A total of 36 CRCs and the corresponding adjacent (2 cm away from the tumor border) surgical specimens in the present study were collected between November 2018 and May 2019, with complete clinicopathological data collected at The Second Hospital of Shandong University (Jinan, China). In the present study, patients had only received surgical treatment, and patients who had received chemotherapy and/or targeted therapy were excluded. The patients ranged in age between 35 and 65 years (mean age, 52 years), with 20 males and 16 females. All studies were approved by the Ethics Committee of The Second Hospital of Shandong University, and informed written consent was obtained from all patients.

The siRNA sequence targeting SCARNA9L was: 5′-CGGUCUACCUGAUGCAUGAUCUCUA-3′. The quantitative PCR primer sequences were as follows: SCARNA9L forward, 5′-ATAAAGGTAGCAGTTGTAGGAATG-3′; SCARNA9L reverse, 5′-CTTCATAGTTACAAAGGTCAGTCG-3′; GAPDH forward, 5′-CGACCACTTTGTCAAGCTCA-3′; and GAPDH reverse, 5′-GGTTGAGCACAGGGTACTTTATT-3′.

CRC gene expression microarrays were searched using the search term ‘Colorectal cancer’ in the gene expression omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). GSE73360 and GSE84984 datasets were used to screen the differentially expressed lncRNAs in CRC (23–27). The process included: Comparison analysis by limma R package (28) (version:3.36.5; http://bioinf.wehi.edu.au/limma); Gene Ontology (GO; http://geneontology.org/); analysis through statistical method, by two side Fisher's exact test (29); pathway analysis through two side Fisher's exact test (30) (https://www.genome.jp/kegg/); coexpression network analysis using R function (Cytoscape; version, 3.6.0; http://cytoscape.org/) (31); and global signal transduction network (32) (version, 3.6.0; https://cytoscape.org/; http://www.genome.jp/kegg/).

Scatter diagrams were plotted. Additionally, the limma package v3.36.5 (http://master.bioconductor.org/packages/release/bioc/html/limma.html) was used to screen differentially expressed genes (DEGs), with P<0.05 and log fold-change (FC) >2 set as the threshold. The heatmap of differentially expressed lncRNAs was drawn using the Heatmap Package v3 (https://CRAN.R-project.org/package=heatmap3). Pathway enrichment analysis and coexpression analysis were performed, according to the GSE73360 and GSE84984 datasets, with a false discovery rate (FDR) <0.05 considered significant. Data on the lncRNA expression were obtained from the Oncomine database (www.oncomine.org). The expression data are available from the GEO database (www.ncbi.nlm.nih.gov/geo) (23–27).

SW480 and SW620 human CRC CRC cells were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. SW480 and SW620 cells were maintained in DMEM culture medium (Gibco; Thermo Fisher Scientific, Inc.), and supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO₂ incubator at 37°C.

Both SW480 and SW620 CRC CRC cells were transfected with control (scrambled siRNA) or the aforementioned siRNA targeting SCARNA9L (50 nM; Guangzhou RiboBio Co., Ltd.) using Lipofectamine™ 3000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The in vitro assays were performed 48 h after transfection.

TRIzol reagent (cat. no. 9108; Takara Bio, Inc.) was used to extract total RNA from both SW480 and SW620 cells. Total RNA was subsequently reverse transcribed at 37°C for 1 h using M-MLV reverse transcriptase (cat. no. M1701; Promega Corporation) and using a cDNA synthesis system (cat. no. RR037A; Takara Bio, Inc.), including oligo DT, Random 6-mers (100 µM), primers, 5X PrimeScript buffer, PrimeScript RT Enzyme Mix I and DEPC water. qPCR was performed using SYBR Premix Ex Taq™ (cat. no. RR820A; Takara Bio, Inc.) with the following thermocycling conditions: 94°C for 3 min, followed by 35 cycles of 94°C for 40 sec, 55°C for 1 min and 72°C for 1 min, and finally 72°C for 10 min. The relative expression level of the indicated RNAs was normalized to the expression of GAPDH using the 2^−ΔΔCq method (33).

Approximately 1,000 NC-transfected cells or human CRC cells treated with the indicated siRNA treatment were seeded into 6-well culture plates and incubated with 5% CO₂ at 37°C for 7 days. The medium was replaced with fresh complete DMEM (with 10% FBS) every two days. After 7 days, the cells were washed with PBS twice, fixed with paraformaldehyde for 30 min at room temperature, and stained with 1% crystal violet at room temperature for 15 min, washed with PBS and observed using the Olympus CX31 light microscope (magnification, ×10). Subsequently, the number of colonies was manually counted.

The cell cycle distribution was detected by using a cell cycle kit (Beijing 4A Biotech Co., Ltd.). In brief, NC-transfected or indicated siRNA-treated CRC cells were fixed with 95% ethanol at −20°C for 24 h and incubated with 0.4 ml of propidium iodide (200 µg/ml) for 30 min at room temperature prior to analysis by flow cytometry.

Both SW480 and SW620 CRC CRC cells were transfected with siRNAs and grown to confluent monolayers. Subsequently, a scratch was generated using a 10-µl pipette tip. Cell debris was washed twice using PBS, and serum-free medium was added to induce wound healing. Images of the wounds were captured at 0 and 24 h using the Olympus CX31 light microscope (magnification, ×10), and the percentage of wound closure was measured. The width of the wound was indicated using lines at the edge of the wound, and the closure percentage was measured as the healing width divided by the original wound width.

SW480 and SW620 cells were transfected with siRNAs for 48 h and then trypsinized and resuspended in serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.). A total of 1×10⁵ cells in 100 µl DMEM without FBS were then added to the upper chambers of the Transwell inserts (Millicell; Merck KGaA) and allowed to migrate toward the bottom of the chambers, which contained DMEM with 10% FBS. After 24 h, the remaining cells in the upper chamber were removed, and cells on the underside were fixed in 4% paraformaldehyde for 30 min at room temperature, stained with 0.1% crystal violet for 30 min at room temperature and captured using an Olympus type light microscope sz30. Quantification of the migrated cells was performed by counting cell numbers.

GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for statistical analysis. All data in this study are presented as the mean ± standard deviation (SD). Statistical significance was calculated using a one-way analysis of variance following by the Fisher's Least Significant Difference post hoc test. Paired t-test was used to compare the expression of lncRNAs in tumor tissues and normal tissues. Additionally, the expression of SLMO2-ATP5E and LOC100132062, and the effects of siRNAs on CRC cell proliferation and migration were analyzed using unpaired t-tests. P<0.05 was used to indicate a statistically significant difference.

Microarray analysis was used to screen CRC-associated DEGs and predict the lncRNAs involved in CRC progression. Analysis of the CRC gene expression datasets GSE73360 and GSE84984 was performed. The microarray data of all lncRNAs in CRC tissues were extracted from the GEO dataset. R language was used to screen for differentially expressed lncRNAs from the CRC gene expression datasets, and the scatter diagrams of all lncRNAs from the GSE73360 and GSE84984 datasets are shown in Fig. 1A. In total, 2233 differentially expressed lncRNAs were screened from GSE73360, and 6749 DEGs were screened from GSE84984, based on P<0.05 and FC >2.

Furthermore, the heatmaps of differentially expressed lncRNAs that were screened from the GSE73360 and GSE84984 datasets are shown in Fig. 1B and C, respectively. According to the results of the heatmaps, SCARNA9L, SLMO2-ATP5E, and LOC100132062 were highly expressed in CRC, in both the GSE73360 and GSE84984 datasets. To the best of our knowledge, there are no studies on the effects of these lncRNAs on CRC; therefore, the differential expression of SCARNA9L, SLMO2-ATP5E, and LOC100132062 and their possible regulatory mechanisms in CRC progression were investigated in the present study.

Interestingly, according to the KEGG pathway analysis of GSE73360 and GSE84984, several pathways were notably affected (including those upregulated and downregulated) in CRC tissues (Fig. 2A and B). Several genes in the indicated pathways, such as tyrosine metabolism and fatty acid degradation pathways, were downregulated, suggesting effects on cell metabolism and CRC progression (Fig. 2A). Additionally, in the analysis of enrichment pathways for upregulated genes in the GSE73360 and GSE84984 datasets, the cell cycle, adherent junctions, and mismatch repair pathways, which have the potential to direct affect CRC progression, were notably upregulated (Fig. 2B).

Subsequently, further coexpression network analysis on GSE73360 and GSE84984 datasets were performed to identify the key lncRNAs involved in the regulation of CRC progression (Fig. 3). Interestingly, the data revealed that multiple core lncRNAs affected the expression of key proteins involved in the regulation of cancer progression, such as lnc-MFAP4-2, lnc-TFAP-2A-4, and lnc-KRTAP9-1 (Fig. 3), and the precise mechanisms need further study. Furthermore, three obviously upregulated lncRNAs, SCARNA9L, SLMO2-ATP5E, and LOC100132062, were identified from the coexpression network analysis (Fig. 3).

Taken together, three lncRNAs, SCARNA9L, SLMO2- ATP5E, and LOC100132062, were notably upregulated and have the potential to be involved in CRC progression.

The potential involvement of the three identified lncRNAs, SCARNA9L, SLMO2-ATP5E and LOC100132062, in the progression of CRC was further explored.

siRNAs targeting the indicated lncRNAs were designed and transfected into two human CRC cell lines, SW480 and SW620 cells, to inhibit its expression. Cell proliferation is critical in cancer progression. Therefore, colony-formation assays were performed to investigate whether these three lncRNAs affected the proliferation of CRC cells. As was confirmed by RT-qPCR assays, the expression levels of SLMO2-ATP5E and LOC100132062 in human CRC tissues were notably higher compared with normal tissues (Fig. S1). The transfection of siRNAs targeting SLMO2-ATP5E and LOC100132062 decreased the expression of these lncRNAs, as confirmed by RT-qPCR assays (Fig. S2). However, the depletion of SLMO2-ATP5E and LOC100132062 had no obvious effects on CRC cell proliferation (Fig. S3), as shown by the colony-forming assays.

Subsequently, the effects of SCARNA9L on CRC progression were studied. Firstly, high mRNA expression levels of SCARNA9L were found in 36 human CRC tissues collected at The Second Hospital of Shandong University (Fig. 4A). Also, through RT-qPCR assays, it was found that the transfection of siRNAs targeting SCARNA9L effectively decreased the expression levels of the indicated lncRNAs in both SW480 and SW620 cells compared with the control group or the NC group (Fig. 4B). Interestingly, the depletion of SCARNA9L dramatically suppressed the proliferation of SW480 and SW620 cells, with a notable decrease in the number of colonies (Fig. 5A and B). The aforementioned results suggest that SCARNA9L could contribute to the proliferation of CRC CRC cells.

The control of cell cycle is essential to maintain the proliferation of cells, and disruption of the cell cycle will result in abnormal proliferation and further promote tumorigenesis. Therefore, differences in the cell cycle distribution between cells with SCARNA9L knockdown and the control groups were detected. Notably, the data indicated that the ablation of SCARNA9L dramatically resulted in an increased percentage of cells at the G₁ phase and a decreased proportion of cells at the G₂/M phases (Fig. 6); suggesting the arrest of cell cycle at G₁ phase. Thus, the ablation of SCARNA9L led to a significant cell cycle arrest and may further block CRC CRC cell proliferation. However, the depletion of SLMO2-ATP5E and LOC100132062 had no obvious effects on cell cycle (data not shown).

Wound-healing and transwell assays were performed to evaluate the effects of SCARNA9L on the migration and invasion of SW480 and SW620 cells. Interestingly, the depletion of SCARNA9L significantly suppressed the extent of wound closure in both SW480 and SW620 cells (Fig. 7). Additionally, according to the transwell assays, depletion of SCARNA9L significantly inhibited the migration of SW480 and SW620 cells through membranes (Fig. 8). On the other hand, SLMO2-ATP5E and LOC100132062 had no effects on CRC cell migration (data not shown).

Overall, the lncRNA SCARNA9L contributes to cell migration and invasion of CRC in vitro.