Profiling of long non-coding RNAs identifies LINC00958 and LINC01296 as candidate oncogenes in bladder cancer

Abstract

Aberrant expression of long non-coding RNAs (lncRNAs) has been regarded as a critical component in bladder cancer (BC) and lncRNAs have been associated with BC development and progression although their overall expression and functional significance is still unclear. The aim of our study was to identify novel lncRNAs with a functional role in BC carcinogenesis. RNA-sequencing was used to identify aberrantly expressed lncRNAs in 8 normal and 72 BC samples. We identified 89 lncRNAs that were significantly dys-regulated in BC. Five lncRNAs; LINC00958, LINC01296, LINC00355, LNC-CMC1-1 and LNC-ALX1-2 were selected for further analyses. Silencing of LINC00958 or LINC01296 in vitro reduced both cell viability and migration. Knock-down of LINC00958 also affected invasion and resistance to anoikis. These cellular effects could be linked to direct/indirect regulation of protein coding mRNAs involved in cell death/survival, proliferation and cellular movement. Finally, we showed that LINC00958 binds proteins involved in regulation and initiation of translation and in post-transcriptional modification of RNA, including Metadherin, which has previously been associated with BC. Our analyses identified novel lncRNAs in BC that likely act as oncogenic drivers contributing to an aggressive cancerous phenotype likely through interaction with proteins involved in initiation of translation and/or post-transcriptional modification of RNA.

Figure 1

Non-protein coding RNA expression in bladder cancer. (a) Overall biotype distribution of non-protein coding transcript (i.e. lincRNAs, antisense RNAs, miRNAs, snoRNAs, snRNAs and miscRNAs) identified using Cufflinks and Cuffmerge and annotated according to Gencode v19. (b) Biotype distribution of the significantly differentially expressed non-protein coding genes using CuffDiff analysis. (c) Heat map of 89 significantly differentially expressed lincRNAs between normal urothelium and BC. Rows represent individual lincRNAs, columns represent individual tissue samples (sorted according to tumor stage as indicated). Yellow indicates up-regulation and blue indicates down-regulation. Genes are mean centered (black color) and normalized. LincRNAs selected for functional characterization are highlighted in red. *LncRNAs up/down regulated in the TCGA dataset (FC_log2 > 0.15 or <−0.15, p < 0.01). (d) The expression of the five selected lincRNAs in normal (n = 8) urothelium compared to BCs (n = 72). Significance was tested using Student’s t-test. FPKM: Fragment Per Kilobase of exon per Million mapped reads.

Figure 2

Subcellular distribution and knock-down efficiency of LINC00958 and LINC01296 in FL3 cells. (a) Percentage of total RNA found in the nuclear and cytoplasmic fraction in FL3 cells determined by RT-qPCR. Left panel: lincRNA-candidates. Right panel: nuclear (MALAT1) and cytoplasmic (GAPDH) markers for fraction purity. (b and c) FL3 cells were transfected with the indicated LNA longRNA GapmeR (50 nM) followed by total (b) or fractionated (c) RNA extraction 48 hours post-transfection. Knock-down-efficiency of lncRNA-candidates was analyzed by RT-qPCR. Relative total RNA expression levels were normalized to GAPDH and compared to Scr. Relative subcellular RNA expression levels were normalized to Scr. Columns represent the average of two independent experiment measured in triplicates. Bars: standard deviation (SD), ns: not significant and *p < 0.05 and **p < 0.01.

Figure 3

Functional effects of LINC00958 or LINC01296 knock-down in FL3 cells. FL3 cells were transfected either with a non-targeting LNA^TMGapmeR (Scr) or 2 different LNA^TMGapmeR separately targeting LINC00958 or LINC01296 (LNA-1 and LNA-2) at the indicated concentrations (10, 25 or 50 nM). (a) Dose-dependent knock-down efficiency was verified by RT-qPCR 48 hours post-transfection. Results were normalized to Scr. (b) Dose-dependent viability was assessed by MTT reduction 24, 48 and 72 h post-transfection and expressed as percentage compared to viability of untransfected cells. (c) Dose-dependent cell death was determined by the LDH release assay 24, 48 and 72 h post-transfection and depicted as percentage of released LDH out of total cellular LDH. (d) Cell morphology was examined upon candidate knock-down by phase contrast microscopy 72 h post-transfection (25x magnification, scale bar 40 μm). Cellular migration (e) and invasion (f) of transfected FL3 cells (25 nM LNA^TMGapmeR). (g) Anoikis resistance upon candidate knock-down (25 nM LNA^TMGapmeR). Cell viability was assessed by both automatic and manual cell counting. Results were normalized to Scr. FCS: Fetal calf Serum. Columns in (b,c and g) represents the average of two or three independent experiments, each measured in three to five replicates. Bars: SD and *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 4

Ingenuity Pathway Analysis. The most significantly biological functions and diseases associated with altered intracellular levels of LINC00958 or LINC01296 in FL3 cells 24 h post transfection are shown. Only RNAs demonstrating log2 ratios <−0.6 or >0.6, when comparing cells transfected with LNA^TMGapmeR (Scr) to cells transfected with LNA^TMGapmeRs targeting either LINC00958 or LINC01296 (LNA-1 and LNA-2), were included in the analysis (LINC00958: n = 1933 genes and LINC01296: n = 915 genes). Threshold value; p = 0.05. The analyses were based on knowledge from all species, all tissues, and all data sources.

Figure 5

In vitro pull-down and mass spectrometry analysis identified LINC00958 interacting proteins. (a) Ingenuity Pathway Analysis. The most significantly pathways and biological functions associated with proteins binding biotin-tagged LINC00958. Only proteins demonstrating FCs ≥ 3, when comparing biotin-tagged LINC00958 to controls were included in the analysis (n = 330). Threshold value; p = 0.05. The analyses were based on knowledge from all species, all tissues, and all data sources. (b) Anti-MTDH RIP using FL3 cell lysates. Detection of endogenous LINC00958 was performed using RT-qPCR (upper panel). The RT-qPCR was performed in triplicates. The experiment was repeated twice and the result of one representative experiment ±SD is shown. IP with a c-Myc antibody was included as negative control. Western blotting analysis (lower panel) was used to evaluate IP of MTDH in the analyzed samples. Loading control input samples: β-actin. FT: flow-through and IP: immunoprecipitation.