LncRNA GAS6-AS1 facilitates tumorigenesis and metastasis of colorectal cancer by regulating TRIM14 through miR-370-3p/miR-1296-5p and FUS

Long non-coding RNAs (lncRNAs) are essential regulators of tumorigenesis and the development of colorectal cancer (CRC). Here, we aimed to investigate the role of lncRNA GAS6-AS1 in CRC and its potential mechanisms. Bioinformatics analyses evaluated the level of GAS6-AS1 in colon cancer, its correlation with clinicopathological factors, survival curve and diagnostic value. qRT-PCR were performed to detect the GAS6-AS1 level in CRC samples and cell lines. The CCK8, EdU, scratch healing, transwell assays and animal experiments were conducted to investigate the function of GAS6-AS1 in CRC. RNA immunoprecipitation (RIP) and dual-luciferase reporter gene analyses were carried out to reveal interaction between GAS6-AS1, TRIM14, FUS, and miR-370-3p/miR-1296-5p. GAS6-AS1 was greatly elevated in CRC and positively associated with unfavorable prognosis of CRC patients. Functionally, GAS6-AS1 positively regulates CRC proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and induces CRC growth and metastasis in vivo. Moreover, GAS6-AS1 exerted oncogenic function by competitively binding to miR-370-3p and miR-1296-5p, thereby upregulating TRIM14. Furthermore, we verified that GAS6-AS1 and TRIM14 both interact with FUS and that GAS6-AS1 stabilized TRIM14 mRNA by recruiting FUS. Besides, rescue experiments furtherly demonstrated that GAS6-AS1 facilitate progression of CRC by regulating TRIM14. Collectively, these findings demonstrate that GAS6-AS1 promotes TRIM14-mediated cell proliferation, migration, invasion, and EMT of CRC via ceRNA network and FUS-dependent manner, suggesting that GAS6-AS1 could be utilized as a novel biomarker and therapeutic target for CRC.


Clinical samples
Paired CRC and para-carcinoma samples were obtained from 40 patients who underwent tumor resection at the First Affiliated Hospital of Soochow University (November 2019 to May 2020). Each patient was CRC-positive at diagnosis and had not received preoperative chemoradiotherapy. The age of patients was range from 40 to 75-year-old. Informed consent was obtained from all patients. This study was approved by the ethics committee of the First Affiliated Hospital of Soochow University (No.2019138).

Cell culture
The human CRC cell lines HT29, LoVo, RKO, SW620, the human normal colon epithelial cells (NCM460), and the human embryo kidney cell line HEK-293T were purchased from the Cell Bank of the Chinese Academy of Sciences and the American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 (Hyclone, USA) or DMEM medium (Hyclone, USA) with 10% fetal bovine serum in a cell incubator at 37 °C and 5% CO 2 .

Real-time RT-PCR
Total RNA was extracted using a TRIzol reagent kit (Invitrogen, USA). Primers were designed by Sangon Biotech (Shanghai, China) and were listed in Additional file 1: Table S1. Quantitative real-time PCR was conducted with either 2X SYBR Green qPCR Master Mix (Abm, Canada) or miDETECT A Track miRNA qRT-PCR Starter Kit (RiboBio, China). β-actin (for mRNAs and lncRNAs) and U6 (for miRNAs) served as controls.

Subcellular fractionation
The PARIS Kit (Invitrogen, USA) was utilized to extract the cell nuclear and cytoplasmic RNA, for subsequent qRT-PCR. We used β-actin (for cytoplasm) and U6 (for nuclear) for normalizations.

Cell proliferation assay
Cells were grown in 96-well plate with 5 × 10 3 per well. Cell proliferation was detected by Cell Counting Kit (Beyotime, China) after transfected or not. Then the absorbance at 450 nm was measured.

EdU assays
BeyoClick EdU-555 Kits (Beyotime, China) were used for EdU assays. Cells were cultivated in medium containing 10 μM EdU before fixing with 4% paraformaldehyde and subsequent stained with EdU reaction buffer. To visualize the DNA, the cells were stained with Hoechst and observed with fluorescence microscope. The EdU-positive cells were counted.

Scratch healing assay
Cells were seeded into 24-well plates and cultured in the incubator. 10 μl pipette tips were used to scratch on monolayer cells at multiple sites. The area of scratch was observed at 0 h, 24 h and 48 h after scratching. Resulting images were processed with ImageJ software.

Transwell assays
Transwell chambers (Corning, USA) with (migration) and without (invasion) Matrigel (BD Biosciences, USA) were applied to perform transwell assays. Briefly, 1 × 10 5 cells were cultured in the upper wells with 100 µL serumfree medium. The lower chamber was infused with 600 µL complete medium. After 48 h, the cells on the lower side of the membrane were fixed in 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified under a microscope.

Experimental animals
Animal experiments were conducted following the principles of the Animal Management and Use Committee of Soochow University and approved by the Medical Ethics Committee of The First Affiliated Hospital of Soochow University (Approval No.2017213). 24 BALB/c nude mice (4-5 weeks) were obtained from SLAC Laboratory Animal Center (Shanghai, China) and reared in a specificpathogen-free environment at 23-25 °C. Xenograft tumors were established by subcutaneously injecting 5 × 10 6 cells (HT29-GAS6-AS1, HT29-Vector, LoVo-shGAS6-AS1, and LoVo-shControl cells). The tumor size was determined weekly. The mice were euthanized after four weeks, and the tumors were excised and weighed.

RNA fluorescent in situ hybridization
Fluorescent in situ hybridization (FISH) kit (RiboBio, China) was applied for the in-situ detection of GAS6-AS1 in HT29 and LoVo cells following the guidelines. Cells were observed by the fluorescence microscope.

RNA immunoprecipitation (RIP)
RIP was performed using EZ-Magna RIP kits (Millipore, USA). Cells were collected and incubated with anti-Ago2 antibody (Abcam, UK) 24 h after transfection. IgG was used as the negative control. Quantitative RT-PCR was used to assess the co-precipitated RNAs.

Statistical analysis
All data were analyzed using Prism 7.0 or SPSS24.0. T-test or ANOVA analyses were applied to examine the differences among the groups. P < 0.05 denoted a statistical difference.

GAS6-AS1 is overexpressed in CRC and correlates with poor clinical outcome
Data on lncRNA GAS6-AS1 expression and corresponding clinical information were obtained from the TCGA-COAD dataset in the TCGA database, including 41 normal colon tissues and 446 colon cancer tissues. Detailed information on colon cancer samples is shown in Additional file 2: Table S2, including sex, age, T stage, N stage, M stage, clinical stage, and microsatellite instability (MSI) status (MSH: high microsatellite instability; MSS: microsatellite instability low). Expression differences and clinical prognostic analyses were performed using R software. The GAS6-AS1 level in colon cancer tissues was significantly higher than that in normal tissues (Fig. 1A). In addition, the relative GAS6-AS1 level of patients with T4 stage disease was higher than that of patients with T1-3 stage disease (Fig. 1B). GAS6-AS1 levels were higher in lymphatic metastasis-positive patients than those in lymphatic metastasis-negative patients (Fig. 1C). Furthermore, GAS6-AS1 levels in patients with distant metastasis were significantly higher than those in patients without distant metastasis (Fig. 1D). Although no statistical significance was detected between GAS6-AS1 in stage III and stage IV patients, stage IV patients exhibited higher GAS6-AS1 levels than stage I and II patients. GAS6-AS1 expression in patients with stage III-IV was higher than that in stage I and II patients (Fig. 1E). Patients with MSS and MSL exhibited higher GAS6-AS1 expression levels than patients with MSH (Fig. 1F). Survival analysis revealed that patients with relatively high GAS6-AS1 expression had shorter survival times than those with relatively low GAS6-AS1 expression (P = 0.028) (Fig. 1G). Receiver operating characteristic (ROC) analysis of GAS6-AS1 was performed and the area under the curve (AUC) was 0.929 (Fig. 1H), which indicates the diagnostic value of GAS6-AS1. After examining GAS6-AS1 levels in CRC, 40 pairs of operable CRC and para-cancer matched samples were collected and analyzed via qRT-PCR. The results indicated remarkably higher GAS6-AS1 levels in the CRC samples than in the para-cancerous samples (Fig. 1I). These findings suggested that GAS6-AS1 expression was elevated in CRC and that GAS6-AS1 was positively associated with tumor progression and poor prognosis.
To further uncover the function of GAS6-AS1 in CRC cell mobility, scratch and transwell assays were performed. In scratch assays, we found that GAS6-AS1 upregulation significantly increased the migration of HT29 cell (Fig. 2H), whereas GAS6-AS1 downregulation reduced the migration of LoVo cell (Fig. 2I). In line with these results, transwell migration experiments revealed that the number of migrating HT29 cells increased after GAS6-AS1 overexpression (Fig. 2J), while the number of migrating LoVo cells was reduced after GAS6-AS1 knockdown (Fig. 2K). Furthermore, transwell invasion assays demonstrated that GAS6-AS1 overexpression promoted the invasion of HT29 cells (Fig. 2L), whereas downregulating GAS6-AS1 impeded LoVo cell invasion (Fig. 2M). These results indicated that GAS6-AS1 promoted CRC cell viability and mobility in vitro.

GAS6-AS1 promotes in vivo tumorigenesis and CRC metastasis
To further evaluate the role of GAS6-AS1 in CRC tumorigenicity in vivo, xenograft models were established. Notably, GAS6-AS1 overexpression promoted subcutaneous xenograft tumor growth (Fig. 3A), whereas GAS6-AS1 knockdown significantly reduced tumor volume (Fig. 3B). Tumors of the GAS6-AS1 overexpression group weighed more than control tumors (Fig. 3C). Lower tumor weight was observed in the GAS6-AS1 knockdown group compared to the control group (Fig. 3D). To further reveal the effect of GAS6-AS1 on CRC metastasis in vivo, we developed lung metastasis models of CRC and lung metastatic foci were counted. Compared to the control group, GAS6-AS1 overexpression resulted in more metastatic pulmonary nodules (Fig. 3E). GAS6-AS1 knockdown resulted in fewer metastatic pulmonary nodules (Fig. 3F). Collectively, these observations demonstrated that GAS6-AS1 overexpression facilitated CRC growth and metastasis in vivo.

TRIM14 contributes to GAS6-AS1-mediated cell proliferation, migration, and invasion
To evaluate the correlation between GAS6-AS1 and TRIM14 expression in CRC tissues, TRIM14 levels in 40 CRC cases were analyzed by qRT-PCR for correlation analysis. The results showed a positive correlation between GAS6-AS1 and TRIM14 (Fig. 7A). Further rescue experiments were performed to verify whether GAS6-AS1 promoted tumor progression in a TRIM14dependent manner. CCK8 and EdU assays revealed that TRIM14 downregulation partially reversed the effects of GAS6-AS1 overexpression on CRC cell proliferation (Fig. 7B, C). Scratch, transwell migration, and transwell invasion experiments demonstrated that TRIM14 knockdown rescued the effects of GAS6-AS1 overexpression on CRC cell migration and invasion (Fig. 7D-F). These Fig. 7 GAS6-AS1 promotes CRC development via regulating TRIM14. A The correlation between GAS6-AS1 and TRIM14 was analyzed by Spearman's correlation analysis. B, C CCK8 and EdU assays revealed that GAS6-AS1 overexpressing-caused proliferation promoting could be reversed by silencing TRIM14. D-F Scratch and transwell assays revealed that the migration and invasion effects of GAS6-AS1 on CRC cells could be reversed by silencing TRIM14. (scale bar: 200 μm for EdU assay, 50 μm for wound healing assay, 100 μm for Transwell assay). * P < 0.05, ** P < 0.01, *** P < 0.001