LncRNA SLCO4A1-AS1 facilitates growth and metastasis of colorectal cancer through β-catenin-dependent Wnt pathway

Human colorectal samples were collected from the First Affiliated Hospital of Harbin Medical University. And the clinicopathological features were listed in Table 1. These samples were divided into SLCO4A1-AS1 high or low group (the median value of SLCO4A1-AS1 expression as the cutoff), followed by survival rate analysis. Informed consent allowing use of these samples was obtained from each patient. Samples were processed according to the standard procedures with appropriate ethical approval by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University.

Human normal colorectal mucosa cell FHC and CRC cell lines (HCT116, HCT8, HT29, SW480, LOVO and SW620) were purchased from the American Type Culture Collection (ATCRC) and maintained according to the standard procedures.

shRNAs (shSLCO4A1-AS1: 5’-GCCTGAGCTTGTTCACAAA-3′) were designed using Clontech RNAi Target Sequence Selector and constructed into the pSiCoR plasmid according to the instructions. Two hundred ninety-three T cells transfected with pSiCoR as well as VSVG, RRE and RSV-REV were used to generate virus. HCT116 and SW480 cells were infected with virus supernatants. Stable cell lines were isolated by GFP sorting.

Antibodies for Vimentin (1:2000; #5741, Cell Signaling Technology), E-cadherin (1:1000; #14472, Cell Signaling Technology), AXIN2 (1:2000; #5863, Cell Signaling Technology), MYC (1:5000; #13987, Cell Signaling Technology), β-catenin (1:1000; #8480, Cell Signaling Technology), phospho-β-catenin (T41, S45) (1:1000; #9565, Cell Signaling Technology), GSK3β (1:1000; #12456, Cell Signaling Technology) and GAPDH (1:5000; #5014, Cell Signaling Technology) were bought from Cell Signaling Technology. α-catenin (1:2000; #610194, BD) was from BD Transduction Laboratories. Fibronectin (1:1000; SAB4500974, Sigma) and LGR5 (1:1000; SAB2700211, Sigma) were from Sigma.

Cell apoptosis were analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (BD Biosciences).

This assay was carried out as described before [24]. In brief, HA-ubiquitin vector were transfected into CRC cells. Forty-eight h after transfection, cells were treated with MG132 (10 μM/L) for 10 h. Then cell lysates were pulled down using anti-β-catenin or IgG. Eluates were separated by SDS-PAGE and immunoblotted with anti-K48-Ub.

Animal experiments were s approved by the Medical Experimental Animal Care Commission at the First Affiliated Hospital of Harbin Medical University. To assess the effect in vivo of SLCO4A1-AS1 on tumorigenesis, shSLCO4A1-AS1 or control SW480 cells (2×10⁶ cells per mouse) were injected into the flanks of 5-week-old athymic nude BALB/c mice were manipulated nude recipients subcutaneously. Tumor volumes weights were determined at indicative time points. To evaluate the effects of SLCO4A1-AS1 on tumor metastasis in vivo, shSLCO4A1-AS1 or control SW480 cells (4×10⁶ cells per mouse) were injected into the spleen subcapsular of each BALB/c nude mice. Six weeks after injection, metastatic nodules in livers were counted under a dissecting microscope.

CCK-8 and colony formation assays were used for evaluation of cell proliferation ability. For CCK-8 assay, cells were seeded into a 96-well plate. At indicative time points, 10 μl CCK-8 solution was added into each well and incubated for 2 h at 37 °C. Then, the absorbance at 450 nm was determined. For the colony formation assay, 2×10³ cells per well were seeded in a six-well plate and cultured for 2 weeks. Colonies were fixed with methanol and stained with 0.1% crystal violet (1 mg/ml).

For migration assay, 2×10⁴ cells were seeded in the top chamber (8-μm pore; BD Biosciences) with serum-free medium and the lower chamber was added with 10% fetal bovine serum medium. Cells were incubated for 24 h and then non-migrated cells were removed and the migrated cells on the lower side were fixed, stained with crystal violet and photographed with an IX71 inverted microscope (Olympus Corp., Tokyo, Japan). For invasion assay, 8×10⁴ cells were placed in the upper chamber coated with 100 μl Matrigel (BD Biosciences, MA). And other steps were the same as migration assay.

TRIzol solution was used to extract total RNAs from sample tissues or cell lines according to the manufacturer’s protocol. The M-MLV reverse transcriptase (Promega) was used for cDNA synthesis. qRT-PCR was performed as previously described [25]. Gene expression was normalized to U6 or GAPDH and calculated according to the 2^−ΔΔCT method. Specific primer sequences were available if requested.

ISH were conducted as previously described [26]. The probe sequences for SLCO4A1-AS1 were as follows: 5′-GAAGCTAGATGCRCAGCTAAT-3′ and 5′-TTGCGTTCATCGGAACRCAGG-3′.

Cells were lysed with RIP buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.1% NP40, 5% glycerol and 0.5 mM DTT) supplemented with RNase inhibitor, followed by addition of specific antibody. RNA–protein complexes were enriched by Protein A/G beads. Then precipitated RNAs were eluted and used for cDNA synthesis.

Biotin-labeled RNAs were obtained by the MaxiScript T7 kit (Ambion) with biotinylated CTP. Biotin-labeled RNAs in refolding buffer (10 mM Tris pH 7.5, 0.1 M KCl and10 mM MgCl2) were added into cell lysates and incubated for 4 h at 4 °C, followed by addition of beads. After washed 4 times, beads were boiled and precipitated proteins were checked with western blot.

SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Data was expressed as mean ± SD. Survival curves were calculated using the Kaplan-Meier curve followed by log-rank test. One-way ANOVA analysis or two-tailed Student’s t-tests were performed for p-value analysis, as appropriate. P < 0.05 was considered statistically significant.

To identify CRC-related lncRNAs, we analyzed an online non-coding RNA profiling according to Li’s cohort (GSE104836) consisting of 10 pairs of tumor and adjacent normal tissues. Among all differentially expressed lncRNAs between tumor and normal tissues, SLCO4A1-AS1 was the most upregulated in tumor samples (Fig. 1a). Previous study showed that copy number amplification (CNA) is linked to upregulation of oncogene expression and subsequent tumorigenesis [27]. According to TCGA database, we found that the frequency of CNA of SLCO4A1-AS1 was very high in various cancers, including about 9% in CRC (Fig. 1b). To validate it, we collected 50 CRC samples and detected the copy number of SLCO4A1-AS1 by qPCR. We found that 8 samples had copy number amplification including 6 3-copy and 2 4-copy samples (Fig. 1c), which implied that SLCO4A1-AS1 may be involved in CRC development. To further validate it, we measured the expression of SLCO4A1-AS1 in CRC samples and adjacent normal tissues. We found that SLCO4A1-AS1 was significantly upregulated in CRC tissues compared with paired normal tissues (Fig. 1d and e). ISH assay also showed that the expression of SLCO4A1-AS1 was higher in CRC samples (Fig. 1f). Consistently, the expression of SLCO4A1-AS1 was higher in CRC cell lines (HCT116, HCT8, HT29, SW480, LOVO and SW620) than that in human normal colorectal mucosa cell FHC (Fig. 1g). To further determine the relationship between SLCO4A1-AS1 expression and tumor malignance, we checked the expression of SLCO4A1-AS1 in different stages of CRC samples and found that SLCO4A1-AS1 expression is positively correlated with clinical grade (Fig. 1h). Furthermore, by Kaplan–Meier survival analysis, we found that CRC patients with higher expression of SLCO4A1-AS1 showed poorer prognosis (Fig. 1i). In addition, receiver operating characteristic (ROC) curve was performed to evaluate the sensitivity and specificity of SLCO4A1-AS1 expression in predicting CRC tissues from normal tissues. The area under curve (AUC) was 0.924, which indicated SLCO4A1-AS1 might be a good predictor in CRC (Fig. 1j). Collectively, above results implied that SLCO4A1-AS1 was significantly upregulated and might serve as a biomarker for prognosis in CRC.

To investigate the role of SLCO4A1-AS1 in CRC, we knocked down SLCO4A1-AS1 with two independent siRNAs in HCT116 and SW480 cells (Fig. 2a). CCK-8 assays showed that SLCO4A1-AS1 knockdown led to growth retardation of HCT116 and SW480 cells (Fig. 2b). Colony formation assay similarly indicated that SLCO4A1-AS1-depleted CRC cells formed fewer colonies than the controls (Fig. 2c). Predictably, SLCO4A1-AS1 significantly inhibited the percent of CRC cells in S phase (Fig. 2d). In addition, Transwell assays showed that SLCO4A1-AS1 silencing decreased CRC cell migration (Fig. 2e) and invasion (Fig. 2f). Besides, we performed western blot to evaluate whether SLCO4A1-AS1 regulates pithelial-mesenchymal transition (EMT) in CRC cells. Results demonstrated that knockdown of SLCO4A1-AS1 upregulated the expression of epithelial markers such as E-cadherin, α-catenin while decreased the expression of mesenchymal markers such as Vimentin and Fibronectin (Fig. 2g). Finally, we assessed the effect of SLCO4A1-AS1 on cell apoptosis. By staining with Annexin V/PI, we found that SLCO4A1-AS1 knockdown dramatically promoted the apoptosis (Fig. 2h). Taken together, our data demonstrated that SLCO4A1-AS1 knockdown could inhibit CRC proliferation and invasion in vitro.

To further determine SLCO4A1-AS1-mediated molecular mechanism, we performed analysis in bioinformatics according to the Li’s cohort (GSE104836). We divided the 10 CRC samples in Li’s cohort into two groups based on SLCO4A1-AS1 expression (median value as the cutoff). Gene Set Enrichment Analysis (GSEA) showed that SLCO4A1-AS1 highly expression was positively related to cell division and G1/G2 phase transition (Fig. 3a), which supported our above results that SLCO4A1-AS1 promoted CRC cell proliferation. Some signaling pathways such as Wnt/β-catenin signal and NOTCH signal were reported to be involved in human cancers [28, 29]. We analyzed the relationship of SLCO4A1-AS1 with cancer-related signaling pathways and found that SLCO4A1-AS1 expression was positively correlated with Wnt/β-catenin signaling pathway (Fig. 3b). As shown, many target genes of Wnt/β-catenin signaling was downregulated in SLCO4A1-AS1^low CRC samples compared with SLCO4A1-AS1^high samples (Fig. 3c). Similarly, SLCO4A1-AS1 knockdown significantly decreased the mRNA and protein levels of these target genes in HCT116 and SW480 cells (Fig. 3d-f). What’s more, the expression levels of AXIN2, LGR5, SOX4 and MYC were positively correlated with that of SLCO4A1-AS1 in the 50 CRC sample tissues (Fig. 3j), which indicated that SLCO4A1-AS1 regulates Wnt/β-catenin signaling in CRC.

We above showed that SLCO4A1-AS1 activated Wnt/β-catenin signaling in CRC. To determine how SLCO4A1-AS1 activates Wnt/β-catenin signaling, we performed RNA pulldown assays, followed by silver staining and mass spectrum (MS) identification. We identified β-catenin as an interactive protein of SLCO4A1-AS1 (Fig. 4a). To verify it, we performed pulldown assay and found that biotin-labeled SLCO4A1-AS1 precipitated endogenous β-catenin in HCT116 and SW480 cells (Fig. 4b). Furthermore, β-catenin antibody also enriched endogenous SLCO4A1-AS1 in HCT116, SW480 and CRC sample cells (Fig. 4c and d). In addition, SLCO4A1-AS1 co-localized with β-catenin in CRC sample cells (Fig. 4e). To further determine the essential region in SLCO4A1-AS1 for the interaction with β-catenin, we conducted domain mapping. We found that SLCO4A1-AS1 (nt 900~ 1200) directly bound to β-catenin (Fig. 4f). Moreover, deletion of this region (nt 900~ 1200) abrogated this interaction between SLCO4A1-AS1 and β-catenin (Fig. 4g). Furthermore, we performed RNA electrophoretic mobility shift assay (RNA-EMSA) with biotin-labeled probe (nt 900~ 1200) and demonstrated their direct association (Fig. 4h).

We have confirmed the interaction between SLCO4A1-AS1 and β-catenin. Then we performed western blot and found that SLCO4A1-AS1 knockdown significantly decreased the protein level of β-catenin in HCT116 and SW480 cells (Fig. 5a). On the contrary, overexpression of full-lengthen or nt 900~ 1200 dramatically upregulated the protein level of β-catenin in HCT116 and SW480 cells (Fig. 5b). Additionally, we validated the elevated β-catenin ubiquitination signals using β-catenin immunoprecipitates from SLCO4A1-AS1–depleted HCT116 cells through (Fig. 5c) and consequently decreased β-catenin stability (Fig. 5d). A previous study showed that β-catenin phosphorylation by GSKβ promotes its ubiquitination-mediated degradation [30]. We then assessed the effect of SLCO4A1-AS1 on β-catenin phosphorylation and found that SLCO4A1-AS1 knockdown significantly increased β-catenin phosphorylation in HCT116 and SW480 cells (Fig. 5e). Moreover, SLCO4A1-AS1-overexpressed CRC sample tissues showed lower β-catenin phosphorylation (Fig. 5f). Besides, we found that SLCO4A1-AS1 knockdown enhanced the interaction between β-catenin and GSKβ in HCT116 and SW480 cells (Fig. 5g) while overexpressing SLCO4A1-AS1 abrogated their interaction (Fig. 5h). To further determine whether SLCO4A1-AS1 activated Wnt/β-catenin signaling by enhancing the stability of β-catenin, we restored the protein levels of β-catenin in HCT116 and SW480 cells (Fig. 5i). By qRT-PCR, we found that restoration of β-catenin rescued the activation of Wnt/β-catenin signaling in HCT116 and SW480 cells (Fig. 5j). Summarily, our results indicated that SLCO4A1-AS1 stabilized β-catenin by preventing the association between β-catenin and GSKβ, and consequently activated Wnt/β-catenin signaling in CRC.

Whether the SLCO4A1-AS1-mediated augment of CRC cell growth and metastasis relied on activation of Wnt/β-catenin signaling was assessed in SLCO4A1-AS1-silenced HCT116 and SW480 cells transfected with β-catenin-overexpressing plasmid or empty control. Results showed that decreased proliferation, colony formation, migration and invasion potentials of SLCO4A1-AS1-silenced cells were rescued by ectopic expression of β-catenin in HCT116 and SW480 cells (Fig. 6a-d). What’s more, SLCO4A1-AS1 knockdown delayed tumor growth in vivo while overexpression of β-catenin in the meantime reversed it (Fig. 6e and f). Then we measured the activation of Wnt/β-catenin signaling in formed tumor tissues. As shown, the Wnt/β-catenin signaling was also downregulated in vivo after SLCO4A1-AS1 depletion (Fig. 6g). Finally, we evaluated the effect of SLCO4A1-AS1 on tumor metastasis in vivo, and found that SLCO4A1-AS1 knockdown severely reduced the metastatic nodules in the liver while β-catenin overexpression reversed this trend (Fig. 6h and i). Taken together, above data suggested that SLCO4A1-AS1 exerted functions dependent on activation of Wnt/β-catenin signaling in CRC.