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The effect of splicing MST1R in gastric cancer was enhanced by lncRNA FENDRR
- Authors:
- Published online on: May 25, 2021 https://doi.org/10.3892/etm.2021.10230
- Article Number: 798
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Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Gastric cancer (GC) is one of the leading causes of cancer-related mortality worldwide, demonstrating over 50% of morbidity and mortality (1). Despite significant advances, the overall survival rate of patients with GC has not improved in the past 20 years (2). Although numerous molecular targets have been reported to be involved in the progression of GC (3,4), the mechanisms underlying GC development remain to be further investigated.
Serine/arginine rich splicing factor (SRSF)1 is a 35-kDa serine/arginine-rich splicing factor. SRSF1 can regulate pre-mRNA alternative splicing and maintain genome stability by interacting with transcription factor E2F transcription factor 1 in the cell cycle (5). Furthermore, SRSF1 facilitates transcriptional elongation by recruiting positive transcription elongation factor b kinase and promoting the subsequent phosphorylation of serine 2 on the C-terminal domain of RNA polymerase II at the post-transcriptional level (6).
Macrophage stimulating 1 receptor (MST1R) is a membrane tyrosine kinase of the MET family, which has been reported to be a novel potential target for cancer treatment (7). MST1R has been found to be positively associated with the development and progression of multiple types of epithelial cancer, including GC (8-10). Moreover, several preclinical studies have revealed that MST1R exhibited oncogenic properties, including the promotion of cellular proliferation, migration, invasion and survival in several human cancer cell lines (11,12). Despite exhibiting diverse functions in numerous malignancies, the alternatively spliced products of MST1R show considerable sequence homology with each other (11). In human cancer, the alterative skipping of MST1R exon 5 and exon 6 promotes the loss of 106 amino acids in the MST1R β-chain, which produces a 109 kDa MST1R splicing variant known as recepteur d'origine nantais (RON) Δ160. This isoform was identified to be involved in the activation of the PI3K/AKT signaling pathway and cellular transformation in vitro (13). Previous studies found that SRSF1 played a key role in the regulation of MST1R pre-mRNA splicing (8,13). Therefore, further investigation into the association between SRSF1 and MST1R isoform splice variants in GC may provide novel insights for targeted therapies.
Materials and methods
Cell lines and culture
The GC cell lines Kato III and MKN-45 and the immortalized gastric epithelium cell line GES-1 were obtained from the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China). Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and penicillin, and maintained at 37˚C in 5% CO2.
Vector construction and cell transfection
Small interfering RNA (siRNA) targeting SRSF1 (SRSF1-siRNA; 10 nM), FENDRR-siRNA (10 nM), pcDNA3.1 vector for MST1R overexpression (pcDNA3.1-MST1R, MST1R OE) and the corresponding empty vector [(referred to as negative control (NC)] were purchased from Sangon Biotech (Shanghai) Co., Ltd. SRSF1- and FENDRR-siRNA were transfected into GC cells using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The transfection efficiency was analyzed using reverse transcription-quantitative PCR (RT-qPCR). The sequences of the siRNAs were as follows: NC siRNA (siRNA-ctrl), 5'-UUCUCCGAACGUGUCACGUTT-3'; SRSF1-siRNA, 5'-GGAAUGAAGCAACUGAGAUUU-3'; and FENDRR-siRNA, 5'-GGGTTACGATTGCCCAGAT-3'. For MST1R overexpression, GC cells were plated into 60-mm plates at a density of 4x105 cells/well and incubated overnight. Upon cells reaching 50-60% confluence, supernatants containing the pcDNA3.1 vector carrying the MST1R gene were added directly to the cell culture and incubated for 24 h. Following the incubation, GC cells were plated in selection medium containing 2.5 µg/ml puromycin for a further 3 days.
Cell Counting Kit-8 (CKK-8) assay
A CCK-8 assay (Beyotime Institute of Biotechnology) was used to determine cell viability. Briefly, GC cells were plated into 96-well plates at a density of 5x103 cells/well and transfected with NC, siRNA-FENDRR or siRNA-FENDRR + MST1R pcDNA3.1 vector for 72 h. Following the incubation, cells were incubated with 10 µl CCK-8 reagent for another 2 h at 37˚C. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).
Cell apoptosis
GC cells were seeded into six-well plates (5x104 cells/well). Following centrifugation at 200 x g for 5 min at 4˚C, the cell pellet was resuspended in 100 µl binding buffer and incubated with 5 µl Annexin V-FITC (BD Biosciences) and propidium iodide (BD Biosciences) at room temperature for 15 min. The cell apoptotic rate was measured using a flow cytometer (BD Biosciences) and the data were analyzed using WinMDI 2.9 software.
Agarose electrophoresis
Vectors at a concentration of 0.03 µg DNA/µl were subjected to electrophoresis on an ethidium bromide-containing gel (1% agarose). Subsequently, bands were photographed with a Vilber E-BOX (Vilber Lourmat Sté). Meanwhile, 1 unit DNase and 1.2 µg DNA (Sigma-Aldrich; Merck KGaA) were incubated at 37˚C with the vectors and complexes for 30 min. Subsequently, 2% SDS solution was added as a DNA release reagent. Samples were subjected to agarose gel electrophoresis and compared with untreated DNA. The data were quantified using Image Pro Plus (version 6.0; Media Cybernetics, Inc.).
RT-qPCR
Total RNA was extracted from cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was subsequently analyzed using a SYBR Premix Ex Taq II kit (Takara Bio, Inc.) on a 7900HT system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the following conditions: 60˚C for 1 min, 90˚C for 15 min, followed by 40 cycles of application at 90˚C for 15 sec and 55˚C for 60 sec. The following primer sequences were used for the qPCR: FENDRR forward, 5'-TTCATCGGCTGCGTATTCG-3' and reverse, 5'-TTGCCTTCTAGTCGCCTCC-3'; and β-actin forward, 5'-GTCCACCGCAAATGCTTCTA-3' and reverse, 5'-TGCTGTCACCTTCACCGTTC-3'. Expression levels were quantified using the 2-ΔΔCq method (14). β-actin was used as the internal control for normalization.
Transwell assay
A Transwell assay was performed to analyze cell migration and invasion. The upper chambers of the Transwell plates were pretreated with 100 µl Matrigel (BD Biosciences) for 4 h at 37˚C, while Matrigel was not used in the migration assay GC cells were seeded into the upper chamber of the plates in media supplemented with 1% FBS at a density of 1x106 cells/chamber. RPMI-1640 medium supplemented with 10% FBS was added into the lower chambers. Following 24 h of incubation at 37˚C, the Transwell chamber was rinsed twice with PBS (5 min each time), then the cells were fixed with 5% glutaraldehyde at 4˚C and stained at 37˚C with 0.1% crystal violet for 30 min. The Transwell chamber was washed twice with PBS and observed under a light microscope (magnification, x200). Three random fields were selected. The number of cells invading the Matrigel was a reflection of the invasive ability.
Western blotting
Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnnology) and quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology). Proteins (40 µg per lane) were separated via 10% SDS-PAGE, then transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4˚C overnight: Anti-AKT (cat. no. ab18785; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. ab38449; 1:1,000), Anti-p-ERK (cat. no. ab201015; 1:1,000), anti-ERK (cat. no. ab32081; 1:1,000), anti-MST1R (cat. no. ab52927; 1:1,000), anti-cleaved caspase-3 (cat. no. ab32042; 1:1,000), anti-SRSF1 (Thermo Fisher Scientific, Inc.; cat. no. 32-4500, 1:1,000) and anti-β-actin (cat. no. ab8226; 1:1,000; all Abcam except for SRSF1). Following the primary antibody incubation, the membranes were incubated with an anti-rabbit secondary antibody (HRP-conjugated; Abcam; cat. no. ab7090, 1:5,000) at room temperature for 1 h. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) was used to visualize the protein bands. ImageJ software (version 2.0; National Institutes of Health) was used to quantify the intensity of the bands.
Bioinformatics prediction
The LncRNA2Targetv2.0 tool (http://123.59.132.21/lncrna2target/index.jsp) was used to determine the interaction between long non-coding RNAs (lncRNAs) and upstream of the SRSF1 coding region.
RNA pull-down
For the RNA pulldown assay, Biotin RNA Labeling Mix (Roche Diagnostics) was used to transcribe and label probe-control or probe-FENDRR in vitro. An RNA structure buffer (Thermo Fisher Scientific, Inc.) was used to induce secondary structure formation from the biotin-labeled RNAs. The biotinylated FENDRR (Shanghai GenePharma Co., Ltd.) and negative control (bio-NC; GenePharma) were coated with streptavidin-conjugated magnetic beads (Roche Diagnostics). GC cells were lysed using lysis reagent (Roche Diagnostics) and then incubated with the magnetic beads for 6 h. The enrichment level of SRSF1 was detected by western blotting.
Immunofluorescence staining
GC cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then fixed with pre-cooled methanol at 4˚C for a further 10 min. Subsequently, cells were incubated with an anti-RON ∆160 primary antibody (Abcam; cat. no. ab124671, 1:1,000) overnight at 4˚C. Following the primary antibody incubation, the cells were incubated with a goat anti-rabbit IgG secondary antibody (Abcam; cat. no. ab6721, 1:5,000) for 1 h at room temperature. DAPI (Beyotime Institute of Biotechnology) was used to counterstain the nuclei. Samples were visualized using a fluorescence microscope (model no. CX23; Olympus Corporation, magnification, x200). Three random fields were selected.
Co-immunoprecipitation (co-IP) assay
The co-IP assay was performed as previously described with modifications (5). Briefly, Kato III cell protein supernatants were pretreated with 50 µl A/G (Protein A/Protein G) beads (Selleck Chemicals) prior to immunoprecipitation and then with 5 µg control IgG (Santa Cruz Biotechnology, Inc.), anti-MST1R or anti-SRSF1 magnetic beads (Shanghai ZE YE Biological Technology Co., Ltd.) overnight at 4˚C. Following further incubation with 50 µl A/G beads at 4˚C for 6 h, the immunoprecipitates were eluted with ice-cold PBS supplemented with 0.2% NP-40 five times. Subsequently, the immunoprecipitated proteins were separated via SDS-PAGE and visualized using western blotting as aforementioned.
Statistical analysis
Data are presented as the mean ± SD. Statistical differences between two groups were determined using unpaired Student's t-test. Comparisons among multiple groups were made using an ANOVA followed by a Tukey's post hoc test. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
Results
Expression levels of MST1R and RON ∆160 are upregulated in Kato III cells
To determine the biological function of MST1R and RON ∆160 in GC, western blotting was performed. As indicated in Fig. 1A, the expression levels of MST1R were significantly upregulated in Kato III cells compared with the other cell lines. The expression levels of RON ∆160 in Kato III cells were higher compared with GES-1 cells (Fig. 1B). Moreover, the mRNA expression levels of MST1R and RON ∆160 were also notably upregulated in Kato III cells compared with GES-1 cells (Fig. 1C). Therefore, Kato III cells were selected for use in subsequent experiments. These results revealed that the expression levels of MST1R and RON ∆160 may be upregulated in Kato III cells.
Knockdown of SRSF1 significantly downregulates MST1R and RON ∆160 expression levels in Kato III cells
To determine the transfection efficiency of SRSF1-siRNA, RT-qPCR was performed. As shown in Fig. 2A and B, SRSF1 expression levels were significantly downregulated in Kato III cells following SRSF1 knockdown. Moreover, Kato III cells were more sensitive to SRSF1-2-siRNA. Thus, SRSF1-2- siRNA was selected for use in subsequent experiments. Similarly, the expression levels of RON ∆160 were significantly downregulated following the transfection with SRSF1-2- siRNA in Kato III cells (Fig. 2C). The expression levels of MST1R were also downregulated in GC cells following the knockdown of SRSF1 (Fig. 2D). As expected, the mRNA expression levels of MST1R and RON ∆160 in GC cells were also notably downregulated following transfection with SRSF1-2-siRNA (Fig. 2E). Taken together, these findings indicated that the knockdown of SRSF1 may significantly downregulate MST1R and RON ∆160 in Kato III cells.
Knockdown of FENDRR suppresses the expression levels of MST1R and alters the distribution of RON ∆160 in Kato III cells
FENDRR was identified to directly interact with SRSF1. RT-qPCR was used to analyze the transfection efficiency of FENDRR-siRNA transfection. As shown in Fig. 3A, the expression levels of FENDRR were downregulated in Kato III cells in the presence of FENDRR-siRNA. In addition, the knockdown of FENDRR significantly downregulated the expression levels of SRSF1 and MST1R in Kato III cells (Fig. 3B). Immunofluorescence staining revealed that RON ∆160 expression was significantly upregulated in Kato III cells in the presence of FENDRR-siRNA (Fig. 3C). mRNA expression levels of MST1R and RON ∆160 in Kato III cells were also markedly upregulated following the transfection with FENDRR-siRNA (Fig. 3D). Altogether, these findings suggested that the silencing of FENDRR may regulate the expression levels of MST1R and alter the distribution of RON ∆160 in Kato III cells by suppressing SRSF1 expression.
Silencing of FENDRR suppresses the proliferation of GC cells by regulating MST1R
To determine the effect of FENDRR siRNA on interaction between SRSF1 and MST1R, co-IP assays were performed. As demonstrated in Fig. 4A, SRSF1 was found to directly bind with MST1R, while FENDRR siRNA reversed this phenomenon. Meanwhile, FENDRR could bind with SRSF1 (Fig. 4B). A CCK-8 assay was used to analyze cell viability. The results revealed that the knockdown of FENDRR notably inhibited the viability of Kato III cells, while the reduction in viability induced by FENDRR-siRNA was partially rescued by the overexpression of MST1R (Fig. 5A). Similarly, the apoptosis of Kato III cells was markedly increased following the genetic silencing of FENDRR, which was significantly reversed by the overexpression of MST1R (Fig. 5B). In addition, Kato III cell migration and invasion levels were suppressed in the presence of FENDRR-siRNA. However, the inhibitory effect of FENDRR-siRNA on cell invasion was significantly abrogated by MST1R overexpression (Fig. 5C and D). Altogether, these findings suggested that the knockdown of FENDRR may suppress the proliferation of GC cells by regulating MST1R expression.
Knockdown of FENDRR significantly inhibits the progression of GC in vitro via inactivation of PI3K/AKT signaling
To further investigate the mechanism through which FENDRR mediated the progression of GC, western blotting was performed. As shown in Fig. 6A-E, the protein expression levels of MST1R and the p-AKT/AKT and p-ERK/ERK ratios were notably downregulated in Kato III cells following the knockdown of FENDRR, and were partially reversed following MST1R overexpression. In contrast, the genetic silencing of FENDRR markedly upregulated the ratio of cleaved caspase-3/procaspase-3 in GC cells, while the promoting effect of FENDRR-siRNA on the cleaved caspase-3/procaspase-3 ratio was notably abrogated following MST1R overexpression. Altogether, these results suggested that the knockdown of FENDRR may inhibit the progression of GC in vitro by inhibiting PI3K/AKT signaling.
Discussion
MST1R has been reported to play a key role in the pathogenesis of GC (9). The alternative splicing variant of precursor MST1R mRNA, RON ∆160, has been identified as an oncogenic transcription factor that regulates numerous signaling pathways associated with tumorigenesis, including cellular transformation activities such as focus formation and anchorage-independent growth (8). A previous study demonstrated that the upregulation of variant RON Δ160 in GC altered the phenotype and enhanced the invasive ability of GC cells. Furthermore, RON Δ160 was found to be closely associated with tumorigenesis, and both regional lymph node and widespread metastasis (13). The findings of the present study revealed that the expression levels of MST1R were regulated by FENDRR knockdown. These findings further validated the results of previous studies (8,13), indicating that FENDRR and SRSF1 may promote the tumorigenesis of GC by regulating RON ∆160 expression.
SRSF1 is known to serve roles in RNA splicing and genome stability (6,15,16). The RNA recognition motif of SRSF1 for RNA binding can promote spliceosome assembly at adjacent splice sites to facilitate appropriate exon inclusion (17,18). The aberrant spliceosome function of SRSF1 was previously associated with the mis-splicing of multiple genes, including MST1R and enhancer of zeste 2 polycomb repressive complex 2 subunit, which have been implicated in the pathogenesis of myeloid neoplasms (19,20). The results of the current study demonstrated that the knockdown of SRSF1 could lead to the inactivation of MST1R and RON Δ160 in GC cells. In addition, the results of the co-IP assay found that SRSF1 directly bound to MST1R. These data provided novel insights into the biological function of SRSF1, suggesting that SRSF1 may regulate MST1R and RON Δ160 expression. According to Bonomi et al (21), the involvement of SRSF1 in epithelial-to-mesenchymal transition derives from its ability to affect the splicing program of the proto-oncogene MST1R. Previous research has shown that SRSF1 could promote the production of MST1R through skipping of exon 11. More specifically, SRSF1 acts by directly binding to an exonic splicing enhancer (ESE) located in the constitutive exon 12(22). Therefore, SRSF1 may interact with MST1R in GC.
An increasing number of studies have reported the important role of non-coding RNAs in cellular biological functions (23,24). Previous research indicated that FENDRR was associated with the development of osteosarcoma (25). The present study found that the knockdown of FENDRR downregulated MST1R and RON ∆160 expression levels in GC cells. Based on these findings, it was hypothesized that the knockdown of FENDRR expression may result in the downregulation of RON ∆160 in GC.
Furthermore, the current research revealed that the genetic silencing of FENDRR inactivated the PI3K/AKT signaling pathway in GC cells. PI3K/AKT signaling is known to be involved in the tumorigenesis of cancer (26,27). A previous study reported that PI3K/AKT signaling played a key role in cancer progression, drug resistance and treatment (28). Xu et al (29) found that PI3K/AKT signaling led to reduced apoptosis and increased proliferation in GC cells. The findings of the present study were consistent with the aforementioned studies. Moreover, the present study demonstrated that MST1R overexpression partially rescued the inhibitory effect of FENDRR-siRNA on the PI3K/AKT signaling pathway in vitro. Ling et al (30) demonstrated that MST1R promoted PI3K/AKT signaling during the development of colorectal cancer. These findings were consistent with the present data, suggesting that FENDRR may mediate the expression levels of MST1R and RON ∆160 by inhibiting PI3K/AKT signaling.
In conclusion, the results of the present study suggested that lncRNA FENDRR may function as an oncogene during the progression of GC by mediating the alternative splicing of MST1R and SRSF1 expression. Therefore, lncRNA FENDRR may serve as a potential target for the diagnosis and treatment of GC.
Acknowledgements
Not applicable.
Funding
Funding: This study was supported by a grant from the National Natural Science Foundation of China (grant no. 81272680) and Zhejiang Provincial Department of Science and Technology (grant no. 2020C03112).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
DZ and XW conceived and supervised the study. DZ, XZ and XW designed the study. LT, JZ and YZ performed the experiments and analyzed the data. DZ, XZ and XW were responsible for confirming the authenticity of all the raw data. All authors reviewed the results and approved the final version of the manuscript.
Ethics approval and consent to participate
Not applicable
Patient consent for publication
Not applicable.
Competing interests
These authors declare that they have no competing interests.
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