Background

As one of the most prevalent cancers, colorectal cancer (CRC) has become the third-leading cause of cancer-related death worldwide every year [1]. Hyperactivation of some signaling pathways, including wnt/β-catenin, PI3K/AKT, JAK/STAT signaling pathways and so on, often contributes to the development, progression, metastasis and resistance to chemotherapy of CRC [5,6,7]. Emerging evidence shows that lncRNAs are critical regulators involved in various biological processes via multiple mechanisms [8, 9], such as development, immune regulation and especially tumorigenesis [10,11,12]. Importantly, accumulating studies have proven that aberrant expression of lncRNAs is closely related to various human cancers [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).

Fig. 4
figure 4

SLCO4A1-AS1 interacts with β-catenin. a RNA pulldown using biotin-labeled probe or intron control and sample lysates, followed by SDS-PAGE electrophoresis, silver staining and MS identification. b RNA pulldown showed that SLCO4A1-AS1 interacted with β-catenin in HCT116 and SW480 cells. SLCO4A1-AS1 was labeled with biotin. c RNA IP showed that β-catenin enriched SLCO4A1-AS1 in HCT116 and SW480 cell lysates. d β-catenin enriched SLCO4A1-AS1 in CRC sample cell lysates. e SLCO4A1-AS1 co-localized with β-catenin in CRC sample cells as shown by RNA fluorescence in situ hybridization (RNA-FISH). The scale bar was 10 μm. f, g Domain map** showed that SLCO4A1-AS1 (nt: 900~ 1200) interacted with β-catenin and was indispensible. d900~ 1200 represented deletion of nt 900~ 1200. h RNA electrophoretic mobility shift assay (RNA-EMSA) showed that biotin-labeled SLCO4A1-AS1 (nt: 900~ 1200) directly bond to β-catenin. *p < 0.05 and **p < 0.01

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SLCO4A1-AS1 increased the stability of β-catenin by inhibiting its phosphorylation

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.

Fig. 5
figure 5

SLCO4A1-AS1 increased the stability of β-catenin by inhibiting its phosphorylation. a WB analysis showed that siRNA-induced SLCO4A1-AS1 knockdown decreased the protein level of β-catenin in HCT116 and SW480 cells. H3, nuclear marker; EEA1, cytoplasmic marker. b Overexpression of SLCO4A1-AS1 (full-length or nt 900~ 1200) promoted the protein level of β-catenin in HCT116 and SW480 cells. H3, nuclear marker; EEA1, cytoplasmic marker. c Knockdown of SLCO4A1-AS1 enhanced the ubiquitination of β-catenin in HCT116 cells. d SLCO4A1-AS1 knockdown accelerated the degradation of β-catenin in HCT116 and SW480 cells. Chx, cycloheximide. e SLCO4A1-AS1 knockdown promoted the phosphorylation of β-catenin in HCT116 and SW480 cells as shown by western blotting. f WB analysis showed that the protein levels of β-catenin were higher in SLCO4A1-AS1high CRC samples while the phosphorylation of β-catenin was lower. g SLCO4A1-AS1 knockdown enhanced the interaction between β-catenin and GSK3β in HCT116 and SW480 cells. h WB analysis indicated that overexpression of SLCO4A1-AS1 (full-length or nt 900~ 1200) abrogated the interaction between β-catenin and GSK3β in HCT116 and SW480 cells. i, j Restoration of the protein levels of β-catenin by ectopic expression of β-catenin (i) rescued SLCO4A1-AS1 knockdown-induced inactivation of wnt/β-catenin signaling in HCT116 and SW480 cells (j)

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SLCO4A1-AS1 promotes CRC proliferation, migration and invasion by activating wnt/β-catenin signaling in vitro and in vivo

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.

Fig. 6
figure 6

SLCO4A1-AS1 promotes CRC proliferation, migration and invasion by activating wnt/β-catenin signaling in vitro and in vivo. a, b Ectopic expression of β-catenin rescued the decreased proliferation ability of HCT116 and SW480 cells induced by SLCO4A1-AS1 knockdown as shown by CCK-8 and colony formation assays. c, d Overexpression of β-catenin restored the migration and invasion potential of HCT116 and SW480 cells as indicated by a Transwell and Matrigel assay, respectively. Scale bar, 50 μm. e, f SLCO4A1-AS1 knockdown delayed the tumor propagation in vivo while overexpressing β-catenin rescued it. Tumor volumes were measured at indicative time points. Tumor weights were measured at the end point of experiments. g Total RNAs were extracted from tumor tissues in f and the activation of wnt/β-catenin signaling was assessed by qRT-PCR. Results showed that SLCO4A1-AS1 knockdown downregulated wnt/β-catenin signaling while ectopic expression of β-catenin upregulated it in vivo. h Knockdown of SLCO4A1-AS1 expression remarkably reduced the metastatic nodules in the liver. i The liver sections were shown via H&E staining. Scale bar, 50 μm. *p < 0.05 and **p < 0.01

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Discussion

In recent years, great efforts have been made to search cancer-related lncRNAs and determine their molecular mechanisms in tumor development and progression [31]. Here we identified the physiological functions of an uncharacterized lncRNA SLCO4A1-AS1 and determined its molecular mechanism. SLCO4A1-AS1 was highly expressed in CRC tissues and may act as a biomarker for CRC diagnosis. Notably, we detected an unbelievable high AUC, which might be due to the limited size of CRC samples. SLCO4A1-AS1 was found to promote the proliferation and invasion of CRC cells, indicating that it may be implicated in the process of tumorigenesis. Moreover, SLCO4A1-AS1 knockdown induced CRC cell apoptosis, which implied that SLCO4A1-AS1 may be important for the functional maintenance of normal cancer cells.

Further analysis showed that the activation of Wnt/β-catenin signaling was affected by SLCO4A1-AS1. As one of the most essential intracellular signaling pathways, Wnt/β-catenin signaling mediates diverse cellular processes, including embryonic development, cell proliferation, differentiation, migration, survival and so on [32,33,34]. Hyperactivation of the Wnt/β-catenin signaling often leads to various cancers such as liver cancer and CRC [35,36,37]. For instance, CRCAT-1-mediated activation of Wnt signaling pathway promotes cell proliferation and inhibits apoptosis in cervical cancer cells [38]. Additionally, activation of Wnt/β-catenin signaling by TGFβ promotes CRC development [39]. In our study, we found that SLCO4A1-AS1 knockdown severely decreased the protein level of β-catenin but not mRNA level by the mechanism that SLCO4A1-AS1 inhibited the phosphorylation and consequently ubiquitylation-mediated degradation of β-catenin. Through interacting with β-catenin, SLCO4A1-AS1 impaired the binding of GSK3β to β-catenin and inhibited β-catenin phosphorylation by GSK3β. Emerging evidence shows that lncRNAs can exert functions by regulation in trans [40]. lncRNAs may associate with proteins to regulate their stability, activity or other properties [11, 41, 42]. Based on above evidence, we proposed that SLCO4A1-AS1 may bind to β-catenin and then shield the interactive domain of β-catenin with GSK3β.

β-catenin level plays a pivot role in the canonical Wnt pathway [43]. Increase of β-catenin protein level may lead to abnormal cell proliferation and human diseases [44]. The regulation of β-catenin protein level is complicated and delicate. Phosphorylation and ubiquitylation of β-catenin are all reported to participate in the regulation of β-catenin stability [45]. For example, Liu et al. demonstrated that phosphorylation of β-catenin by CKIα in vivo is indispensible for subsequent phosphorylation of β-catenin by GSK3β, which finally leads to degradation of β-catenin [45]. Besides, other studies showed that phosphorylated β-catenin is ubiquitylated by E3 ubiquitin ligase β-TrCP and then degraded by the ubiquitin–proteasome pathway [46, 47]. Abrogation of β-catenin degradation promotes the accumulation of β-catenin in cells and induces tumor occurrence. For instance, inactivating mutation of APC, a pivot subunit of the degradation complex of β-catenin, gave rise to spontaneous CRC in mice [48]. So far, the regulatory mechanism of β-catenin turnover is not fully understood. Our study revealed that SLCO4A1-AS1 regulated the stability of β-catenin by weakening the association between β-catenin and GSK3β.

Continuous mutations of genes are popularly considered as a cause of tumors [49]. Gene copy number alterations or mutations are the common aberrances in cancers, and some studies have demonstrated the relevance between gene copy-number alterations and tumor formation and progression [50]. Previous study shows that DNA copy-number gain was observed on chromosome 20q in primary colorectal tumor [51]. Notably, SLCO4A1-AS1 is also located on chromosome 20q. Moreover, SLCO4A1-AS1 is really substantially amplified in CRC according to TCGA database and our experiment (Fig. 1b and c). However, how copy-number amplifications on chromosome 20q affect the expression and functions of SLCO4A1-AS1 in CRC remains further investigation.

Conclusion

In summary, we found that lncRNA SLCO4A1-AS1 was highly expressed in CRC tissues. Upregulated SLCO4A1-AS1 promoted CRC progression through inhibiting the degradation of β-catenin by attenuating the interaction between β-catenin and GSK3β. This study revealed the vital significance of SLCO4A1-AS1 in CRC development.