LINC02418 promotes colon cancer progression by suppressing apoptosis via interaction with miR-34b-5p/BCL2 axis

Abstract

Background: LncRNAs act as functional regulators in tumor progression through interacting with various signaling pathways in multiple types of cancer. However, the effect of LINC02418 on colorectal cancer (CRC) progression and the underling mechanisms remain unclear.

Methods: LncRNA expression profile in CRC tissues was investigated by the TCGA database. The expressional level of LINC02418 in CRC patients was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Kaplan-Meier analyses was used to investigate the correlation between LINC02418 and overall survival (OS) of CRC patients. Cell proliferative, migratory and invasive abilities were detected by CCK-8 assays, colony formation assays and trans-well assays in HCT116 and LoVo cells which were stably transduced with sh-LINC02418 or sh-NC. The binding between LINC02418 and miR-34b-5p, and the interaction between miR-34b-5p and BCL2 were determined by dual-luciferase assays. Western blot experiments were conducted to further explore the effect of miR-34b-5p on BCL2 signaling pathway. Rescue experiments were performed to uncover the role of LINC02418/miR-34b-5p/BCL2 axis in CRC progression.

Results: LINC02418 was upregulated in human colon cancer samples when compared with adjacent tissue, and its high expressional level correlated with poor prognosis of CRC patients. LINC02418 promoted cancer progression by enhancing tumor growth, cell mobility and invasiveness of colon cancer cells. Additionally, LINC02418 could physically bind to miR-34b-5p and subsequently affect BCL2 signaling pathway. Down-regulation of LINC02418 reduced cell proliferation, while transfection of miR-34b-5p inhibitor or BCL2 into LINC02418-silenced CRC cells significantly promoted CRC cells growth.

Conclusions: LINC02418 was upregulated in human CRC samples and could be used as the indicator for prediction of prognosis. LINC02418 acted as a tumor driver by negatively regulating cell apoptosis through LINC02418/miR-34b-5p/BCL2 axis in CRC.

Keywords: BCL2; Colorectal cancer; LINC02418; ceRNA; microRNA-34b-5p.

Fig. 1

LINC02418 expression in CRC tissues and cell lines. a The differential expression of LINC02418 in CRC samples (n = 478) and adjacent normal colon tissues (n = 41) was analyzed based on TCGA; **P < 0.01, compared to adjacent tissue group. b Quantification analysis of LINC02418 level was conducted in 20 pairs of CRC samples and adjacent normal colon tissues. c LINC02418 levels in SW620, HCT116, LoVo, Colo205, HT-29, SW480 and normal colon epithelial cell line NCM460 were determined by qRT-PCR; **P < 0.01, compared to NCM460. d Kaplan–Meier analyses of the correlations between expression of LINC02418 and overall survival of patients with CRC in stage 1–2 and stage 3–4 (stage 1, 76 patients; stage 2, 168 patients; stage 3, 127 patients; stage 4, 66 patients). The error bars stand for standard deviation (SD). Data were representatives of three independent experiments with similar results

Fig. 2

Down-regulation of LINC02418 inhibited tumor growth, cell mobility and cell invasion. a HCT116 and LoVo cells were transfected with three shRNAs targeting LINC02418 and control sh-NC. The cells were harvested at 48 h post transfection, and qRT-PCR was conducted to measure LINC02418 level. b, c Cell proliferation of HCT116 and LoVo cells were determined via CCK-8 assays (b) and colony formation assays (c). d, e Tumor volume curve and tumor wet weight of mice subcutaneously injected with HCT116 (d) and LoVo (e) cells which were stably transduced with sh-LINC02418 or sh-NC. f Transwell assay were performed to determine the effect of LINC02418 on the migration and invasion ability of HCT116 and LoVo cells. The results were expressed as the number of invaded cells per field. The error bars stand for standard deviation (SD). Data were representatives of three independent experiments with similar results; **P < 0.01, compared to sh-NC group; ns, no difference, compared to control group

Fig. 3

LINC02418 was a ceRNA for miR-34b-5p in CRC cells. a The putative binding sites of miR-34b-5p to wild type LINC02418 (LINC02418-WT), and the mutant LINC02418 (LINC02418-MUT). b Luciferase activities of HCT116 and LoVo cells co-transfected with miR-34b-5p mimic/miR-NC and luciferase reporters harboring LINC02418-WT or LINC02418-MUT were measured by dual luciferase assays; **P < 0.01; ns, no difference, compared to miR-NC group. c The effect of knockdown of LINC02418 on miR-34b-5p expression in CRC cells were quantified by qRT-PCR; **P < 0.01, compared to sh-NC group. d The differential expression of miR-34b-5p in CRC and normal tissues were analyzed by qRT-PCR. e, f The negative correlation between miR-34b-5p and LINC02418 levels was presented and determined by Pearson’s correlation curve and FISH experiments, respectively. The error bars stand for standard deviation (SD). Data were representatives of three independent experiments with similar results

Fig. 4

BCL2 was the target of miR-34b-5p in CRC cells. a The putative binding sites of miR-34b-5p to wild type BCL2 (BCL2-WT) and the mutant BCL2 (BCL2-MUT). b, c Luciferase activity detection of HCT116 (b) and LoVo (c) cells co-transfected with miR-34b-5p mimic/miR-NC and luciferase reporters expressing BCL2-WT or BCL2-MUT. d Endogenous protein level of BCL2 in HCT116 and LoVo cells transfected with miR-NC, miR-34b-5p inhibitor or miR-34b-5p mimic were detected by western blotting. e The expressional level of BCL2 mRNA in CRC and normal tissues were examined by qRT-PCR. f Pearson’s correlation curve revealed the positive relevance between BCL2 and LINC02418 levels. g, h IHC and WB assays were performed to detect the protein level of BCL2 in CRC and normal tissues. The error bars stand for standard deviation (SD). N, normal tissue; T, tumor tissue. Data were representatives of three independent experiments with similar results; **P < 0.01; ns, no difference, compared to miR-NC group

Fig. 5

LINC02418 indirectly regulated BCL2 expression through sponging miR-34b-5p. a Endogenous protein level of BCL2, caspase 9, caspase 3 and cleaved-caspase 9, cleaved-caspase 3 were detected in HCT116 and LoVo cells by western blotting. b, c Cell growth of HCT116 and LoVo cells were determined by CCK-8 assays and colony formation assays. d Apoptosis rates of HCT116 and LoVo cells were analyzed by flow cytometry assays. Each independent experiment contained 3 repeated samples per group. The error bars stand for standard deviation (SD). Data were representatives of three independent experiments with similar results; **P < 0.01, compared to sh-NC group; ^{^^}P < 0.01, compared to sh-LINC02418 group