Background
Gastric carcinoma is one of the most common malignancies worldwide and the fourth leading cause of cancer death [
1]. Approximately 40% of patients with gastric carcinoma present with metastases, and only approximately 5% of these patients exhibit 5-year survival [
2]. The prognosis of GC patients with metastatic disease remains poor due to the lack of effective therapies. New therapeutic options will become available only if we improve our understanding of the mechanisms underlying metastatic spread.
LncRNAs are transcripts longer than 200 nucleotides without protein-coding potential [
3]. Tens of thousands of lncRNAs are expressed in human cells, but the function of most lncRNAs remains unknown [
4]. An increasing number of studies have demonstrated the importance of lncRNAs for regulating a wide range of processes, including development, differentiation, cell proliferation, cell death and cancer development [
5,
6]. Recently, several GC-implicated lncRNAs have been identified, and their functions and mechanisms have been clarified [
7‐
11]. For instance, the lncRNA GClnc1 promotes gastric carcinogenesis and may act as a scaffold for WDR5 and KAT2A complexes to specify the histone modification pattern [
9]. The lncRNA GMAN enhances the translation of ephrin A1 mRNA by competitively binding GMAN-AS and, thus, promotes GC invasion and metastasis [
10]. However, the well-characterized lncRNAs involved in GC are merely the tip of the iceberg, and an even larger number remain unknown.
Here, we demonstrate that the lncRNA SGO1-AS1 (also known as SGOL1-AS1), which is downregulated in gastric carcinoma and associated with tumor progression and patient prognosis, prevents gastric carcinoma EMT, invasion and metastasis in vitro and in vivo. Mechanistically, SGO1-AS1 reduces the stability of TGFB1/2 mRNA by competitively binding the PTBP1 protein, resulting in reduced TGFβ production. In turn, TGFβ inhibits SGO1-AS1 transcription by inducing ZEB1. Thus, in this study, we identified a novel metastasis-suppressive lncRNA, i.e., SGO1-AS1, with crucial biological, mechanistic and clinical impacts on GC that mediates a double-negative feedback loop with TGFβ via ZEB1.
Methods
Clinical specimens
Five pairs of snap-frozen GC tissues and matched adjacent normal mucosa tissues were obtained for the lncRNA microarray analysis. Furthermore, the following two cohorts of frozen samples were collected for the qRT-PCR assay: a small GC cohort (Cohort 1) containing 18 pairs of GC tissues and corresponding adjacent normal mucosa tissues to confirm 13 lncRNAs with more than a 4-fold difference in the microarray analysis and a large GC cohort (Cohort 2) including 92 pairs of GC tissues and matched adjacent normal samples to detect the expression levels of SGO1-AS1, TGFB1/2 and ZEB1. Additionally, GC tissue microarrays containing 95 GC tissues and 80 adjacent tissues (Cohort 3) were included in this study for the ISH analysis. All tissues were collected immediately after surgery from the Affiliated Cancer Hospital of Guangzhou Medical University (Guangzhou, Guangdong, China). All procedures carried out in this research involving human participants were performed in accordance with the ethical standards of the Institutional Review Board of the Affiliated Cancer Hospital of Guangzhou Medical University. The clinical and histopathological characteristics of the patients are described in Additional file
1: Table S1–2.
Microarray analysis
The total RNA was extracted from 5 paired GC tissues and corresponding adjacent normal mucosa tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA was amplified and reverse-transcribed into fluorescent cDNA. Then, the labeled cDNA was hybridized onto the LncRNA+mRNA Human Gene Expression Microarray V4.0 (Agilent, Palo Alto, CA), and after washing, the arrays were scanned with an Agilent Scanner G2565CA (Agilent). Agilent Feature Extraction software (version 10.7.3.1) was used to analyze the acquired array images and the Agilent qRT-PCR results. The data are available via Gene Expression Omnibus (GEO) under accession number GSE157289.
qRT-PCR
The total RNA was isolated from patient tissues and cultured cells using TRIzol reagent (Invitrogen), and cDNA was synthesized using a PrimeScript RT Reagent Kit (Takara, Otsu, Japan). Subsequently, quantitative polymerase chain reaction (qPCR) analyses were performed using a SYBR Premix Ex Taq Kit (Applied Biosystems, Foster City, CA, USA). β-actin was used as the endogenous control to normalize gene expression. The mRNA expression of SGO1-AS1, TGFB1, TGFB2 and PTBP1 in the human tissues is presented as -∆Ct, and the gene expression in cells with different treatments is presented as 2
-∆∆Ct. The ∆Ct was calculated by subtracting the Ct of β-actin from the Ct of the gene of interest. The ∆∆Ct was calculated by subtracting the ∆Ct of the control sample from the ∆Ct of the treatment sample. The primer sequences for each gene are provided in Additional file
1: Table S3.
In situ hybridization (ISH)
The ISH analysis was performed using a kit from Boster (Wuhan, Hubei, China). Tissue microarray slides were deparaffinized, digested with proteinase K, hybridized with DIG-labeled probes for SGO1-AS1 and U6 (positive control) at 52 °C overnight and subsequently visualized with an anti-DIG-POD antibody and DAB complex. The SGO1-AS1 probe was 5′-CCGCCTCCCAGCCAACCAATGGAGGAGCGAGGCG-3′. The results were evaluated by two individuals in a blinded fashion, and the SGO-AS1 expression levels were quantified according to its positive percentage and staining intensity.
Rapid amplification of cDNA ends (RACE) analysis
We used 5′-RACE and 3′-RACE analyses to determine the transcriptional initiation and termination sites of SGO1-AS1 using a SMARTer™ RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) following the manufacturer’s instructions. Nested PCR products were cloned into the pMD20-T vector and then sequenced. The sequences of the SGO1-AS1-specific primers used in the nested PCR of the RACE assay are shown in Additional file
1: Table S4.
Subcellular fractionation
Nuclear and cytoplasmic separation was performed using a PARIS Kit (Life Technologies, USA) according to the manufacturer’s instructions, and then, a qRT-PCR analysis was conducted.
Cell culture
The GC cell lines SGC7901, BGC823, AGS, MGC803, MKN45 and MKN28 were obtained from the Chinese Academy of Medical Science (Beijing, China), and the gastric epithelial cell line GES-1 was obtained from the Beijing Institute for Cancer Research (Beijing, China). The GC cell line NCI-N87 and the HEK293T cell line were obtained from the American Type Culture Collection (Manassas VA, USA). The cell lines involved in our experiments were reauthenticated by a short tandem repeat analysis every 6 months after resuscitation in our laboratory. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in 5% CO2.
RNAi, plasmid construction and cell transfection
The recombinant lentiviral vectors used for SGO1-AS1 overexpression or knockdown were purchased from RiboBio (Guangzhou, Guangdong, China), and the PTBP1 short hairpin RNA (shRNA) lentiviral vectors were obtained from GeneChem (Shanghai, China). The target sequences for SGO1-AS1 and PTBP1 were as follows: shAS1#1, 5′-GCTATCTTCCTCCTCCTCACA-3′; shAS1#2, 5′-CTACCGCCGCCACATTCGAAA-3′; shAS1#3, 5′-GCCTCCCTCTTGTGAGAAGAA-3′; shAS1#4, 5′-AGCTTGCAACGCGGAAGCAGC-3′; and shPTBP1, 5′-GCGGCCAGCC CATCTACATC-3′. To establish the cell lines that stably overexpress or deplete SGO1-AS1, SGC-7901 cells were infected with recombinant SGO1-AS1 lentiviruses, while MKN28 cells were infected with SGO1-AS1 shRNA lentiviruses. Then, the infected cells were selected with 1 mg/L puromycin (InvivoGen, San Diego, CA, USA) for 2 weeks to obtain cells with stable overexpression or knockdown of SGO1-AS1. siRNAs targeting ZEB1 or AGO2 were designed and synthesized by Sangon Biotech (Shanghai, China), and their sequences are as follows: siZEB1#1 sense, GGCAAGUGUUGGAGAAUAAUC, antisense, UUAUUCUCCAACACUUGCCUU; siZEB1#2 sense, GGACAGCACAGUAAAUCUACA, antisense, UAGAUUUACUGUGCUGUCCUG; siAGO2 sense, GGUUGAUACUUAAGCUCUAUU, antisense, UAGAGCUUAAGUAUCAACCUG. To construct the reporter vectors for SGO1-AS1 promoter activity, the wild-type SGO1-AS1 promoter sequence (1 kb sequence upstream of the transcription start site) and its ZEB1-binding site mutated sequences were chemosynthesized by Huada (Shenzhen, Guangdong, China) and inserted into the vector pGL3 basic (Promega) upstream of the firefly luciferase gene.
PTBP1 knockout by CRISPR/Cas9
A small guide RNA (sgRNA) targeting the genome sequence of PTBP1 was cloned into LentiCRISPRv2 (Addgene), and lentivirus particles were generated by cotransfecting the recombinant vector and packaging plasmids into HEK293T packaging cells. MKN28 cells were infected with lentiviruses, and single cells were isolated 48 h after infection by FACS (BD FACS Aria III) into 96-well plates. Independent clones were allowed to grow for 3 weeks. The PTBP1 knockout cells were identified by Western blotting and targeted Sanger sequencing. The sgRNA targeting PTBP1 was 5′-CAGAGCAGACCCGCGGGGGA-3′.
Western blotting analysis
The Western blotting analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-PTBP1 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-PTBP2 antibody (Abcam, Cambridge, UK), anti-PTBP3 antibody (Sigma-Aldrich, St. Louis, MO, USA), anti-HNRNPK antibody (Abcam), anti-HNRNPM antibody (Sigma-Aldrich), anti-FUBP3 antibody (Abcam), anti-CPSF2 antibody (Abcam), anti-G3BP2 antibody (Atlas Antibodies), anti-TGFβ1 antibody (Proteintech Group), anti-TGFβ2 antibody (Abcam), anti-p-SMAD2 antibody (Cell Signaling Technology), anti-SMAD2 antibody (Cell Signaling Technology), anti-p-SMAD3 antibody (Cell Signaling Technology), anti-SMAD3 antibody (Cell Signaling Technology), anti-SMAD5 antibody (Abcam), anti-ID2 antibody (Abcam), anti-ZEB1 antibody (Abcam), anti-SNAI antibody (Abcam), anti-E-cadherin antibody (Proteintech Group), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology) and anti-GAPDH antibody (Sigma-Aldrich). The blots were incubated with a goat anti-rabbit or anti-mouse secondary antibody (Sigma-Aldrich) and visualized with a commercial ECL kit (Pierce, Rockford, IL).
RNA pull-down assay
The RNA pull-down assays were carried out as previously described. Briefly, the SGO1-AS1 sequences were cloned into the pMD20-T vector with the T7 promoter and transcribed in vitro with biotin RNA labeling mix and T7 RNA polymerase (Invitrogen) according to the manufacturer’s instructions. The RNA pulldown assay was performed using a Pierce Magnetic RNA-Protein Pull-Down Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. Finally, the retrieved proteins were measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) gels for mass spectrometry or a Western blot analysis.
RNA immunoprecipitation (RIP) assay
The RIP assays were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. Briefly, 100 μL of cell extract were incubated with magnetic bead-antibody complex. Antibodies were used for RIP, and IgG served as a negative control. The precipitated RNAs were isolated using TRIzol (Invitrogen) for the RNA sequencing (RNA-seq) and qRT-PCR analyses.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assays were performed using a Chromatin Immunoprecipitation Assay Kit (Millipore, Bedford, MA, USA). MKN28 cells were exposed to TGFβ1 or vehicle for 24 h and then crosslinked, lysed and sonicated. Immunoprecipitation was performed using an anti-ZEB1 antibody (Abcam, Cambridge, UK) and IgG. The precipitated DNA was quantified using qPCR and normalized to the respective 2% input.
RNA-seq analysis
To identify the differentially expressed genes upon PTBP1 knockout, the total RNA was isolated from the PTBP1 knockout or control MKN28 cells using TRIzol reagent, and PolyA RNA was subsequently purified from the total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq was performed to detect the mRNA expression profiles at GENTED (Shanghai, China) using HiSeq3000 (Illumina, USA). The differentially expressed genes with a fold change > 2 and a P-value < 0.05 were selected. To reveal the PTBP1-bound mRNAs, RIP experiments were conducted using a PTBP1 antibody (Cell Signaling Technology) or IgG. The total RNA was isolated with TRIzol (Invitrogen), and ribosomal RNA was removed from the total RNA. RNA-seq was performed at CLOUDSEQ (Shanghai, China) using HiSeq3000 (Illumina, USA). The data are available via GEO under accession numbers GSE157582 and GSE157941.
Luciferase reporter assay
HEK293T cells were seeded in 24-well plates and transfected with SGO1-AS1 promoter reporter constructs with wild-type or mutated ZEB1 binding sites. The pTK-Cluc vector was used as an internal transfection control. The transfected cells were treated with TGFβ1 (5 ng/mL) or vehicle control for 48 h, and firefly and Renilla luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. The SBE4 promoter luciferase reporter vector (Addgene) was transfected into the PTBP1 knockout or control MKN28 cells. In addition, HEK293T cells were transfected with SBE4 promoter reporter vectors and then treated with conditioned medium from cells with SGO1-AS1 knockdown or overexpression. The firefly and Renilla luciferase activities were measured 48 h after the transfection using a dual luciferase system.
Cell invasion, migration and proliferation assays
For the cell invasion assay, starved cells suspended in serum-free DMEM were seeded into the upper chamber with Matrigel in the insert of a 24-well culture plate (Corning Costar). Medium containing 15% fetal bovine serum was added to the lower compartment as a chemoattractant. After incubation for 48 h, the invasive cells adhering to the lower membrane of the inserts were fixed, stained, counted and imaged. The cell migration ability was measured using a wound-healing assay. The cells were placed in 6-well plates and cultured until reaching 90% confluence. An artificial scratch was created using a 10 μL pipette tip, and the cells were cultured in serum-free medium for 36 h or 48 h. Wound closure images were captured in the same field under magnification. Cell proliferation was examined using cell counting. The cells were seeded into 6-well plates, and the cell numbers were counted after 1, 2, 3, 4, 5, 6 and 7 days of culture in DMEM supplemented with 10% fetal bovine serum using a Coulter Counter.
Sphere culture
Cells were seeded into ultralow attachment 6-well plates (Corning Costar) and cultured in DMEM/F12 medium (Gibco) supplemented with 2% B27 (Life Technologies), 20 ng/ml FGF (R&D Systems, MN, USA), 20 ng/ml EGF (R&D Systems) and 5 μg/ml insulin (R&D Systems). Two weeks later, sphere pictures were obtained, and the sphere formation ratios were calculated.
Animal experiments
Subsequently, 6- to 8-week-old female BALB/c nude mice were purchased from the Experimental Animal Center of Guangdong (Foshan, Guangdong, China). To investigate the role of SGO1-AS1 in tumor metastasis and growth in vivo, luciferase-labeled SGC7901 cells overexpressing SGO1-AS1 or the control vector (2 × 106 cells per mouse) were injected into the tail vein or stomach of the BALB/c nude mice. The luciferase signal intensity was monitored in vivo using an In Vivo Imaging System (FX PRO, Bruker, Billerica, MA, USA). Then, the mice were sacrificed, and the metastatic foci in the abdominal cavity and lung were evaluated. In addition, SGC7901 cells with SGO1-AS1 overexpression or control cells were subcutaneously injected into nude mice The mice were sacrificed 28 days after implantation, and the tumors were excised and weighed.
To confirm the inhibitory effects of SGO1-AS1 on metastasis activity via TGFβ signaling in vivo, we orthotopically implanted luciferase-labeled MKN28 cells stably expressing shSGO1-AS1 or control shRNA into the stomach of nude mice and treated the mice with saline or SB431542 (20 mg/kg body weight, i.p.) three times per week for 3 weeks. The luciferase signal intensity was monitored in vivo by bioluminescence imaging. All animal studies were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University, and the animals were treated ethically and humanely.
Statistical analysis
A Student’s t-test or chi-square test was used for the two-sample comparisons. The differences among three or more groups were analyzed with a two-way analysis of variance. The overall survival curves were plotted using the Kaplan-Meier method, and the survival differences were evaluated with a log-rank test. A Cox regression was utilized to estimate the hazard ratio and 95% confidence intervals of survival. The pairwise expression correlations were analyzed using Pearson correlation tests. P-values < 0.05 were considered statistically significant.
Discussion
Most patients with GC die from metastatic disease, but knowledge regarding the mechanisms of metastasis in gastric tumors is limited [
2]. In this study, we identified a metastasis-suppressive lncRNA, i.e., SGO1-AS1, which is decreased in progressed gastric cancer and inversely correlated with gastric tumor metastasis. We further revealed that SGO1-AS1 interacts with the protein PTBP1, and their interaction competitively reduces TGFB1/2 mRNA binding to PTBP1. In turn, the decreased binding of TGFB1/2 mRNA to PTBP1 leads to a reduction in TGFB1/2 mRNA stability and reduced TGFβ production, thus preventing the EMT and metastasis. In addition, TGFβ represses SGO1-AS1 transcription by inducing ZEB1. Thus, SGO1-AS1 and TGFβ form a double-negative feedback loop via ZEB1 to regulate the EMT and metastasis (Fig.
8h).
LncRNAs often exert their effects through the proteins with which they interact [
32]. Here, we identified PTBP1 as an SGO1-AS1-interacting protein. PTBP1 has been shown to be involved in tumorigenesis by regulating alternative splicing [
33,
34], controlling mRNA stability [
17,
35] and determining mRNA localization [
19]. For example, PTBP1 enhances the PKM2 isoform and reduces the PKM1 isoform by controlling PKM alternative splicing, which promotes aerobic glycolysis and provides a selective advantage for tumor formation [
16,
36]. PTBP1 mediates MCL1 mRNA stability and regulates cellular apoptosis induced by antitubulin chemotherapeutics [
23]. Notably, several lncRNAs have been reported to be associated with PTBP1 [
17,
37,
38]. The hypoxia-induced lncRNA LUCAT1 interacts with PTBP1 in CRC cells, facilitating the association between a set of DNA damage-related genes and PTBP1 and resulting in altered alternative splicing of these genes, thereby conferring resistance to chemotherapeutic drugs in CRC cells [
39]. The lncRNA MEG3 can recruit PTBP1 to regulate small heterodimer partner mRNA stability and cholestatic liver injury [
17]. Recruiting PTBP1 to target mRNAs appears to be a common mechanism among these lncRNAs. However, we found that the interaction between SGO1-AS1 and PTBP1 reduces the enrichment of this protein in TGFB1/2 mRNA to facilitate their decay. In addition to PTBP1, it is possible that SGO1-AS1 might bind other proteins, such as G3BP2, to regulate GC metastasis as G3BP2 was found to be an SGO1-AS1-interacting protein in the mass spectrum analyses and verification analyses in our study. The role of this and other proteins bound by SGO1-AS1 in gastric carcinoma deserves further investigation.
Identifying TGFβ-induced ZEB1 as a potent transcriptional repressor of SGO1-AS1 is another important finding of this study. Here, we demonstrate that a reciprocal negative feedback loop exists between SGO1-AS1 and TGFβ/ZEB1. Although the double positive feedback loop between TGFβ and lncRNA is well documented [
40‐
42], to the best of our knowledge, the reciprocal repressive loop between TGFβ and lncRNA has rarely been observed. Our current study provides evidence of a reciprocal repressive loop between TGFβ and the lncRNA SGO1-AS1 in GC metastasis. We show that ZEB1 induced by TGFβ transcriptionally inhibits SGO1-AS1 expression; in turn, SGO1-AS1 inhibits TGFβ expression by reducing TGFB mRNA stability, which mediates the reciprocal repressive loop between TGFβ/ZEB1 and SGO1-AS1 in GC.
TGFβ signaling is highly conserved in multicellular organisms and is involved in multiple cellular processes, such as cell growth, stemness, migration, invasion, the EMT, ECM, remodeling and immune regulation [
43]. The activation of canonical TGFβ signaling is caused by the binding of TGFβ ligands (TGFβ1, TGFβ2 and TGFβ3) to heteromeric TGFβ type I and II receptors, which phosphorylate SMAD2 and SMAD3, resulting in complex formation with SMAD4 and nuclear translocation to regulate target gene transcription [
44]. TGFβ plays a critical role in tumorigenesis and tumor progression in a complex and pleiotropic manner; in early tumor initiation, it plays a tumor-suppressive role by inhibiting cell proliferation and stimulating apoptosis; however, in advanced tumors, it promotes tumor progression by inducing the EMT, which is correlated with increased invasiveness, metastasis and chemoresistance in tumor cells [
45,
46]. Because of its role in advanced tumors, TGFβ is considered a therapeutic target. Several strategies have been proposed to inhibit TGFβ signaling to combat malignant tumors (e.g., small-molecule inhibitors of receptor kinases, TGFβ neutralizing antibodies and antisense compounds) [
47]. Our finding that SGO1-AS1 and TGFβ/ZEB1 form a double-negative feedback loop hints at the possibility of new therapeutic approaches to block the TGFβ signal by introducing SGO1-AS1 or using the interference of ZEB1, although this possibility remains to be confirmed by future studies.
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