Clinical samples
A total of 33 pairs of CRC tissues and peritumoral tissues were obtained from patients who underwent surgical resection in the Affiliated Cancer Hospital of Nanjing Medical University (Nanjing, China). None of these patients underwent radiotherapy, preoperative chemotherapy or other tumor-specific therapies. All fresh tissues were stored in −80°C until use.
Arraystar human SE-LncRNA microarray
Arraystar human SE-LncRNA microarray is global profiling of SE-lncRNAs and protein-coding mRNA, which about 7753 SE-lncRNAs and 7040 coding mRNA. The microarray analysis was performed by Kangcheng Biology Engineering (Shanghai, China) following the arraystar standard protocol. Briefly, four CRC tissues and peritumoral tissues were selected to profiled the expression of SE-lncRNAs. The dysregulation of SE-lncRNAs were identified and analyzed according to the criteria of fold change >2 and P-value < 0.05. The raw data have been uploaded to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). The GEO accession numbers are GSE15102.
Cell culture
The CRC cell lines HCT-116 (RRID: CVCL_0291), SW480 (RRID: CVCL_0546), SW620 (RRID: CVCL_0547), HCT-8 (CVCL_2515), HT-29 (RRID: CVCL_0320), LoVo (RRID: CVCL_0399), HCT-15 (RRID: CVCL_0292) and normal human colon epithelial cell line (FHC, RRID: CVCL_3688 ) were purchased from the American Type Culture Collection (ATCC). These cells were cultured in 90% Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Invitrogen, USA) at 37 °C incubator containing 5% CO2.
RNA extraction and reverse transcription
Total RNA was extracted from tissues and cells with the TRIzol reagent (Qiagen, USA). The RNA quantity and quality were assessed by NanoDrop ND-2000 spectrophotometer (Thermo, USA). The integrity of RNA was confirmed by 1% agarose gel electrophoresis. And then RNA was reversely transcribed as cDNA using the Prime-Script RT reagent with gDNA Eraser (TaKaRa, Japan) according to the manufacturer’s instructions.
Fluorescence quantitative real-time PCR analysis
The fluorescence quantitative real-time PCR (qRT-PCR) analysis was performed using the SYBR Green Master Mix kit (TaKaRa, Japan) on a life Quantstudio 6 Flex system (Applied Biosystems, USA)with the following conditions: 95°C for 10 min and 40 cycles of 95°C 15 s, 60°C 60s. The relative mRNA expression was calculated by 2 -ΔΔCT method, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control of CT value. The primer sequences were showed in Supplementary Table S1.
Protein-coding potential
The protein-coding potential of AC005592.2 isoform (ENST00000510311.1,ENSG000002231185.2) was assessed by using Coding Potential Assessment Tool (CPAT, http://lilab.research.bcm.edu/cpat/), Coding Potential Calculator (CPC, http://cpc.cbi.pku.edu.cn/)[13] and PhyloCSF[14, 15]. Here, the UCSC genome browser may serve as an alternative to viewing PhyloCSF scores for AC005592.2 by copying the URL.
Subcellular localization analysis
The separation of nucleus and cytoplasm fractions was performed with the PARISTM kit (Invitrogen, USA) according to the manufacturer’s instructions. Then mRNA expression of AC005592.2 in the nucleus and cytoplasm was testified by qRT-PCR. CT values of AC005592.2 were compared to GAPDH in the nucleus, and were compared to U6 in cytoplasm.
siRNA transfection
The three siRNAs targeting AC005592.2 (siRNA-78 sense strand: GGAAGCUAGUAGAAGAUUUTT and anti-sense strand: AAAUCUUCUACUAGCUUCCTT; siRNA-273 sense strand: GAAUGGCACUUUGGACAAUTT and anti-sense strand: AUUGUCCAAAGUGCCAUUCTT; siRNA-402 sense strand: GGAGUAGGCUGACCAGUUATT and anti-sense strand: UAACUGGUCAGCCUACUCCTT) and scrambled negative control siRNA (siRNA-NC, siRNA-NC sense strand: UUCUCCGAACGUGUCACGUTT and anti-sense strand: ACGUGAGCACUUCGGAGAATT) were synthesized and purchased from GenePharma (Shanghai, China). The Lipofectamine RNAi MAX kit (Invitrogen, USA) was used to transfected siRNA into CRC cells according to the manufacturer’s instructions. Two of the three siRNA sequences were selected for further studies based on the knockdown efficiency, as confirmed by qRT-PCR.
Construction and infection of vectors for AC005592.2 over-expressing lentivirus
The vectors for AC005592.2 over-expressing lentivirus and negative control were designated and constructed as LV5-AC005592.2 and LV5-NC by GenePharma (Shanghai, China). CRC cells were infected with LV5-AC005592.2 and LV5-NC in the presence of 5 μg/mL polybrene. After 24 h, the supernatant was replaced with fresh culture medium, then cultured for 48-72 h. the expression of AC005592.2 infected cells was validated by qRT-PCR.
CCK8 assay
Cell proliferation was examined with the CCK-8 detection kit (Dojindo, Japan) according to the manufacturer protocol. Briefly, the CRC cells with different treatments were replanted in 96-well plates at a density of 5×103 cells/well, and then incubated with 10 µl CCK8 solution for 37°C 2 h. The proliferation index was measured every 24 h to 96 h at 450 nm absorbance.
Transwell assay
Cell migration and invasion assays were performed using Falcon Cell Culture Insert (BD Biosciences, USA), the 8.0 µm pore polycarbonate membranes of invasion assays coated with Matrigel (BD Biosciences, USA). Briefly, approximately 4×104 cells with different treatments were seeded into the upper chamber with 0.2 mL serum-free DMEM, and the lower chamber was added 0.6 mL DMEM containing 10% FBS as a chemoattractant. After further incubation for 24–48 h, CRC cells that penetrate to the other side of membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of cells was counted under a light microscope as cell migration or invasion ability
Cell apoptosis analysis
Cell apoptosis was performed using the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Multi Sciences, China) according to the manufacturer’s instructions. Briefly, CRC Cells were harvested and washed in cold phosphate-buffered saline (PBS). The washed cells were centrifuged and resuspended using 1X annexin-binding buffer to obtain a cell density of 1 × 106 cells/ml. Then add Alexa Fluor 488 annexin V and 100 µg/mL PI working solution to the cell suspension. After incubation at room temperature for 15 minutes, add 1X annexin-binding buffer. Finally, the stained cells were analyzed by flow cytometry.
Western blot analysis
The CRC cells were collected in the radio-immunoprecipitation (RIPA) lysis buffer (Biovision, USA) to extract cellular protein, and the protein concentration was detected with BCA protein assay kit (Thermo, USA). Total lysis was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked in 5% nonfat dry milk for 1 h at room temperature, and incubated with 1:1000 Human Olfactomedin-4 (OLFM4) antibody (Affinity Biosciences, USA, RRID: AB_2846459) or 1:5000 GAPDH antibody (Abcam, USA, RRID: AB_2049706) at 4°C overnight. After being incubated with Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (Abcam, USA, RRID: AB_955417) for 1 h at room temperature, the protein bands were detected by ECL method (Millipore, USA)
RNA sequencing array and bioinformatics analysis
Three siRNA ACC00552.2 transfected HT-29 cells and three negative control were selected for RNA-Sequencings (RNA-seq) to find downstream target genes of AC005592.2. Whole RNA-seq was performed by Guangzhou RiboBio (Guangzhou, China) using Illumi-naHiSeq 3000 platform. All the differentially expressed genes (fold change > 2, P-value < 0.05) were used for hierarchical clustering, volcano plots, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses. P-value < 0.05 was considered as the threshold to define significant enrichment of the genes GO and KEGG enrichment analysis.
Statistical analysis
Statistical analyses were performed by GraphPad Prism v6.0 (GraphPad Software, Inc. La Jolla, CA, USA) and SPSS Statistics Version 20.0 (SPSS, Chicago, IL, USA). The unpaired 2-tailed student's t-test was used to evaluate significant significance between two groups. Statistical differences for more than two groups were determined by two-way ANOVA and Multiple t-test. All data were represented as the mean ± SD for triplicate independent measurements. The value of P < 0.05 was considered to be a statistical difference.