Introduction
Gastric cancer (GC), a malignancy primarily originating from the epithelium of the gastric mucosa, presents a significant global health challenge due to its intricate etiology and high mortality rate [
1‐
4]. This complex disease is characterized by its close association with genetic mutations and irregular gene expression. Moreover, the pathogenesis and progression of GC have been directly linked to infections from certain viruses and microorganisms, most notably the Epstein-Barr virus (EBV) and
Helicobacter pylori (
H. pylori) [
5‐
7]. On a global scale, GC ranks fifth in incidence and fourth in mortality rates [
2], underlining the critical importance of investigating the molecular mechanisms underpinning its development. Understanding these mechanisms is pivotal for improving early diagnosis and therapeutic strategies for GC [
8]. The disease is disproportionately prevalent in developing nations [
9]. Notably, approximately half of these cases are found in Eastern Asia, with China representing a significant proportion. In fact, the incidence of GC in China constitutes 42.6% of the global total, while the mortality rate is at 45.0%, making China fifth in incidence and sixth in mortality among 183 nations [
4,
10‐
13].
Long noncoding RNAs (lncRNAs), a category of noncoding RNAs exceeding 200 nucleotides in length that lack protein-coding capability, have emerged as critical players in a plethora of biological processes [
14]. LncRNAs are involved in the regulation of gene expression, species evolution, embryonic development, metabolic processes, and even tumorigenesis. Certain lncRNAs are identified as potential tumor suppressors or promoters in various cancers, with their dysregulated expression often tied to the biological features of tumor cell proliferation, invasion, and metastasis [
14,
15]. Moreover, lncRNAs have been implicated in a variety of other diseases, such as cardiovascular conditions, neurological disorders, and metabolic diseases, often as a result of abnormal expression patterns [
16]. Recent studies have also demonstrated the involvement of specific lncRNAs in the immune system's biological processes, such as immune cell differentiation, cell cycle regulation, and apoptosis [
17,
18]. Therefore, the potential to harness the regulatory capabilities of lncRNAs in treating various diseases represents a burgeoning area of research.
The intricate interplay between GC and lncRNAs pivots around the complex regulation of gene expression and diverse cellular processes [
19]. Emerging evidence has implicated dysregulation of certain lncRNAs in the pathogenesis and progression of GC [
20,
21]. This dysregulation appears to interfere with critical signaling pathways implicated in cellular differentiation and apoptosis. Moreover, a subset of these lncRNAs has shown potential as diagnostic and prognostic indicators for GC [
22,
23]. However, a comprehensive understanding of the roles and therapeutic potential of lncRNAs in GC is yet to be fully elucidated. Our current study contributes to this knowledge base by identifying a novel lncRNA, XLOC_004787, demonstrating its significant upregulation in GC tissues and cell lines. Zhu H had reported that XLOC_004787 was aberrantly expressed in human gastric cells and tissues infected with
H. pylori when compared to the control group, and the aberrant expression of XLOC_004787 may contribute to the pathological response and development of
H. pylori-related diseases [
24]. Yao M had also reported that XLOC_004787 was a upstream regulatory factor of miR-107 and was logically involved in inhibiting CVB3 replication and release, as well as the resulting inflammatory responses [
25]. The development of gastric cancer is closely related to
H. pylori infection. Therefore, we explored its functional role through both silencing and overexpression in GC cells. Our results indicate that XLOC_004787 plays a key role in cell migration and proliferation and impacts EMT, metastasis, proliferation, and the expression of various signaling pathway proteins. Furthermore, we provide evidence that XLOC_004787 may regulate the progression of GC by modulating the nuclear entry of P-Smad2/3 and β-catenin. An additional observation was a decrease in the expression of mir-203a-3p, suggesting XLOC_004787's role in mediating the migration and proliferation of GC cells. This is particularly significant as previous studies, such as those by Wang Z, et al., have reported that mir-203a-3p inhibits GC cell proliferation by targeting IGF-1R [
26]. Therefore, our findings hint at the potential of XLOC_004787 as a novel therapeutic target for GC, necessitating further investigation in this promising area.
Materials and methods
Tumor tissue collection
In this study, both GC tissues and their corresponding non-tumor tissues were procured from the Affiliated People's Hospital of Jiangsu University during the period from 2017 to 2020. This study was conducted in strict accordance with the principles outlined in the Helsinki Declaration. All procedures pertaining to tissue sample collection and subsequent experimental protocols were duly approved by the Ethics Committee of Jiangsu University (Zhenjiang, China) and the Ethics Committee of the Affiliated People's Hospital, Jiangsu University. Post-collection, the GC samples were promptly immersed in TRIzol reagent and subsequently stored at -80 °C until further experimental use.
Cell culture
This study utilized normal gastric mucosal epithelial cells (GES-1) in combination with five GC cell lines (BGC-823, HGC-27, MKN-45, SGC-7901, MGC-803). These cell lines were sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and the American Type Culture Collection (ATCC, Manassas, VA, USA), and were preserved in liquid nitrogen at the School of Medicine, Jiangsu University. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) enriched with 10% fetal bovine serum (FBS; Gibco). The cell culture process was carried out in a humidified incubator set at 37 °C and supplied with 5% CO2.
Fluorescence in situ hybridization analysis (FISH) assay
Gene Pharma (Suzhou, China) was responsible for designing and synthesizing the XLOC_004787 probe conjugated with Cy3. The RNA-FISH kit was purchased from Gene Pharma. Cells were seeded onto glass coverslips, cultured overnight, and then fixed with 4% paraformaldehyde at room temperature for 30 min. After a 15-min treatment with 0.1% Triton X-100, cells were washed twice with PBS, and 200μL of 1 × hybridization buffer was added to each well, followed by incubation at 37 °C for 30 min. The liquid was discarded, and 200μL of 2 × buffer C was added and incubated at 37 °C for 30 min. The probe was diluted to 1 μM, denatured for 10 min in a water bath at 75 °C, and then 2μL of 1 μM biotin-labeled probe, 4μL of 1 μM SA-Cy3/FAM, and 14μL of PBS were added and incubated at 37 °C for 30 min. 180μL of buffer E was added afterwards. The cells were mixed with the probe mixture and incubated at 37 °C for 12–16 h for hybridization. The next day, cells were washed with 0.1% buffer F at 37 °C for 10 min, washed three times with 2 × buffer C, washed at 60 °C for 10 min each, and washed at 42 °C for 10 min each (three times in total). Finally, diluted DAPI-stained cells were added in the dark for 15 min, washed, sealed, and then observed with a fluorescence microscope (Leica, Mannheim, Germany).
RNA Isolation and qRT-PCR Assays
The extraction of total RNA from GC cells and tissues was accomplished using the TRIzol reagent (Invitrogen, ThermoFisher Scientific), with the procedure executed in accordance with the provided guidelines.The isolated total RNA was then reverse transcribed into cDNA via the Superscript Reverse Transcriptase Kit (Vazyme, Nanjing, China). The real-time PCR amplification was performed using the ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), in conjunction with the TransStart Top Green qPCR Super Mix (TRAN, China). The primers for qRT-PCR were as follows:
-
(1) GAPDH-Forward, 5′-AGGTGAAGGTCGGAGTCAAC-3′;
-
GAPDH-Reverse, 5′-GGGTGGAATCATATTGGAACA-3′;
-
(2) XLOC_004787-Forward, 5’-CTATTACTTTTCCTTTCAAGCACAC-3’;
-
XLOC_004787-Reverse, 5′-TGACAAGTATTTGCTGAGTGCCTA-3’.
The qRT-PCR protocol included an initial step at 95 °C for 5 min, followed by 40 cycles of: 95 °C for 5 s; 60 °C for 20 s, 65 °C for 1 min, and 95 °C for 15 s. The 2−ΔΔt method was subsequently employed for data quantification, designating GAPDH as the internal control.
Cell transfection
Cell transfections were carried out using siRNA-XLOC_004787 (GenePharma, Shanghai, China) and pcDNA- XLOC_004787 (Sangon biotech, Shanghai, China), implemented with Lipofectamine 3000 (Invitrogen, ThermoFisher Scientific) following the manufacturer's recommended procedures. The efficiency of the cell transfections was subsequently evaluated using qRT-PCR. The siRNA-XLOC_004787 sequences used were as follows:
-
(1) 5’-GCACACAUCUCAGUUGUUATT-3’,
-
3’-UAACAACUGAGAUGUGUGCTT-5’;
-
(2) 5’-GCAACUCUUCACUUUACUATT-3’,
-
3’-UAGUAAAGUGAAGAGUUGCTT-5’;
-
(3) 5’-GCUGCUCACACUCAUACUUTT-3’,
-
3’-AAGUAUGAGUGUGAGCAGCTT-5’.
The human gastric carcinoma cell line SGC-7901 was subjected to knockdown of XLOC_004787 via transfection with siRNA-XLOC_004787, while HGC-27 cells were transfected with pcDNA-XLOC_004787 plasmids and control plasmids. Post-transfection, the cells were detached using 0.25% trypsin, resuspended, and then enumerated. Subsequently, a population of 1 × 103 cells was seeded into six-well plates and cultured under standard conditions, with the medium being refreshed every three days over a period of 10 to 14 days. After incubation, cells were fixed with 4% paraformaldehyde and stained with crystal violet. Additionally, 1 × 103 cells were seeded into 96-well plates and maintained at 37 °C in a 5% CO2 atmosphere. The subsequent day, 10 μl CCK-8 reagent (Tongren, Shanghai, China) was added to each well at 24, 48, 72, and 96-h intervals and incubated for one hour. The absorbance of each well was recorded via enzyme-linked immunosorbent assay at a wavelength of 450 nm, with the resultant data averaged to plot a growth curve. This entire experimental procedure was performed in triplicate.
Cell migration assay
The cell migration assays were performed using Transwell chambers (pore size, 8 µm; Corning, Costar, NY, USA). Both SGC-7901 and HGC-27 cells were transfected with siRNA-XLOC_004787, pcDNA-XLOC_004787, and corresponding controls, respectively. After 48 h, the cells were resuspended in serum-free DMEM medium and counted. A volume of 600 µL of medium containing 10% FBS was added into the lower chamber as a chemoattractant. Meanwhile, a total of 1 × 105 cells in 200 µL of medium were uniformly seeded into the upper chamber. After incubation for 12 to 24 h, the chambers were immersed in 4% paraformaldehyde for 30 min at room temperature, rinsed with PBS, and the cells stained with crystal violet. The chamber was then washed again with PBS. Under a microscope, five random fields were chosen to image and count cells that migrated to the lower surface of the Transwell chamber membrane, for subsequent statistical analysis.
Western blotting analysis and antibody utilization
SGC-7901 and HGC-27 cells were lysed utilizing RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) enriched with phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitors. Uniform protein quantities (100 µg) were isolated using 10% SDS-PAGE gels, after which they were transferred onto PVDF membranes. Membranes were then blocked using 5% non-fat milk, and subsequently incubated with primary antibodies (as detailed in Table
1). Following a period of incubation, the membranes were washed and further incubated with HRP-conjugated anti-rabbit IgG (H + L) goat secondary antibodies (Fcmcs, Nanjing, China) for an hour under ambient conditions. Upon another washing cycle, protein detection was facilitated using the ECL system (Image Quant LAS 4000 mini, Pittsburgh, PA, USA) in accordance with the manufacturer's instructions.
Table 1
The antibodies used in this study
E-cadherin | 1:500 | Wanleibio |
N-cadherin | 1:500 | Wanleibio |
mmp2 | 1:500 | Wanleibio |
mmp9 | 1:500 | Wanleibio |
Snail | 1:500 | Wanleibio |
Vimentin | 1:500 | Wanleibio |
β-catenin | 1:500 | Wanleibio |
GSK3β | 1:500 | Wanleibio |
P-GSK3β | 1:400 | Wanleibio |
C-Myc | 1:400 | Wanleibio |
Cyclin D1 | 1:500 | Wanleibio |
TGF-β | 1:1000 | Wanleibio |
Smad2/3 | 1:500 | Wanleibio |
P-Smad2/3 | 1:400 | Wanleibio |
GAPDH | 1:2000 | Abcam |
Immunofluorescence analysis
Subsequent to transfection (48 h post-procedure), SGC-7901 and HGC-27 cells (both at a density of 2 × 104) were dispersed into 24-well plates that contained cell slides. Following a period of 24 h post-inoculation, the cells were fixed using a 4% poly-methyl fermentation solution for 30 min and rinsed with PBS. Permeabilization of cells was achieved with 0.5% TritonX-100 (Sigma–Aldrich, Hong Kong, China) for a period of 10 min, after which the cells were rinsed and blocked using 5% BSA for 30 min. Primary antibodies for P-Smad2/3 and β-catenin were added, followed by overnight incubation at 4 °C. The following day, cells were rinsed with PBS, then treated with Cy3 labeled goat anti-rabbit IgG antibody (Huabio, Hangzhou, China) and incubated for 45 min at 37 °C in dark conditions. A final rinse with PBS was done before adding 0.5 ng/ml DAPI for 10 min. Upon washing with PBS, cells were sealed with an anti-fluorescence quencher, followed by observation under a confocal laser scanning microscope.
Dual luciferase reporter assay
GenePharma Co. (Suzhou, China) constructed both the wild type (WT) and mutant (MUT) XLOC_004787 plasmids. Human embryonic kidney (HEK 293 T) cells, at a density of 1.0 × 105 cells/well, were cultured in a 24-well plate. Subsequently, the cells were co-transfected with either the WT or MUT plasmids and mir-203a-3p mimics (GenePharma, Shanghai, China) using Lipofectamine 3000 as a transfection reagent. After a co-transfection period of 36 h, both renilla and firefly luciferase activities were quantified. The ratio of these activities was then used to evaluate the interaction between XLOC_004787 and mir-203a-3p.
Statistical analysis
Statistical analyses for this research were conducted using GraphPad Prism 8.2 software (La Jolla, USA). Image Pro Plus software (Media Cybernetics, USA) was used to obtain the relative gray-scale value of the bands and to perform cell counting. Independent t-tests were utilized for comparisons among different groups. Each experiment was independently conducted three times. All statistical tests were two-tailed, with a threshold of p < 0.05 set as the criteria for statistical significance.
Discussion
The interplay between lncRNAs and various diseases, particularly cancers, is intricate and multifaceted [
16,
18]. A surge of research within the last decade has unveiled the dual role of lncRNAs, acting as potential oncogenes or tumor suppressors in the development of cancer [
27]. Their dysregulation is often associated with pivotal biological characteristics including tumor cell proliferation, invasion, and metastasis [
18]. Specific lncRNAs such as MALAT1, HOTAIR, and UCA1, are found to be highly expressed in GC and are implicated in lymph node metastasis, pathological grading, and recurrence rates [
28‐
30]. Conversely, lncRNAs like SNHG5 and LINC00675 are known to behave as suppressors of GC, with their diminished expression linked to an increase in GC cell proliferation, invasion, and metastasis [
31,
32]. While we have gained substantial insight into lncRNA regulatory mechanisms and their various biological roles in the human body, the discovery and characterization of novel functional lncRNAs remain a vibrant area of research. In this study, we identified a novel lncRNA, XLOC_004787, through chip-based screening and subsequently verified its full length using sequencing techniques. Previous studies on XLOC_004787 have focused on its role in certain viruses, such as the Coxsackie B3 virus (CVB3) [
25], but its function and specific involvement in GC remain unclear. Our findings indicate elevated expression of XLOC_004787 in both GC tissues and cell lines. Moreover, we observed that an upregulated expression of XLOC_004787 in HGC-27 cells promoted cell migration and proliferation. Conversely, the suppression of XLOC_004787 in SGC-7901 cells led to the inhibition of these same processes.
The heightened expression of XLOC_004787 could potentially modulate mmp9 and mmp2, thereby influencing the migratory patterns and proliferation of GC cells. We observed the phenomenon of EMT in numerous malignant tumors, which gradually morphs tightly bound epithelial cells into more invasive mesenchymal cells. It should be noted that early onset of EMT in tumors has the potential to modify the tumor cell microenvironment, including factors like inflammation, fibrosis, and neovascularization [
33]. In malignant tumors, alterations in epithelial cells are typically marked by the reduction of E-cadherin, an epithelial cell marker protein, coupled with an increase in the expression of N-cadherin, Snail, and Vimentin [
33,
34]. Our findings indicate that overexpression of XLOC_004787 enhances the levels of N-cadherin, Snail, and Vimentin, while concurrently lowering the expression of E-cadherin in HGC-27 cells.
Additionally, the TGF-β signaling pathway, known to influence cell cycle, differentiation, extracellular matrix synthesis, and tumor immune response, plays a pivotal role [
35]. The Wnt/β-catenin signaling pathway has been demonstrated to be integral in embryonic development, tissue regeneration, and particularly in cancer development. Aberrant expression of the Wnt/β-catenin signaling pathway has been associated with various cancers, including colon, breast, and liver cancers [
36,
37]. This abnormal activation of the Wnt/β-catenin signaling pathway can foster proliferation, invasion, and metastasis of cancer cells during cancer progression [
38]. Our results show that XLOC_004787 may mediate the proliferation of GC cells via the TGF-β and Wnt/β-catenin signaling pathways. The findings of our study revealed that knockdown of XLOC_004787 substantially attenuated the migration, proliferation, and EMT of GC cells. Conversely, overexpression of XLOC_004787 resulted in an inverse outcome. In HGC-27 cells overexpressing XLOC_004787, the concentration of TGF-β, P-Smad2/3, C-myc, CyclinD1, P-GSK3β, and β-catenin increased, with higher nuclear localization of P-Smad2/3 and β-catenin. In contrast, in SGC-7901 cells exhibiting downregulated XLOC_004787 expression, the levels of TGF-β, P-Smad2/3, C-myc, CyclinD1, P-GSK3β, and β-catenin diminished, and P-Smad2/3 and β-catenin were less abundant in the nucleus.
In the human body, the regulation of lncRNAs operates within an intricate network, and their interaction with microRNAs (miRNAs) is multifaceted. LncRNAs have been observed to function as “miRNA sponges [
27],” serving to absorb and thereby modulate miRNA expression—an interaction denoted as the “miRNA sponge effect.” This interaction impedes miRNA binding to their intended target RNAs, thereby influencing their expression [
39]. Conversely, miRNAs are capable of altering the expression and function of lncRNAs by influencing their transcription or splicing processes [
40]. In the scope of this study, it was predicted and substantiated that the lncRNA XLOC_004787 targets the downstream miRNA, miR-203a-3p. The results showed that XLOC_004787 and miR-203a-3p share sequence complementarity and exhibit reciprocal regulation. Upon overexpressing miR-203a-3p, we noted a reduction in GC cell proliferation and migration, accompanied by the inhibited expression of EMT-related proteins, the TGF-β signaling pathway, and the Wnt/β-catenin signaling pathway. Simultaneously, there was an augmentation in E-cadherin expression. Conversely, the inhibition of miR-203a-3p produced the inverse results. Mir-203a-3p has been previously reported as a tumor suppressor gene targeting IGF-1R [
26], thus this study further clarifies this relationship.
The main limitations of this study are its small inclusion of clinical subjects and the lack of in vivo experiments due to existing laboratory and equipment constraints. For a more comprehensive understanding of this lncRNA's applicability as a potential biomarker, it would be prudent to involve larger patient groups in subsequent clinical trials. Furthermore, the application of inhibitors or inducers related to the Wnt/β-catenin and TGF-β signaling pathways could further elucidate the role of XLOC_004787 in promoting GC cell proliferation and migration.
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