LINC01315 accelerates the growth and epithelial-mesenchymal transition of colorectal cancer cells via activating the Wnt/β-catenin signal

ABSTRACT The pathological roles of long non-coding RNAs (lncRNAs) in colorectal carcinoma (CRC) have been corroborated. To date, the pathological contributions of LINC01315 in the epithelial-mesenchymal transition (EMT) property of CRC are still ambiguous. By silencing LINC01315, we disclosed that LINC01315 promoted the growth, metastatic characteristics, and the EMT of CRC cells in vitro. Mechanistically, LINC01315 activated Wnt/β-catenin signaling. LINC01315 bound to the β-catenin promoter and activated its transcription. In rescue experiments, ectopic overexpression of β-catenin counteracted the inhibiting effector-triggered by LINC01315 deletion. In summary, this preliminary study brings new insights to the pathological significance of the LINC01315/Wnt/β-catenin signaling pathway in CRC.


Introduction
Colorectal carcinoma (CRC), one of the most frequent gastrointestinal malignancies, remains the primary trigger of malignancy-related mortality [1,2]. The major cause of CRC-caused mortality is distant metastasis, and approximately 60% of patients with CRC are expected to develop metastases [3]. The presence of metastasis confers a worse prognosis to the clinical outcome of CRC-affected patients [4]. An extensive body of studies has already focused on oncogenes and tumor suppressor genes that involve metastasis [5][6][7]. It is well-accepted that cancer metastasis is a highly complicated, multistep process and tightly linked with epithelial-to-mesenchymal transition (EMT) [8]. Focusing on the agents involved in the EMT of CRC cells might aid the comprehension of metastatic dissemination [9].
Herein, we reported that inhibition of LINC01315 dramatically retards CRC cells' growth and EMT event. Mechanistically, LINC01315 regulates β-catenin transcription via direct interaction with its promoter. We collectively establish that LINC01315 acts as a pro-oncogenic factor in CRC by activating the canonical Wnt/β-catenin signal.

CRC tissues
31 pairs of paraffin-embedded colorectal cancer tissues and matched paracancerous tissues were purchased from Xian Ailina Biotechnology Co., Ltd (Xian, China). The clinical characteristics are provided in Table 1. Informed consent from all participants was obtained. The study protocol was granted by the Ethical Committee from Guangrao County People's Hospital (No. GR2019-11). The ethical approvement can be found in the Supplementary material.

Migration and invasion assay
HCT116 or SW480 cells (1 × 10 5 ) were seeded in 6-well dishes and cultured overnight to reach 90% confluency, and an artificial was made using a 200 µL tip. After washing with PBS, cells were recorded at 0 h or 48 h. The migration rate (%) = (0 h wound width −48 h wound width)/ 0 h wound width ×100. In the invasion assay, 200 µL of cell suspension (2 × 10 4 ) were plated in the Transwell top compartment (8-μm membrane; BD Biosciences) with an FBS-free DMEM medium. 600 µL of 10% FBS medium was added into the lower compartment. After 24 h, the invading cells across the membrane were fixed and stained with crystal violet. The number of invading cells was counted under an inverted microscope.

Real-time quantitative PCR (qRT-PCR)
The total RNAs were extracted using TRIzol solution (Invitrogen). cDNA synthesis was implemented using a Primescipt RT reagent kit (TaKaRa, Dalian, China). qRT-PCR was performed using an SYBR Premix ExTaq Real-Time PCR Kit (TaKaRa). GAPDH was used as the internal reference, and data were calculated using the 2 −ΔΔCT method.

RNA immunoprecipitation (RIP)
RIP assay was carried out using a Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore). Cell lysates were incubated with β-catenin of IgG antibody. Co-precipitated RNAs were enriched by Protein A/G beads. Finally, the coprecipitated RNAs were subjected to qRT-PCR assay.

RNA pull-down
LINC01315 sequence was labeled in Biotin, and RNA pull-down was performed using a Pierce Magnetic RNA-Protein Pull-Down Kit (ThermoFisher, MA, USA). Cell lysates were incubated with the biotin-labeled LINC01315 for 4 h at 4°C and treated with magnetic beads for 30 min. After being washed three times, the beads were boiled and precipitated proteins were detected using immunoblotting.

Statistical analysis
Statistical analysis was done using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). Data are shown as Mean ± SD. Kaplan-Meier curve followed by the log-rank test was applied to evaluate the prognosis of patients. Oneway ANOVA tests or two-tailed Student's t-test was used for statistical analysis, where indicated. P value<0.05 is considered significant.

Results
In this study, we focused on the pro-cancer characteristics of LINC01315 in CRC. LINC01315 promotes the growth, metastatic properties, and the EMT of CRC cells in vitro. Mechanistically, LINC01315 binds to the β-catenin promoter and activates the Wnt/β-catenin signaling.

LINC01315 is overexpressed in CRC
First, we assessed the aberrant lncRNAs expressions using the GEO database (GSE39582 and GSE41328) based on the filter conditions of P < 0.05 and |fold change| > 1 (Figure 1(a)) [25,26]. Venn diagram showing the intersection of the differential lncRNAs in the two GEO datasets (Figure 1(b)). The pathological roles of LINC00152, LINC00675, and LINC01207 in CRC have been delineated previously. Nonetheless, the pro-oncogenic action of LINC01315, especially in CRC EMT, has not been fully addressed [27][28][29][30]. In the public The Cancer Genome Atlas Program (TCGA) dataset, in contrast to normal, high LINC01315 was found in CRC (Figure 1(c)). We evaluated the expressions of LINC01315 in 31 pairs of CRC specimens and paracancerous tissues. LINC01315 was profoundly elevated in CRC compared with paracancerous tissues (Figure 1(d)). Notably, Kaplan-Meier survival analysis revealed that patients with high LINC01315 expression levels had poorer overall survival than patients with low LINC01315 expression levels (Figure 1  (e)). Comparing the normal colorectal mucosa cell FHC, CRC cells (SW480, LOVO, HCT116, and HT29) exhibited higher LINC01315 expression (Figure 1(f)). Finally, LINC01315 is mainly located in the cytoplasm, as revealed by the lncATLAS website (https://lncatlas.crg.eu) (Figure 1(g)). Collectively, the above results implied that LINC01315 might act as a predictor of prognosis for CRC.

Deletion of LINC01315 restrains CRC cell growth and mobility
To figure out the biological role of LINC01315 in CRC progression, shLINC01315 was applied to knock down the endogenous LINC01315 expression in HCT116 and SW480 cells (Figure 2(a)). CCK-8 assays showed that LINC01315 silencing caused dramatic growth inhibition in CRC cells (Figure 2(b)). A similar suppressive action was observed in the clonal formation ability of CRC cells up LINC01315 knockdown (Figure 2(c)). In addition, wound healing and transwell assays implied that attenuation of LINC01315 expression impaired CRC cells' migration ( Figure 2(d)) and invasive abilities (Figure 2(e)). Besides, epithelial marker E-cadherin expression was raised by LINC01315 deficiency, while the protein expression of the mesenchymal marker, N-cadherin, was lessened (Figure 2(f)). All these data validated that attenuation of LINC01315 expression prohibits CRC EMT and invasion.

LINC01315 activates Wnt/β-catenin in CRC
Wnt/β-catenin signal is implicated in cancer metastasis and EMT [31]. The Western blot results indicated that the silencing of LINC01315 substantially reduced the protein expression of β-catenin (Figure 3(a)). To probe whether LINC01315 activated Wnt/β-catenin, the TOP/FOP flash luciferase reporter assay in CRC cells was conducted. The result indicated that the introduction of LINC01315 activated the Wnt/β-catenin pathway (Figure 3(b)). Next, we assayed the nuclear/ cytoplasmic localization of β-catenin in LINC01315 overexpressing CRC cells. As presented in Figure 3(c), force expression of LINC01315 increased β-catenin nucleus ectopic expression. Similarly, the mRNA levels of Wnt/βcatenin target genes, Cyclin D1 and C-myc, were raised in CRC cells upon LINC01315 overexpression (Figure 3(d)). c. Colony formation assays were performed to determine the proliferation of shLINC01315-transfected or shNC-transfected CRC cells. d. Wound-healing assay of LINC01315 knockdown HCT116 and SW480 cells. e. Transwell invasion assay analyzed the effect of LINC01315 knockdown on CRC cells invasion. f. Western blot assay was performed to test the expression of EMT markers (E-cadherin and N-cadherin) in the LINC01315 knockdown cells. **P < 0.05 compared with shNC.

LINC01315 interacts β-catenin in CRC cells
LncRNAs have been reported to regulate genes expression at the transcriptional level, the impact of LINC01315 on β-catenin was further examined. As shown in Figure 4(a), upregulation of LINC01315 resulted in an increased expression of β-catenin at the mRNA level. Subsequently, the luciferase reporter assay proved the activating effect of LINC01315 in the β-catenin promoter (Figure 4  (b)). To seek the mechanism by which LINC01315 regulates β-catenin transcription, the pull-down assay was executed, and we discovered that LINC01315 precipitated the endogenous β-catenin in CRC cells (Figure 4(c)). Similarly, the RIP assay suggested that the endogenous LINC01315 was enriched by β-catenin antibody (Figure 4(d)). Finally, serial truncated promoter promoters were constructed. As indicated in Figure 4(e), the luciferase activities of P1 (−2000 to +44) and P2 (−1756 to +44) promoters were increased upon LINC01315 overexpression. The luciferase activities of P3 (−1472 to +44), P4 (−1188 to +44), P5 (−888 to +44), and P6 (−484 to +44) were not upregulated by LINC01315. These data implied that the sequences (−1756 and −1473) of the β-catenin promoter contain a LINC01315 binding site. b. TOP/FOP luciferase reporter assays were performed to detect the β-catenin activity. c. Altered nuclear translocation of β-catenin in response to LINC01315 overexpression. d. Upregulation of LINC01315 raised the RNA levels on Wnt/β-catenin direct-target genes. **P < 0.05 compared with vector.

LINC01315 promotes CRC invasion and EMT via activating wnt/β-catenin
To confirm LINC01315-induced augment of CRC cells invasion and EMT was dependent on βcatenin. LINC01315-silenced CRC cells were transfected with pcDNA3.1-β-catenin plasmid to restore the expression of β-catenin ( Figure 5(a)). In Figure 5(b-e)), the proliferation, clonogenic ability, migration, and invasiveness suppressed by LINC01315 inhibition were rescued by pcDNA3.1-β-catenin in CRC cells. Inhibition of LINC01315 decreased N-cadherin and increased the expression of E-cadherin. However, this tendency was countered by β-catenin overexpression ( Figure 5(f)). These above observations implied that LINC01315 exerts pro-oncogenic functions is dependent on Wnt/β-catenin signaling in CRC.

Discussion
Emerging studies have focused on the cancerspecific lncRNAs and illuminated their imperative roles in cancer progression [14,15]. In this work, we elucidated the pathomechanism of LINC01315 in CRC EMT. LINC01315 is abnormally high in CRC and acts as an unfavorable prognostic marker for CRC. LINC01315 silencing suppresses the migration and invasion of CRC cells and diminishes the EMT process.
Ectopic lncRNAs expressions have been implicated in cancer metastasis through the downstream effector molecules deregulation [32]. LINC01315 elevates the expression of discs large homolog 3 (DLG3) via sponging miR-211 and then delays oral squamous cell carcinoma (OSCC) progression [33]. Patients with a high LINC01315 expression level are associated with worse clinical outcomes, and LINC01315 might be an independent prognostic predictor in breast cancer [19]. As a ceRNA, inhibition of LINC01315 attenuates the aggressive phenotypes of CRC through sponging the miR-205-3p [21]. In papillary thyroid cancer (PTC), LINC01315 sponges miR-497-5p and fortifies the metastatic behaviors of PTC cells [20]. The way of lncRNA exerts the function mainly depends on its subcellular localization. Except for the ceRNA network of lncRNAs-miRNAs, lncRNAs serve as oncogenic regulators through alternative mechanisms, including transcriptional, posttranscriptional regulation, and chromatin modification [34,35]. Although early studies show LINC01315 participates in the regulation of CRC cells' mobility and invasion, the potential involvement of LINC01315 in CRC EMT has never been proposed before [21]. Deregulation of the Wnt/β-catenin signaling axis plays a significant role in the EMT and is required for colorectal tumor metastasis [1]. Herein, we disclosed that LINC01315 positively regulates EMT and activates the Wnt/β-catenin signaling. The expressions of mesenchymal markers and the downstream targets (C-myc and Cyclin D1) of the Wnt/β-catenin signal are raised in CRC cells upon LINC01315 overexpression. Subsequently, we found that LINC01315 inhibition substantially declined the level of β-catenin at both protein and mRNA levels, indicating that LINC01315 transcriptionally regulates β-catenin expression.
Mechanistically, LINC01315 directly binds to the promoter site of β-catenin, as demonstrated by the luciferase reporter gene assay. Further experiment with truncated constructs of β-catenin promoter indicates that LINC01315 binding site exists the sequence between the nucleotides −1756 and −1473 in the β-catenin promoter. Notably, our results showed that LINC01315 knockdown repressed CRC EMT and invasion, while β-catenin overexpression partially reversed the inhibition effects caused by LINC01315 knockdown.
There are some deficiencies in the presented study. The results obtained are solely based on Figure 5. Overexpression of β-catenin reverses the effects of LINC01315 on CRC cells EMT. a. HCT116 and SW480 cells were cotransfected with shLINC01315 and pcDNA3.1-β-catenin. The expression of β-catenin was determined by Western blot. b. CCK-8 assay showing proliferation of HCT116 and SW480 cells. C. Colony formation assays were performed to determine the proliferation of shLINC01315 and pcDNA3.1-β-catenin co-transfected CRC cells. d. Wound-healing assay of HCT116 and SW480 cells. e. Transwell invasion assay analyzed CRC cells invasion. f. Western blot assay was performed to test the expression of EMT markers (E-cadherin and N-cadherin) in HCT116 and SW480 cells. **P < 0.05 compared with shNC, ## P < 0.05 compared with shLINC01315.
in vitro experiments. For the most part, in vitro experiments will still need to be followed up with in vivo experiments. In the follow-up work, we will proceed with a xenograft tumor model to explore the anti-metastasis of LINC01315 in vivo. In fact, the degree of β-catenin phosphorylation impacts the stability of β-catenin and directly alters the functions of the Wnt/β-catenin signal. Moreover, an increased Glycogen Synthase Kinase-3β (GSK-3β) stability diminishes the Wnt/β-catenin pathway via phosphorylating β-catenin [12]. Further analysis is required to determine the impact of LINC01315 on GSK-3β and β-catenin phosphorylation.

Conclusion
Collectively, we delineated that LINC01315 was exceptionally high in CRC and increased expression of LINC01315 predicted a poor prognosis in human CRC. Loss-of-function experiments demonstrated that LINC01315 fortified CRC cells migration, invasion, and EMT in vitro. Further analysis indicated that LINC01315 exerts prooncogenic functions by promoting the transcriptional activation of β-catenin. This study yields new insights into the role of LINC01315/Wnt/βcatenin in CRC EMT.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.

Data availability statement
The datasets used or/and analyzed during the current study are available from the corresponding author on reasonable request.