LINC01296 promotes tumor growth and progression by sponging miR-5095 in human cholangiocarcinoma

Abstract

The aim of the present study was to elucidate whether, and how, long intergenic non-protein coding RNA 1296 (LINC01296) is involved in the modulation of human cholangiocarcinoma (CCA) development and progression. Microarray data analysis and reverse transcription-quantitative polymerase chain reaction analysis demonstrated that LINC01296 was significantly upregulated in human CCA compared with nontumor tissues. Furthermore, the expression of LINC01296 in human CCA was positively associated with tumor severity and clinical stage. Knockdown of LINC01296 dramatically suppressed the viability, migration and invasion of RBE and CCLP1 cells, and promoted cell apoptosis in vitro. Furthermore, LINC01296 knockdown inhibited tumor growth in a xenograft model. Mechanistically, LINC01296 was demonstrated to sponge microRNA-5095 (miR-5095), which targets MYCN proto-oncogene bHLH transcription factor (MYCN) mRNA in human CCA. By inhibition of miR-5095, LINC01296 overexpression upregulated the expression of MYCN and promoted cell viability, migration and invasion in CCA cells. The results reveal that the axis of LINC01296/miR-5095/MYCN may be a mechanism to regulate CCA development and progression.

Figure 1

LINC01296 is upregulated in human CCA. (A) LINC01296 upregulation in human CCA samples by bioinformatics analysis according to the dataset (GSE61850). (B) Analysis of LINC01296 expression levels in 57 paired human CCA samples and nontumor tissue by RT-qPCR, ^***P<0.001. (C) Fluorescent in situ hybridization and (D) in situ hybridization probing for LINC01296 in CCA and nontumor samples. (E) Expression levels of LINC01296 in CCA cell lines (RBE, CCLP1, HuCCT1 and HCCC-9810) and noncancerous cholangiocyte cell line (HIBEC) were determined by RT-qPCR. ^*P<0.05 and ^**P<0.01 vs. HIBEC. (F) Survival rates of patients with CCA with high and low LINC01296 by Kaplan-Meier survival analysis. Data are presented as the mean ± standard deviation. CCA, cholangiocarcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; LINC01296, long intergenic non-protein coding RNA 1296.

Figure 2

LINC01296 knockdown suppresses cell proliferation and promotes cell apoptosis. (A) RBE and CCLP1 cells were transfected with shLINC01296, followed by reverse transcription-quantitative polymerase chain reaction detection of LINC01296 expression. Cell proliferation was determined in (B) RBE and (C) CCLP1 cells transfected with shCtrl or shLINC01296 by CCK8 assay and colony formation assay. (D) Cell cycle distribution was determined by flow cytometry. (E) Cell apoptosis was measured in RBE and CCLP1 cells transfected with shCtrl or shLINC01296 by staining with Annexin V/PI. Data are presented as the mean ± standard deviation. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. shCtrl. LINC01296, long intergenic non-protein coding RNA 1296; sh, short hairpin RNA; Ctrl, control; PI, propidium iodide.

Figure 3

LINC01296 knockdown inhibits the migration and invasion of RBE and CCLP1 cells. (A) Cell migration was determined by wound healing assay in RBE and CCLP1 cells transfected with shCtrl or shLINC01296. (B) Cell invasion was determined by Transwell invasion assay in RBE and CCLP1 cells transfected with shCtrl or shLINC01296. Data are presented as the mean ± standard deviation. ^**P<0.01 and ^***P<0.001 vs. shCtrl. LINC01296, long intergenic non-protein coding RNA 1296; sh, short hairpin RNA; Ctrl, control.

Figure 4

LINC01296 interacts with miR-5095 in RBE and CCLP1 cells. (A) miR-5095 binding sites in LINC01296 predicted by bioinformatics analysis. (B) Analysis of miR-5095 expression levels in RBE and CCLP1 cells transfected with LINC01296 overexpressing plasmid or miR-5095 inhibitor by RT-qPCR. (C) Analysis of LINC01296 expression levels in RBE and CCLP1 cells transfected with shLINC01296 or miR-5095 mimic by RT-qPCR. ^***P<0.001 vs. oeVec. (D) Luciferase reporter assays were performed using RBE cells co-transfected with the miR-5095 mimic or inhibitor and LINC01296-wt or LINC01296-mut reporter plasmid. ^**P<0.01. (E) The expression levels of miR-5095 were determined in CCA cell lines (RBE, CCLP1, HuCCT1 and HCCC-9810) and noncancerous cholangiocyte cell line (HIBEC) by RT-qPCR. ^*P<0.05 and ^**P<0.01 vs. HIBEC. (F) The expression of miR-5095 in CCA samples and nontumor tissues was determined by RT-qPCR. ^***P<0.001. (G) Spearman's correlation analysis was used to determine the correlations between the levels of LINC01296 and miR-5095 in human CCA (n=57). Data are presented as the mean ± standard deviation. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCA, cholangiocarcinoma; LINC01296, long intergenic non-protein coding RNA 1296; WT, wild-type; Mut, mutated; miR, microRNA; oeVec, overexpression empty vector; oeLINC01296, LINC01296 overexpression vector; sh, short hairpin RNA; Ctrl, control; NC, negative control; inh, miR inhibitor.

Figure 5

MYCN is a target of miR-5095. (A) Conservation of the miR-5095-targeting sites in the MYCN-3′UTR and its mutant sequence that abrogates MYCN binding to target mRNA. (B) Luciferase reporter assays were performed using RBE cells co-transfected with the miR-5095 mimic or inhibitor and MYCN-WT-3′UTR or MYCN-Mut-3′UTR reporter plasmid. (C) mRNA and (D) protein levels of MYCN in RBE and CCLP1 cells transfected with the miR-5095 mimic or inhibitor were determined by RT-qPCR. (E) Expression of MYCN in cholangiocarcinoma samples and nontumor tissues was determined by RT-qPCR. (F) Spearman's correlation analysis was used to determine the correlations between the levels of MYCN and miR-5095 in human CCA (n=57). Data are presented as the mean ± standard deviation. ^**P<0.01 and ^***P<0.001. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MYCN, MYCN proto-oncogene bHLH transcription factor; WT, wild-type; miR, microRNA; Mut, mutated; NC, negative control; inh, miR inhibitor; Ctrl, control.

Figure 6

Restoration of MYCN reverses LINC01296 knockdown-induced inhibition of cell proliferation, migration, and invasion of RBE and CCLP1 cells. (A) Knockdown of LINC01296 or miR-5095 inhibited the level of MYCN protein in RBE and CCLP1 cells as demonstrated by western blot analysis. (B) Cell viability, (C) apoptosis, (D) migration and (E) invasion were determined in RBE and CCLP1 cells transfected with shLINC01296, miR-5095 mimic, shMYCN, miR-5095 inhibitor or MYCN-overexpressing plasmid by CCK8, flow cytometry, wound-healing, and invasion assays, respectively. Data are presented as the mean ± standard deviation. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Ctrl. Ctrl, control; sh, short hairpin RNA; LINC01296, long intergenic non-protein coding RNA 1296; miR, microRNA; MYCN, MYCN proto-oncogene bHLH transcription factor; oe, overexpression vector.

Figure 7

LINC01296 promoted tumor growth in vivo by activation of MYCN. (A) Tumor growth curves were established by measuring tumor volume every 7 for 28 days after injection. (B) Tumor weights isolated from nude mice in each treatment group were determined on day 28 after injection. (C) Relative LINC01296 and miR-5095 expression in xenograft tumors was determined by reverse transcription-quantitative polymerase chain reaction. (D) MYCN protein expression in xenograft tumors was determined by western blot. GAPDH was served as a loading control. Data are presented as the mean ± standard deviation ^*P<0.05 and ^**P<0.01 vs. shCtrl. sh, short hairpin RNA; Ctrl, control; LINC01296, long intergenic non-protein coding RNA 1296; miR, microRNA; MYCN proto-oncogene bHLH transcription factor; MMP2, matrix metalloproteinase-2.