Background
Gastric cancer (GC) is a one of the most common malignant tumors, and its morbidity ranks the first cancer in China [
1]. Although the early diagnosis of GC as well as the level of surgical techniques and chemoradiotherapy has been significantly improved, the incidence of GC is still rising steadily and becoming younger [
2,
3]. Therefore, it is urgent to explore the molecular mechanism of GC development to provide theoretical basis for the prevention of GC.
Long non-coding RNA (lncRNA) is a class of functional RNA molecules with transcript length of > 200 nucleotides [
4]. Recently, studies have found that lncRNAs are widely involved in almost all physiological and pathological processes of the body, and is closely related to the occurrence and development of many tumors [
5]. LBX2 antisense RNA 1 (LBX2-AS1), transcribed from the intron of chromosome 2p13.1, was a newly discovered lncRNA which was initially reported to act as a tumor promoter [
6]. Studies have shown that LBX2-AS1indicates poor prognosis and promotes cell proliferation and metastasis through Notch signaling in non-small cell lung cancer [
7]. In addition, abnormal expression of LBX2-AS1 is associated with poor prognosis of GC [
8].
MicroRNAs (miRNAs) are a class containing 19 to 25 endogenous non-coding small molecule single-stranded RNA, which is widely found in eukaryotes and highly conserved. miRNAs are complementary to the bases of the 3′ non-coding regions of the target mRNA to promote the degradation of the target mRNA or inhibit its translation, thereby regulating the expression of the target gene and affecting the cell proliferation, differentiation, and apoptosis processes. LncRNAs can serve as the sponge of miRNA, down-regulate the expression of miRNA, and finally change the expression of miRNA target protein at the post-transcriptional level. As a relatively new miRNA, miR-4766-5p has been reported that plasma miR-4766-5p levels are significantly down-regulated in breast cancer [
9]. It was also reported that miR-4766-5p suppressed serine/threonine kinase PAK2 to inhibit tumorigenic ability of colorectal cancer [
10]. Meanwhile, Wei et al. suggested that miR-4766-5p targeted NKAP to inhibit the proliferation and metastasis of GC cells [
11]. Therefore, exploring its mechanism of action can provide more theoretical basis for the treatment of GC.
As a member of the ELR + CXC chemokine family, C-X-C motif chemokine 5 (CXCL5) is an inflammatory mediator cells which recently has been determined to play a core role in some cancer. For example, elevated levels of CXCL5 were detected in human NSCLC that was related to the vascularity of these tumors [
12]. Ando et al. research showed that CXCL5 could promote pancreatic cell migration and invasion by mediating the necroptosis [
13]. Recently, Liu et al. reported that overexpression of CXCL5 dramatically attenuated the suppressive effects of cell proliferation, migration and invasion induced by ROR-α-1 overexpression in hepatocellular carcinoma [
14]. In addition, CXCL5 also has been determined to be associated with late stage of GC [
15]. Therefore, the expression of CXCL5 is closely related to the occurrence of GC.
Our study aimed to investigate the mechanism of LBX2-AS1 in GC progress. Function assays indicated that the decreased proliferation and metastasis of GC cells in response to silenced LBX2-AS1. Bioinformatics analysis and dual luciferase reporter assay was certified the interaction among LBX2-AS1, miR-4766-5p and CXCL5. Rescue assays were performed to prove the functional effects of LBX2-AS1/miR-4766-5p/CXCL5 axis in GC. All the findings are conductive to comprehend the biological functions and molecular mechanisms of LBX2-AS1 in GC progression.
Materials and methods
Clinical samples
78 GC patients were recruited from the Shandong Provincial Hospital Affiliated to Shandong First Medical University. Tumor samples and adjacent tissue were collected and immediately stored at -80 °C until used. The Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University approved this study, and written informed consents were acquired from all enrolled patients.
Cell culture and transfection
Three GC cell lines (MKN-45, BGC-823 and SGC-7901) and the human gastric mucosal epithelial cell line (GES-1) were all obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and seeded into 1640 culture medium containing 10% fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin, and 100 IU/ml streptomycin in 5% CO2 incubator at 37 °C and 95% humidity. Small interfering RNA (siRNA) against LBX2-AS1 (si-LBX2-AS1-1 or 2) and its control (si-NC), pcDNA and pcDNA-LBX2-AS1 overexpression vector (LBX2-AS1) or pcDNA-CXCL5 overexpression vector (CXCL5), miR-4766-5p mimics (miR-4766-5p), miR-4766-5p inhibitor and matched negative controls (miR-inhibitor-NC) were obtained from Guangzhou RiboBio Co., Ltd (Guangzhou, China) and then Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) was used to transfer them following the manufactures’ instructions.
Overexpression of LBX2-AS1 cells
Overexpression of the truncated intracellular form of LBX2-AS1 in GC cell lines (BGC-823 and SGC-7901) was achieved using the plasmid pEGFP-N1 (Generay, Shanghai, China). To eliminate endotoxin contamination, all plasmids were prepared using an Endo-free Plasmid Mini Kit II (Omega, San Carlos, CA, United States). Transient transfection was performed with FuGENE 6 Transfection Reagent (Promega, Sunnyvale, CA, United States). Overexpression of LBX2-AS1 was confirmed with dual-endonuclease digestion and sequencing.
Dual-luciferase reporter assay
In brief, partial fragments of LBX2-AS1 and CXCL5 3’UTR containing miR-4766-5p binding sites were subcloned into the psiCHECK-2 luciferase vector (Promega) to produce The wild-type (WT) LBX2-AS1-3ʹ-UTR (LBX2-AS1-WT) and the wild-type (WT) CXCL5-3ʹ-UTR (CXCL5-WT) reporters, respectively. Also, the mutant LBX2-AS1-3ʹ-UTR (LBX2-AS1-MUT) and the mutant CXCL5-3ʹ-UTR (CXCL5-MUT) reporters with mutant miR-4766-5p binding sites were constructed. Luciferase assay was performed the firefly luciferase 48 h post-transfection.
Cell counting kit-8 (CCK-8) assay
The cultured cells (about 104/96-well) were cultured for 24 h after transfection. Then, 10 µL CCK-8 solution (C0041, Beyotime Institute of Biotechnology, Shanghai, China) was added to each well and cultured for 4 h. The absorbance was measured with a spectrophotometer (Bio-Rad) at 450 nm to measure cells proliferation.
Transwell assay
Transwell assay (Corning, NY, USA) was used to detect the cell migration and invasion abilities following the manufacturer’s protocol. In short, after transfected, about 1 × 105 cells were seeded in an 8 µm polycarbonate membrane filter with or without Matrigel (BD Biosciences, CA, USA) in addition to 100 µl serum-free medium (Thermo Fisher Scientific) to detect the ability of cell migration and invasion in the upper chamber, and the lower chamber added 600 µl medium containing 10% FBS. After 24 h, the membrane was removed and stained at room temperature with 0.1% crystal violet for 15 min. An inverted microscope was further used to count the number of migrated and invasive cells.
qRT-PCR
Total RNA of tissues and cells were extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and qRT-PCR was performed with the SYBR Green PCR kit (Takara, Otsu, Japan) following the manufacturer’s protocol. TaqMan microRNA assays (Thermo Fisher Scientific) were used to measure miR-4766-5p level with U6 small nuclear RNA (U6-snRNA) as the internal control. The relative expressions of LBX2-AS1 and CXCL5 were calculated with GAPDH as the internal control. The primers for LBX2-AS1, CXCL5 and GAPDH were listed as below: LBX2-AS1, Forward: 5′- AGT TTG TCC CAG GTT TGG CA -3′, Reverse: 5′- CAT GCC AGG GTC CTT GTT CT -3′; CXCL5, Forward: 5′-TGT GCA ATT AAC AAA GCT ACT GCA A-3′, Reverse: 5′- AGG CAT CTA AAA AGC TCA GCA ATG-3′; GAPDH, Forward: 5′- CTG GGC TAC ACT GAG CAC C-3′, Reverse: 5′- AAG TGG TCG TTG AGG GCA ATG-3′.
RNA pull down assay
Bio-miR-4766-5p-wt, bio-miR-4766-5p-mut and negative control were constructed in BGC-823 and SGC-7901 cells, and pulldown experiments were undertaken, followed by qRT-PCR to detect LBX2-AS1 enrichment. A total of 1 × 107 GC cells were harvested, lysed and sonicated. The miR-4766-5p probe was used for incubation with C-1 magnetic beads (Life Technologies) at 25 °C for 2 h to generate probe-coated beads. Cell lysate with miR-4766-5p probe or oligo probe was incubated at 4 °C for one night. After washing with wash buffer, the RNA mix bound to the beads was eluted and extracted with an RNeasy Mini Kit (QIAGEN) for qRT-PCR.
Western blot
The process of western blotting analysis was conducted as described by precious studies [
16]. The protein of sample was extracted by using RIPA Lysis Buffer containing PMSF (Solarbio, China). The protein concentration in each extract was determined using a BCA Protein Quantification Kit (Beyotime, Shanghai, China). Samples of total protein were separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes, which were subsequently blocked with 5% skim milk for 2 h. Subsequently, the membranes were incubated with primary antibodies against CXCL5 (1:1000, ab9802, Abcam, MA, USA) or GAPDH (1:2000, ab181602, Abcam, MA, USA) overnight at 4 °C and then incubated with secondary antibody labeled with HRP (Abcam) for 1 h at 37 °C after washed with TBST for four times. ECL chromogenic substrate was used to visualize the bands, and the Image J software was used to determine the gray value of each band.
Statistical analysis
All statistical analyses were analyzed using the SPSS 21.0 statistical software (SPSS, Chicago, IL) in conjunction with Prism 8.0 software (GraphPad, San Diego, CA). All experimental results were expressed as mean ± SD. The data were compared by one-way ANOVA, and a P-value < 0.05 was considered statistically to be statistically significant.
Discussion
GC is a malignant tumor that originates from the gastric mucosal epithelium, and has the highest incidence among various malignant tumors in China [
17]. Recently, more and more papers indicated that GC patients still suffer a diagnosis in late stage and a high mortality [
18]. With the rise of studies on the functional properties of lncRNAs, it has been verified that lncRNAs efficiently affect biological processes of cancers, and exerts pivotal properties [
19,
20], and process important roles in human tumors including GC.
In vivo experiments showed that the expression of LBX2-AS1 contributed to the occurrence of tumors. This suggests that LBX2-AS1 may be involved in tumor development as a tumor promoter.
In terms of mechanism, lncRNA can be used as competing endogenous RNA (ceRNA) sponge cells and play different functions together with target miRNA. LBX2-AS1 was an overexpressed lncRNA in nearly 70% of cancers, implying its huge potential in tumor regulation. In our study, bioinformatics predicted that LBX2-AS1 had a miR-4766-5p binding site, and further experimental verification indicated that miR-4766-5p functioned as a tumor suppressor in GC tissues and cells, this is consistent with previous studies on the role of miR-4766-5p in breast cancer [
9]. Inhibition of miR-4766-5p can reverse the effect of LBX2-AS1 knockout on cell anti-proliferation, anti-migration and anti-invasion. This is consistent with the results of Wei et al. study that miR-4766-5p inhibited growth, migration and invasion in GC [
21]. Our findings demonstrated that miR-4766-5p was a downstream target of LBX2-AS1 in GC.
Many studies have shown that CXCL5 promotes cancer progression. Miyazaki et al. study showed that CXCL5 production results in increased proliferation and invasion in squamous cell carcinomas [
22]. Ma et al. showed that CXCL5 promotes hepatocellular carcinoma cell proliferation, invasion, and intratumoral neutrophil infiltration [
23]. Moreover, colorectal cancer cells has been reported to secret CXCL5, which could promote cell proliferation and migration as well as invasion [
24]. In our study, CXCL5 was proved to be a target of miR-4766-5p and positively related to LBX2-AS1. Meanwhile, LBX2-AS1 knockdown was able to inhibit CXCL5 mRNA and protein expression, which can be reversed by miR-4766-5p inhibitor. CXCL5 improved cell proliferation, migration and invasion of GC cells. Overexpression of CXCL5 overturned these effects of LBX2-AS1 knockdown on proliferation, migration and invasion in GC cells.
Conclusions
Taken together, our study showed that LBX2-AS1 promoted proliferation, migration and invasion via downregulating miR-4766-5p and upregulating CXCL5 in GC cells. This provides some potential therapeutic targets for GC research and provides a basis for us to explore the role of lncRNA-miRNA functional network in cancer. All these findings might actually provide new therapeutic opportunities of GC for this newly identified regulatory signaling pathway.
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