Background
Gastric cancer (GC) ranks in the third responding the mortality related to cancer, posing a tremendous threat to global human health [
1]. With the laparoscopy of endoscope, the efficiency of surgical treatment obtained a huge improvement [
2]. Although substantive achievements were made in the treatment, the morbidity had been keeping rising by years. Meanwhile, the prognosis of GC patients was not satisfactory. The primary reason was attributed to lack of early diagnosis [
3]. Consequently, it was necessary to identify a biomarker for the early diagnosis of GC not only to improve accurateness of diagnosis but also to find a target for treatment.
Lately, the unique functions of long non-coding RNAs (lncRNAs) are discovered in multiple cancers [
4,
5]. LncRNAs are a group of noncoding transcripts whose length is over 200 nucleotides, with limited capacities in coding proteins [
6]. Accumulating evidence suggested that lncRNAs had played vital roles in a broad scale of biological movements among cancers, including apoptosis, metastasis and proliferation as well as chemoresistance [
7‐
9]. Thus, it was exceedingly important to comprehend the pathology of GC by delving into potential mechanism of lncRNAs. LBX2-AS1 is a novel lncRNA and considered to exert oncogenic function in esophageal squamous cell carcinoma by promoting migration and epithelial-mesenchymal transition (EMT). However, the biological function of LBX2-AS1 has not been explored ever in GC.
Competing endogenous RNAs (ceRNA) network attracted more and more attention due to its significant effects on regulating the progression of cancers and non-tumor diseases [
10,
11]. For example, HOTAIR served as a ceRNA to modulate HER2 expression via sponging miR-331-3p in GC [
12]. ZEB1-AS1 repressed the process of GC via ceRNA network. TINCR modulated growth of GC via sponging miR-375 to up-regulate the expression of PDK1 [
13]. This study planned to investigate whether LBX2-AS1 played a role of ceRNA in GC.
Hence, the current study focused on how LBX2-AS1 exerted functions in GC by regulating the downstream targets.
Methods
Cell lines
Human GC cell lines (MGC803, BGC823, HGC27 and SGC7901) and gastric epithelial cell line (GES1) were both procured from ATCC (Rockville, Maryland) and cultivated in the DMEM (Invitrogen, Carlsbad, CA). Cell culture was conducted with 1% Pen/Strep solution (Invitrogen) and 10% FBS (Gibco, Grand Island, NY) at 37 °C in 5% CO2. The culture medium was changed every 3 days.
Total RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNAs from MGC803 and BGC823 cells were isolated in line with the protocol of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) for reverse transcription. SYBR Green PCR Master Mix (Takara, Kyoto, Japan) was then utilized for qPCR. Results were processed by 2−ΔΔCT method and normalized to GAPDH or U6. Primers used here were: LBX2-AS1: Forward: 5′-CGTGGGGAATGGACCCATAG-3′, Reverse: 5′-GGACTTGCCCTTGGTGACTC-3′; miR-491-5p: Forward: 5′-AGTGGGGAACCCTTCCAT-3′, Reverse: 5′-CTCTACAGCTATATTGCCAGCCAC-3′; NFIC: Forward: 5′-TGGCGGCGATTACTACACTTCG-3′, Reverse: 5′-GGCTGTTGAATGGTGACTTGTCC-3′; ZNF703: Forward: 5′-TGCAGCCGCTGTCCTCCACTC-3′, Reverse: 5′-CACCGAGTTGAGTTTGGAGGAG-3′; GAPDH: Forward: 5′-ACCTGACCTGCCGTCTAGAA-3′, Reverse: 5′-GTCAAAGGTGGAGGAGTGGG-3′; U6: Forward: 5′-CTCGCTTCGGCAGCACA-3′, Reverse: 5′-AACGCTTCACGAATTTGCGT-3′.
Transfection
MGC803 and BGC823 cells were collected for 48 h of transfection as per the guidebook of Lipofectamine 2000 (Invitrogen). The shRNAs specific to LBX2-AS1, pcDNA3.1/NFIC, miR-491-5p mimics, miR-491-5p inhibitor, pcDNA3.1/LBX2-AS1, pcDNA3.1/ZNF703 and their relative negative control (NC) sh-NC, pcDNA3.1, NC mimics and NC inhibitor, all these were produced by Genepharma (Shanghai, China). In detail, 2 μg of shRNAs (sh-NC, sh-LBX2-AS1#1/2) or pcDNA3.1 plasmids (pcDNA3.1 vector, pcDNA3.1/NFIC, pcDNA3.1/LBX2-AS1 or pcDNA3.1/ZNF703) were added into each well of a six-well plate. Additionally, cells were transfected with 50 nM mimics (NC mimics or miR-491-5p mimics) or 100 nM inhibitors (NC inhibitor or miR-491-5p inhibitor), respectively.
MGC803 and BGC823 cells were incubated in the 6-well plates for 14 days, then fixed and dyed with 4% paraformaldehyde and 0.5% crystal violet (both for 15 min), severally. Colonies were counted manually.
EdU incorporation assay
4 × 104 GC cells were seeded in the 96-well plates with the ultra-low attachment surface and round bottom (Corning Inc., Corning, NY) for treating with EdU assay kit (Ribobio). After 4 h of incubation with 25 μM EdU medium diluent, cells were fixed via 4% paraformaldehyde for 30 min followed by DAPI staining for another 30 min. Thereafter, cells were pictured by fluorescence microscopy (Olympus, Tokyo, Japan).
Flow cytometry analysis for apoptosis
Flow cytometry analysis for apoptosis was conducted in the MGC803 and BGC823 cells using Annexin V-fluorescein isothiocyanate (FITC)/PI double staining method. In brief, cells were treated with Annexin-V-FITC and propidium iodide (PI) for 15 min in succession. Apoptotic cells were analyzed with FACS Calibur (BD Bioscience, San Jose, CA).
Western blot
Cell protein extracts were quantitated for treatment with electrophoresis on 10% SDS-PAGE and PVDF membranes, then with 5% nonfat milk. Membranes were then mixed with the primary antibodies (Abcam, Cambridge, MA) against Bcl-2 (ab32124, 1:1000 dilution), Bax (ab32503, 1:2000 dilution), Total caspase-3 (ab13847, 1:500 dilution), Cleaved caspase-3 (ab32042, 1:500 dilution), ZNF703 (ab188031, 1:1000 dilution) and control GAPDH (ab181602, 1:10,000 dilution) all night at 4 °C, following incubation with corresponding secondary antibodies tagged with HRP (ab20272, 1:2000 dilution) for 2 h. Protein band was examined by enhanced chemiluminescence (ECL) detection system (Bio-Rad, Hercules, CA).
Transwell assay
GC cells were plated into the upper chamber of transwell inserts (Corning) coating with Matrigel or not for invasion or migration assay. Complete medium was added to the lower chamber. After incubation for 24 h, cells penetrating to the bottom were fixed and stained with crystal violet solution.
Wound healing assay
Cells were cultured in 6-well plates (5 × 105 cells/well) to 90% confluence. Scratches were later made through drawing two parallel lines using a 10 µl sterile pipette tip. Any cellular debris was obliterated through washing the cells thrice with PBS (Sigma-Aldrich, St. Louis, MO, USA). Migration area was detected at 0 and 24 h via a light microscope (Nikon, Tokyo, China).
Dual-luciferase reporter assay
LBX2-AS1 promoter covering the wild-type (WT) or mutated (Mut) NFIC binding sites were cloned into the pGL3-Basic vector (Promega, Madison, WI), then co-transfected with pcDNA3.1/NFIC or pcDNA3.1 for 48 h. Besides, the pmirGLO reporter vector (Promega) containing the WT and Mut miR-491-5p binding sites within LBX2-AS1 or ZNF703 fragment were severally generated for luciferase assays. Finally, the dual-luciferase reporter assay system (Promega) was applied for detection of luciferase intensity.
Chromatin immunoprecipitation (ChIP) assay
Cells of MGC803 and BGC823 treated with formaldehyde, then DNA–protein cross-links were sonicated into fragments of 200-1000 bp and received immunoprecipitate with NFIC-specific antibody and control anti-IgG (Millipore, Bedford, MA) overnight. The precipitated chromatin DNA was retrieved by adding beads and analyzed with RT-qPCR.
Fluorescence in situ hybridization (FISH) assay
The fixed GC cells were rinsed in PBS and mixed with the LBX2-AS1 FISH probe (Ribobio) in the hybridization buffer overnight. Hoechst solution was added for nuclear counterstaining. Cells were measured with fluorescence microscopy.
RNA pull down assay
Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA) was applied for RNA pull down assay in GC cells following the recommendations of supplier. The WT or Mut miR-491-5p sequences containing LBX2-AS1 binding sites were synthesized and biotinylated to Bio-miR-491-5p-WT/Mut for incubation with cell protein samples overnight. The pull-down mixture was collected by beads for detecting the relative enrichment of LBX2-AS1 using RT-qPCR.
RNA immunoprecipitation (RIP) assay
RIP assay was performed in line with the user manual of EZ-Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore) using the specific antibodies against Ago2 and IgG as control. For immunoprecipitation, cell lysates were incubated with anti-Ago2 or IgG for one night. The protein samples being digested, precipitated RNAs were isolated and purified for RT-qPCR analysis.
In vivo assay
3-week-old nude mice were acquired from the National Laboratory Animal Center (Beijing, China). They were subcutaneously injected with MGC803 cells with or without LBX2-AS1 silence. Animal research was conducted upon the approval of the Institutional Animal Care and Use Committee of the 960th Hospital of the PLA. Tumor growth was analyzed by recording tumor volume every fourth day, and 28 days later, tumor volume and weight were examined after mice were sacrificed.
Hematoxylin and Eosin (H&E) staining assay
The collected tissues from xenograft model were fixed with 4% paraformaldehyde at 4 °C, then embedded in paraffin and cut into 4 µm sections. After de-paraffin, sections were rehydrated and stained with H&E (Sigma-Aldrich) at 4 °C for 10 min, finally observed under Olympus light microscope.
Immunohistochemistry (IHC) staining assay
Fresh tissues obtained from in vivo assay were fixed in paraformaldehyde. And then they were dehydrated in ethanol solutions, inset in paraffin and cut into 4-μm thickness. Afterwards, they were cultured with primary antibodies against Ki67 overnight at 4 °C, and then were cultivated with HRP-conjugated secondary antibodies. Finally, all these sections were visualized under microscope (Olympus).
Statistical analysis
Continuous variables of 3 or more biological repeats were displayed as the mean ± SD. PRISM 6 (GraphPad, San Diego, CA) was employed to develop data analysis by Student’s t-test for two groups and by one-way/two-way ANOVA for more than two groups, with p-value less than 0.05 as significant level.
Discussion
A huge body of essays have verified that lncRNAs exerted crucial imparts in the advancement of multiple cancers, including GC [
20,
21]. For illustration, lncRNA HOXA11-AS promoted proliferation and invasion of GC cells by targeting miR-1297/EZH2 axis [
22]. LncRNA DANCR facilitated motility via inhibition of lncRNAs-LET in GC cells [
23]. LncRNA ZEB1-AS1 was reported to forecast unsatisfactory prognosis in GC [
24]. These studies gave encouragement to discover the pathological process in GC. Moreover, LBX2-AS1 displayed unfavorable prognosis and facilitated proliferation and metastasis by Notch signaling in non-small cell lung cancer (NSCLC). Based on the previous researches, our study demonstrated that LBX2-AS1 was expressed in a high level in GC cells. Besides, LBX2-AS1 silence could hinder cell proliferation, migration as well as invasion in GC while promoted apoptosis. Considering regulatory mechanism underlying LBX2-AS1 in GC, we first discovered that cytoplasmic abundance of LBX2-AS1 in GC cells. Furthermore, we discovered that NFIC could stimulate the transcription of LBX2-AS1 in GC cells.
More importantly, lncRNAs are found to be involved in the carcinomas of various cancers through sponging special miRNAs and targeting mRNAs [
25,
26]. For example, ZFAS1 contributed to GC progression through suppressing KLF2 and NKD2 expressions [
27]. GACAT3 severed as a sponge of miR-497 to accelerate the course of GC [
28]. Our study found that miR-491-5p was negatively regulated by LBX2-AS1 in GC and LBX2-AS1 sequestered miR-491-5p to facilitate the growth of GC.
Large quantities of works have certified that miRNAs are powerful regulators of their downstream mRNAs in cancers [
29]. MiR-21 regulated prostaglandin signaling and accelerated GC via targeting 15-PGDH [
30]. MiR-148b repressed glycolysis in GC by targeting SLC2A1 [
31]. TEAD1/4 played an oncogenic role and was negatively modulated by miR-4269 in GC [
32]. In this study, we found that miR-491-5p could negatively regulate the expression of ZNF703. MiR-491-5p constructed a bridge between LBX2-AS 1 and ZNF703.
ZNF703 was discovered to exert oncogenic function in numerous cancers, such as colorectal cancer [
33], breast cancer [
34] and cholangiocarcinoma [
35]. In our study, we found that ZNF703 was positively modulated by LBX2-AS1 and ZNF703 up-regulation could rescue the effects of LBX2-AS1 silence on GC progression.
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