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. 2024 Jan 9;22(23-24):2522–2537. doi: 10.1080/15384101.2023.2289745

The role of lncRNA SNHG14 in gastric cancer: enhancing tumor cell proliferation and migration, and mechanisms of CDH2 expression

Zhou-Tong Dai a,b,c, Yong-Lin Wu d, Tao Xu d, Xing-Rui Li d,✉, Teng Ji a,b,✉
PMCID: PMC10936682  PMID: 38193271

ABSTRACT

LncRNAs are a class of non-coding RNAs that play an important role in regulating gene expression. However, their specific molecular mechanisms in gastric carcinogenesis and metastasis need further exploration. TCGA data showed that the expression of MFGE8, which was closely related to survival, was significantly positively correlated with lncRNA SNHG14. And moreover, the results of high-throughput sequencing and qRT-PCR showed that lncRNA SNHG14 was significantly elevated in gastric cancer. Further, in vitro functional realization showed that lncRNA SNHG14 overexpression significantly increased gastric cancer’s proliferation, invasion and migration. Animal experiments also showed that lncRNA SNHG14 overexpression promoted tumorigenesis and metastasis in vivo. Mechanistically, MFGE8 activates the expression of lncRNA SNHG14, which activates the cellular EMT by stabilizing CDH2. Our study suggests that lncRNA SNHG14 could be a potential target for gastric cancer therapy.

KEYWORDS: MFG8, LncRNA SNHG14, gastric cancer, migration

Simple Summary

Gastric cancer is one of the malignant tumors with a high incidence and high mortality rate worldwide. The current treatment modalities for gastric cancer are surgery, chemotherapy and targeted therapy. However, the 5-year survival rate of gastric cancer patients is still less than 30%. The main reason for the low survival rate of gastric cancer patients is that most cases are already at an advanced disease stage when first diagnosed, with tumor metastasis, tumor heterogeneity and resistance to radiotherapy. TCGA data showed that the expression of MFGE8, which was closely related to survival, was significantly positively correlated with lncRNA SNHG14.We found that lncRNA SNHG14 expression was significantly elevated in gastric cancer by high-throughput sequencing. It was further confirmed in vitro and in vivo that overexpression of lncRNA SNHG14 promoted the proliferation and migration ability of gastric cancer. Mechanistically, lncRNA SNHG14 played an oncogene role by promoting CDH2 expression to activate EMT in tumor cells.

Introduction

Gastric cancer (GC) is the most common malignant tumor of the digestive system that ranks fourth in incidence and second in mortality worldwide [1,2]. However, the survival rate for early detection and treatment of gastric cancer is over 90%. However, due to the lack of specific and effective early diagnosis and screening methods, most patients have developed into middle and late stages by the time they are diagnosed. Moreover, some of them even have distant metastases. They lose the best time for surgical treatment [3,4]. There are many forms of treatment for patients with progressive gastric cancer in clinical practice. The five-year survival rate after treatment is still less than 30% [1,5]. Therefore, an in-depth study of the mechanisms of the development and metastasis of gastric cancer is essential for developing new prevention and treatment measures.

Milk fat globule-epidermal growth factor-factor 8 (MFGE8), also known as lactadherin/BA46/SED1, as a lipophilic glycoprotein on the surface of milk fat globules was first identified in 1990 [6]. It did not attract much attention from researchers at first. However, more and more recent studies have shown the involvement of MFGE8 as an oncogene in tumorigenesis and progression [7–10]. Overexpression of MFGE8 induced the expression of Cyclin D1/D3, which significantly promoted the proliferation of breast cancer cells [8]. Subsequently, MFGE8 was also shown to be elevated in mouse models of melanoma, enhancing tumorigenicity and tumor metastasis through Akt and Twist signal pathways [9]. Besides, Tibaldi et al. [10] identified MFGE8 as a new therapeutic target in ovarian cancer. Based on the above findings, it was speculated that MFGE8 could be closely related to the development and metastasis of gastric cancer. However, there was no report on the relationship between MFGE8 and tumor development in gastric cancer. MFGE8 regulated the biological function of gastric cancer cells and needs to be further elucidated.

Non-coding RNAs are RNA sequences that do not encode proteins, and they were initially thought to be “Transcriptional Noise” [11]. As research progressed, it was discovered that noncoding RNA was an important modulator of cellular life activities [12]. The aberrant expression of non-coding RNAs was associated with the development of many malignancies [13]. As a member of the non-coding RNA family, the abnormal expression of lncRNA SNHG14 has been shown to regulate the development and progression of various malignancies. WANG et al. [14] found that lncRNA SNHG14 expression was significantly increased in endometrial cancer tumor tissues. Its expression was significantly correlated with tumor size, pathological stage and poor prognosis of patients. ZHAO et al. [15] also found that lncRNA SNHG14 enhanced the migration and invasive ability of ovarian cancer cells through up-regulating Dgcr8 expression. DENG et al. [16] found that SNHG14, as a ceRNA of miRNA, inhibited the expression of ANXA2 and promoted pancreatic cancer cell proliferation, invasion, and inhibition of its apoptosis. The role of lncRNA SNHG14 in some cancers has been revealed, but gastric cancer remains unknown.

In this study, our objective was to investigate the role of MFGE8 and lncRNA SNHG14 in gastric cancer and to explore their potential mechanisms. We planned to examine the association between MFGE8 and lncRNA SNHG14 expression and the proliferation and migration of gastric cancer cells. To achieve this, we analyzed the expression of them in gastric cancer tissues and cells, and evaluated their correlation with patient prognosis. Furthermore, we conducted in vivo and in vitro functional experiments to assess the impact of their expression on gastric cancer cell proliferation and migration. Through this research, we hoped to contribute to a better understanding of the roles of MFGE8 and lncRNA SNHG14 in gastric cancer and to provide a foundation for future investigations into the mechanisms of gastric cancer metastasis.

Materials and methods

Cell culture

The human fetal gastric mucosa epithelium cell line GES-1 and the human gastric cancer cell lines MGC-803, SGC-790, AGS and MKN-45 were purchased from the Chinese Academy of Medical Sciences Cell Bank (China). GES-1 and AGS were cultured in DMEM (Gibco, USA) that was supplemented with 10% fetal bovine serum (BI, Australia) and 1% penicillin-streptomycin (Gibco, USA). MGC-803, SGC-790 and MKN-45 were cultured in RPMI-1640 (Gibco, USA) which was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, USA). All cells were cultured at 37°C in an incubator under a 5% CO2 environment.

Clinical sample collection

The 12 gastric cancer and paracancerous tissue specimens were collected from the 2019–2021 specimen bank of the Department of Digestive Surgery, Tongji Medical College, Huazhong University of Science and Technology. All specimens were pathologically confirmed to be gastric cancer or stomach tissue. All patients have obtained informed consent and signed relevant informed consent forms. Meanwhile, it has been certified by the Ethics Committee of Huazhong University of Science and Technology.

Overexpression and knockdown cell lines construction

The three-plasmid lentiviral packaging system was used according to the instructions (pLKO.1, VSVG, GAG-POL). Briefly, lentiviral plasmids were transfected to produce lentiviral particles in HEK-293 T to construct stable cell lines. Then, lentiviral particles were added to the cell lines. Moreover, puromycin was used to screen the target cell lines. The lentiviral plasmids were designed and synthesized by Shanghai Sangon Biotechnology Co., LTD. Meanwhile, the mRNA sequence of CDH2 referred to the Homo sapiens cadherin 2 (CDH2), transcript variant 1, mRNA sequence from the NCBI database (NM_001792.5). The lncRNA SNHG14 sequence was also obtained from the NCBI database (NR_146177.1). The MFGE8 sequence was also obtained from the NCBI database (NP_005919.2).

Western blot assay

Cells were lysed in RIPA lysis buffer (Beyotime, China). The cell lysates were quantified with BCA assay (Beyotime, China). One Step PAGE Gel Fast Preparation Kit (Vazyme, China) is used to prepare SDS-PAGE. Subsequently, Electrophoresis was performed at 100 volts. And the wet transfer to PVDF membranes (Millipore, USA) was performed at 200 mA. 5% skim milk was used to seal PVDF membranes. And the PVDF membranes were incubated with the first antibody and secondary antibody at room temperature. Proteins were visualized using ECL (Beyotime, China). The sequences of used antibodies were indicated as below: β-Actin (AC006, ABclonal, China), Vimentin (A19607, ABclonal, China), CDH1 (A3044, ABclonal, China), CDH2 (A12322, ABclonal, China).

qRT-PCR Assay

Following the manufacturer’s instructions, the RNeasy Mini Kit (QIAGEN, Germany) was used to extract total RNA. HiScript® III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China) was used to transcribe RNA into cDNA reversely. Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China) was used for qRT-PCR. The primers involved were synthesized by Shanghai Sangon Biotechnology Co., LTD. The sequences of used primers were indicated as below: β-Actin F: 5'-GTGGGGCGCCCCAGGCACCA-3'; β-Actin R: 5'-CTCCTTAAT-GTCACGCACGATTTC-3'; MFGE8 F: 5'-CCTGCCACAACGGTGGTTTAT-3'; 5'-CACATTTCGTCTCACAGTGGTT-3'. The lncRNA SNHG14 primers were designed by Shanghai Sangon Biotechnology Co., LTD.

Cell proliferation assay

CCK-8 assay: The CCK-8 kit (Vazyme, China) was used to detect the proliferation capacity of cells. Cells in 96 well plates were added with 10% CCK-8 solution and incubated for 1 h in an incubator. Then, the OD value of cells was recorded at 450 nm with the microplate reader (Thermo Scientific, USA).

Colony formation assay: Briefly, the cells were seeded in 6-well plates at low density. Then, the plate was cultured in an incubator for 14 days. The cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 min and stained with 0.05% crystal violet (Beyotime, China) at room temperature for 30 min. The number of clones larger than 50 was evaluated with the microscope (Olympus, Japan).

Scratch wound healing assay

Cells were seeded at a high density in six well plates. The 20 µL gun head was used to draw a straight line evenly on the cell layer. Meanwhile, the medium was replaced with a serum-free medium. The cells were photographed After continuous culture for 48 hours. The plates were photographed using a microscope (Olympus, Japan).

Transwell assay

For migration assay: Cells were seeded at a medium density in the upper cavity of the transwell chamber (Corning, USA). The serum-free medium was added to the upper chamber. The medium containing fetal bovine serum was added to the lower chamber after 24 h of incubation. The cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 minutes and stained with 0.05% crystal violet (Beyotime, China) at room temperature for 30 minutes. The positively stained cells were evaluated with the microscope (Olympus, Japan). For invasion assay: Matrigel (BD Biosciences, USA) was pre-inoculated in the upper chamber 24 hours prior to the experiment. Subsequent procedures followed the migration assay protocol, with the exception of the Matrigel pre-inoculation step.

Sequencing assay

The sequencing assay was entrusted to Wuhan Sevicebio Co., Ltd. The Illumina HiSeq™ 2000 platform was used for sequencing. The quality control, depth and coverage of sequencing data were provided in the supplementary materials.

RNA immunoprecipitation (RIP)

For RIP, we utilized the RNA Immunoprecipitation (RIP) Kit (Magna, USA) according to the manufacturer’s instructions. In brief, cells were collected and lysed in complete RIP lysis buffer. The cell lysates were then incubated with magnetic beads pre-coated with CDH2 antibodies specific for the protein of interest or IgG as a negative control. After incubation, the beads were washed and the co-precipitated RNA was isolated. The qRT-PCR was used to analyze the co-precipitated RNA.

RNA pull-down assay

The MGC-803 or SGC-7901 cells were subjected to lysis via a protein lysis buffer (Thermo Fisher, USA), and the lncRNA SNHG14 (Bio-SNHG14) was labeled with biotin, using biotinylated random probes as a negative control. The biotin-conjugated probes were then incubated with magnetic beads for a duration of 2 hours. Following this, the probes were allowed to interact with the cell lysis buffer overnight at a temperature of 4°C. Post-incubation, the RNA bound to the probes was washed with washing buffer and subsequently analyzed via qRT-PCR. The biotin-conjugated probes were designed by Shanghai Sangon Biotechnology Co., LTD.

Xenograft mice model

The nude mice (4 weeks) were purchased from the Beijing Huafukang Bioscience Corporation. The purchased nude mice were raised in the SRF barrier system of the Experimental Animal Center of Wuhan University of Science and Technology. The Animal Ethics Committee of Wuhan University of Science and Technology inspected and approved the animal experiments. Briefly, nude mice were randomly divided into negative control and experimental groups. 1 × 106/mL of log phase cells were inoculated into the subcutaneous area on the back of nude mice. A subcutaneous transplantation tumor model was constructed in nude mice. The tumor length and width were measured every 3 days using vernier calipers, and the weight of the nude mice was measured by the analytical balance. The nude mice were euthanized on day 21. Subsequently, the subcutaneous tumor tissue was peeled off. The tumors were weighed and their volumes were calculated. Tumor volume = length × width × width/2. For survival studies, the tumor size and survival of mice were monitored every five days. Ninety days after subcutaneous xenograft tumors were implanted, all mice were euthanized. Each group consisted of seven mice. Survival analysis was conducted using GraphPad Prism software.

Lung metastasis models

To establish a mouse lung metastasis model, cells were resuspended in phosphate-buffered saline (PBS) at a concentration of 1 × 107 cells/mL. The nude mice (4 weeks) were anesthetized, and 100 μL of the cell suspension containing 1 × 106 cells was injected into the lateral tail vein using a BD U-40 needle.

After 14 days, mice were euthanized, and lungs were harvested for analysis. Lungs were fixed in formalin, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was performed to visualize and quantify lung metastases.

Immunohistochemistry assay

The tissues were sequentially fixed, dehydrated, clear, wax-impregnated and embedded. Sections (4–5 μm) were cut from paraffin blocks using a microtome. Next, the tissue sections were dewaxed, hydrated, antigen repaired, antigen repaired, blocking, primary and secondary antibody incubation, and DAB color development. Instruments and reagents used in the experiment were purchased from Wuhan Sevicebio Co., Ltd.

Bioinformatics assay

R software (Version 3.6.3) is used for bioinformatics analysis. Differential genes analysis was performed by limma package. The TCGA data were obtained from the official website (https://tcga-data.nci.nih.gov).

Statistical assay

SPSS software was used for the statistical analysis. Unless indicated in the illustration. All data were presented as mean ± standard deviation from three independent repeats. Student’s t-test and one‐way ANOVA were used to compare the differences. The p value that was less than 0.05 was considered statistically significant.

Result

Expression of MFGE8 is elevated in gastric cancer

Previous studies have documented that MFGE8 was aberrantly expressed in specific cancers [10,17,18]. However, whether MFGE8 was abnormally expressed in gastric cancer is not known. Therefore, the 12 pairs of cancer and paracancerous tissues from patients with gastric cancer were collected. Immunohistochemical staining showed that MFGE8 was up-regulated in the tumor tissue samples (Figure 1a, 1). Next, the expression of MFGE8 mRNA was further analyzed by qRT-PCR. The results also showed the same results (Figure 1c). Unfortunately, the prognostic survival data for the gastric patients in this trial was collected. The expression data and survival data of STAD patients were downloaded from the official website of the TCGA database. The STAD patients from TCGA were subsequently divided into two groups (high expression or low expression group) according to the median value of MFGE8. The results are shown in figure (Figure 1d). The overall survival was worse in the high-expression group. Furthermore, clinical data on gastric cancer patients were obtained from the TCGA database. Univariate Cox analysis was employed to evaluate the potential utility of MFGE8 expression as a prognostic marker in gastric cancer. The results revealed that MFGE8 expression (HR = 1.28, P = 0.03), Stage (HR = 1.54, P < 0.01), T stage (HR = 1.30, P = 0.03), M stage (HR = 2.05, P = 0.02), and N stage (HR = 1.26, P < 0.01) were the primary factors associated with cancer-related mortality. Moreover, in the multivariate Cox analysis, only MFGE8 expression (HR = 1.32, P = 0.03) remained significantly correlated with poor prognosis in gastric cancer (Figure 1e). These results suggested that MFGE8 as an oncogene was involved in the development and progression of gastric cancer.

Figure 1.

Figure 1.

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Expression of MFGE8 Is Elevated in Gastric Cancer. a. Representative images of IHC assays on MFGE8 expression in gastric cancer. b. IHC score for the level of the MFGE8 expression in 12 gastric cancer tissues. c. The relative expression of MFGE8 mRNA in 12 pairs of paraneoplastic and gastric cancer tissue samples. β-actin was served as an internal control. Red represented upregulated expression. Green represented downregulated expression. Gray represented insignificant change. d. The Kaplan-Meier overall survival between high and low expression of MFGE8 group from TCGA. e. The multivariate Cox regression assays of MFGE8 expression with other clinical pathological features (Age, Gender, Grade, T stage, M stage and N stage). *p < 0.05, **p < 0.01 and ***p < 0.001.

MFGE8 promotes gastric cancer migration and invasion in vitro

To investigate the biological function of MFGE8 we initially scrutinized its expression across various cell lines. The results showed that the level of MFGE8 was higher in human gastric cancer cell lines than in human gastric mucosal epithelial cells GES-1 (Figure 2a). Among these gastric cancer cell lines, the highest expression of MFGE8 was found in MGC-803 and SGC-7901 cell lines. Therefore, these two cell lines were selected for subsequent biological function assays. Further, two stable MGC-803 and SGC-7901 human gastric cancer cell lines with MFGE8 knockdown were constructed. The knockdown of MFGE8 reduced the proliferation ability of MGC-803 and SGC-7901 cells by CCK-8 assay (Figure 2b). Besides, the colony formation assays showed the same results (Figure 2c). Next, the effect of altered MFGE8 expression on the migratory capacity of human gastric cancer cells was also evaluated. The transwell and wound healing assays showed that the migration capacity of gastric cancer was significantly reduced after the knockdown of MFGE8 (Figure 2d, 2).

Figure 2.

Figure 2.

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MFGE8 Promotes Gastric Cancer Migration and Invasion in Vitro. a. The expression of MFGE8 mRNA was detected via qRT-PCR assay. b. The cell viability was detected via CCK8 assay. c. The cell viability was detected via colony formation assay. d. The migration ability of cell was detected via scratch wound healing assay. e. The migration ability of cell was detected via transwell assay. *p < 0.05 and **p < 0.01.

MFGE8 promotes gastric cancer migration and invasion in vivo

To further investigate the role of MFGE8 in vivo. The SGC-7901 cell line with stable knockdown of MFGE8 was used to construct a subcutaneous tumor formation assay and lung metastasis model in nude mice so that the influence of MFGE8 on the tumorigenic ability of tumor cells and lung metastasis could be observed. In contrast, the volume and weight of subcutaneous tumors in the control group were significantly larger than those in the MFGE8 knockdown group (Figure 3a-d). Besides, the IHC result showed that MFGE8 and Ki67 expression levels were significantly higher in the control group (Figure 3e). Subsequently, H&E staining was used to determine the number of lung metastases in different groups (figure 3f-h). In our lung metastasis model, the number of metastatic lung nodules in was MFGE8 knockdown group significantly lower than in the control group. In summary, our results showed that the knockdown of MFGE8 significantly reduces the proliferation and metastatic ability of gastric cancer.

Figure 3.

Figure 3.

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MFGE8 Promotes Gastric Cancer Migration and Invasion in Vivo. a. Representative images of subcutaneous tumors formed in nude mice. b. The growth curve of subcutaneous tumors formed in nude mice. c. The weight of subcutaneous tumors formed in nude mice. d. The volume of subcutaneous tumors formed in nude mice. e. Representative images of immunohistochemistry staining for Ki67. f. Representative bioluminescence images of lung metastasis of nude mice model. g. Representative images of H&E stained lung metastatic nodules. h. The number of lung metastatic nodules in each group. *p < 0.05.

MFGE8 regulates LncRNA SNHG14 expression

An increasing number of studies have shown that aberrant lncRNA expression was closely associated with cancer progression or metastasis. However, it was unclear whether MFGE8 could also be involved in regulating the progression and metastasis of gastric cancer through lncRNAs. Therefore, the knockdown of MFGE8 in gastric cancer cell lines combined with high-throughput sequencing was used to explore the possible mechanisms of MFGE8 in regulating gastric cancer proliferation and migration (Figure S1). The differential expression analysis screened a total of 93 lncRNAs, of which 29 lncRNAs were up-regulated and 64 lncRNAs were downregulated (Figure 4a, 4). Moreover, combined transcriptome with GO functional enrichment analysis, it was found that the GO enrichment was in cell-substrate adhesion and extracellular matrix (Figure 4c). KEGG functional enrichment analysis revealed that MFGE8 was involved in the regulation of tumor progression through the following potential pathways: focal adhesion, ECM−receptor interaction, and cell adhesion molecules (Figure 4d). The above results indicated that MFGE8 could regulate the proliferation and metastasis of gastric cancer and may also play this role by regulating the expression of lncRNA. Taking into account the fold change in expression, the top 10 lncRNAs with alterations were identified as potential regulatory lncRNAs of MFGE8. Subsequently, qRT-PCR analysis was performed to validate these candidate lncRNAs. Among them, only the expression of lncRNA SNHG14 was detected and found to be significantly elevated in gastric cancer (Figure 5a). Besides, the clinical information of STAD patients in the TCGA database was combined. The overall survival rate was worse in the lncRNA SNHG14 high-expression group (Figure 5b).

Figure 4.

Figure 4.

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The screening of target lncRNAs for MFGE8. a. The heat map of differential lncRNA. b. The volcano plots of differential lncRNA. c. The functional enrichment map of the GO assay. d. The functional enrichment map of the KEGG assay. The screening conditions for differential lncRNA were set to the p value < 0.05 and logFC value > 1. The GO function enrichment screening condition was set to qvalueFilter < 1 × 10−8. The KEGG function enrichment screening condition was set to qvalueFilter < 0.05.

Figure 5.

Figure 5.

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MFGE8 Regulates LncRNA SNHG14 Expression. a. The relative expression of lncRNAs in the control and MFGE8 knockdown groups. b. The Kaplan-Meier overall survival between high and low expression of lncRNA SNHG14 group from TCGA. c. The correlation analysis for MFGE8 and lncRNA SNHG14. d. The correlation analysis for CDH2 and lncRNA SNHG14. **p < 0.01.

Moreover, we also analyzed the correlation between the expression of lncRNA SNHG14 and both MFGE8 and CDH2 in STAD patients from the TCGA database. The results revealed a significant positive correlation between the expression of lncRNA SNHG14 and both MFGE8 and CDH2 (Figure 5c, 5).

LncRNA SNHG14 promotes gastric cancer migration and invasion

Furthermore, three siRNAs were designed to knock down the expression of lncRNA SNHG14 in gastric cancer cell lines MGC-803 and SGC-7901. The results showed that only siRNA-2 showed a good knockdown effect (Figure 6a). On this basis, the results of in vitro proliferation and migration capacity analysis showed that the knockdown of lncRNA SNHG14 significantly reduced these capabilities of the gastric cell line (Figure 6b-e). To further verify that MFGE8 affects cell proliferation and migration ability by influencing the expression of lncRNA SNHG14. We knocked down the expression of MFGE8 in the cell lines while overexpressing lncRNA SNHG14. The results showed that overexpression of lncRNA SNHG14 in the cell lines partially restored the proliferation and migration inhibition effect caused by MFGE8 knockdown (figure 6f, 6). Simultaneously, similar results were displayed in the subcutaneous xenograft model in nude mice. Knockdown of MFGE8 expression significantly reduced tumor growth, and further overexpression of lncRNA SNHG14 could partially inhibit the effect of MFGE8 knockdown on tumor growth suppression (Figure 6h-j). Additionally, we conducted survival studies in mice. As shown in the Figure 6k (Figure 6k), in the control group, two animals eventually died; in the MFGE8 control group, three animals died; in the lncRNA SNHG14 group, no animal deaths were observed; in the MFGE8 knockdown group, no animal deaths were observed; while in the group with further overexpression of lncRNA SNHG14, one animal died. The above results showed that MFGE8 promotes migration and invasion by activating lncRNA SNHG14 in gastric cancer.

Figure 6.

Figure 6.

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LncRNA SNHG14 Promotes Gastric Cancer Migration and Invasion. a. The efficiency of knockdown of lncRNA SNHG14 was detected via qRT-PCR assay. b. The cell viability was detected via CCK8 assay. c. The cell viability was detected via colony formation assay. d. The migration ability of cell was detected via scratch wound healing assay. e. The migration ability of cell was detected via transwell assay. f. The cell viability was detected via colony formation assay. g. The migration ability of cell was detected via scratch wound healing assay. (Group I, NC-MFGE8 + NC lncRNA; Group II, sh-MFGE8 + NC lncRNA; Group III, NC-MFGE8 + lncRNA SNHG14; Group IV, sh-MFGE8 + lncRNA SNHG14). h. Representative images of subcutaneous tumors formed in nude mice. i. The weight of subcutaneous tumors formed in nude mice. j. The volume of subcutaneous tumors formed in nude mice. k. The Kaplan-Meier survival curve of nude mice. *p < 0.05, **p < 0.01 and ***p < 0.001.

LncRNA SNHG14 promotes the expression of CDH2

A potential binding site between a lncRNA SNHG14 and CDH2 mRNA was identified via bioinformatics analysis (Figure 7a). Following the establishment of a cell line with stable overexpression of lncRNA SNHG14 (Figure 7b), it was observed that the overexpression of lncRNA SNHG14 led to a significant increase in CDH2 expression (Figure 7c). The RIP and RNA pull-down experiments were employed for further investigations (Figure 7d, 7). These experimental results further confirmed the binding of lncRNA and CDH2 mRNA. To further verify that lncRNA SNHG14 affects the proliferation and migration ability of gastric cancer cells by influencing the expression of CDH2. We overexpressed lncRNA SNHG14 in the cell lines while knocking down CDH2 expression using siRNA. The results demonstrated that overexpression of lncRNA SNHG14 significantly increased the proliferation, migration, and invasion capabilities of gastric cancer cell lines, and rescue experiments revealed that overexpression of lncRNA SNHG14 followed by further knockdown of CDH2 led to a decrease these abilities. This implied that CDH2 expression partially contributed to the reduced cell proliferation and migration ability mediated by lncRNA SNHG14 (figure 7f-h). The above results provide theoretical support to explain further the effects of MFGE8 and lncRNA SNHG14 on cell proliferation and migration.

Figure 7.

Figure 7.

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LncRNA SNHG14 Promotes the Expression of CDH2. a. The potential site of prediction of lncRNA SNHG14 binding to CDH2 via bioinformatics analysis. b. The efficiency of overexpression of lncRNA SNHG14 was detected via qRT-PCR assay. c. The expression of CDH2 protein was detected via WB assay. d-e. RNA pull-down and RIP assays were conducted to verify the binding of lncRNA SNHG14 to CDH2 in gastric cancer cells. f. The invasion ability of cell was detected via transwell assay. g. The cell viability was detected via colony formation assay. h. The migration ability of cell was detected via transwell assay. (Group I, NC-CDH2 + NC lncRNA; Group II, NC CDH2 + lncRNA SNHG14; Group III, sh-CDH2 + NC lncRNA; Group IV, sh-CDH2 + lncRNA SNHG14). *p < 0.05, **p < 0.01 and ***p < 0.001.

Discussion

Gastric cancer is usually treated clinically with various methods, including surgery, chemotherapy and radiotherapy [19]. However, most gastric cancer patients were found to be advanced and metastatic and could not be effectively treated with conventional interventions [20]. With in-depth research on the proto-oncogenes and oncogenes of gastric cancer, more and more targeted gene therapy related to gastric cancer have been discovered [21]. Targeted gene therapy has become a hot spot for research in recent years. However, the effect of targeted gene therapy still did not meet the expectation, and several studies did not show significant positive results [22,23]. Therefore, it was essential further to investigate the mechanism of gastric cancer progression and metastasis. In this study, MFGE8 expression was elevated in gastric cancer tissues. Moreover, combined with the clinical information of gastric cancer patients in the TCGA database. It was found that the overall survival rate was lower in the MFGE8 high-expression group. This study helped finds a new therapeutic target for gastric cancer.

MFGE8 was first identified in the milk fat globuli of mouse breast epithelial cells as a lipophilic glycoprotein on the surface of the milk fat glomerule [24,25]. Initially, it was not taken seriously by researchers. It was not until 2002 that a study in Nature succeeded for the first time in finding that MFGE8 enhanced phagocytosis of apoptotic cells by macrophages [26]. Subsequently, more and more recent studies have shown that MFGE8 regulates the life activities of cells. Including regulating the innate immune system [27], angiogenesis [28] and cancer [17]. In fact, the expression of MFGE8 was widely expressed in most organs [29]. Carrascosa et al. [8] found that MFGE8 was expressed in breast cancer cell membranes, cytoplasm, and nuclei through the IHC assay. Moreover, overexpression of MFGE8 significantly promoted the proliferation of breast cancer cells. Sugano et al. [30] also found through IHC that the expression of MFGE8 in bladder cancer was elevated, and its expression amount was closely related to clinical stage and malignancy. In addition, Tibaldi et al. [10] found that blockade with MFGE8-neutralizing antibodies in ovarian cancer significantly inhibited tumor progression. It suggested that MFGE8 is expected to be a new target for tumor therapy. However, the study of MFGE8 and gastric cancer was not yet been reported. Therefore, in this study, the expression of MFGE8 in gastric cancer patients was explored by IHC and qRT-PCR. The results showed that the expression of MFGE8 in gastric cancer tissues was elevated. Subsequently, our analysis integrating GO and KEGG functional enrichment revealed a significant association with cell-substrate adhesion and extracellular matrix, suggesting that MFGE8 may regulate these processes. Moreover, KEGG enrichment analysis indicated potential involvement of MFGE8 in the modulation of tumor progression through pathways such as focal adhesion, ECM-receptor interaction, and cell adhesion molecules. Importantly, these pathways have been well-documented to play critical roles in the regulation of cancer cell migration, invasion, and metastasis. Consequently, we hypothesized that MFGE8 participates in the regulation of cell proliferation and migration processes [31]. Subsequently, the effect of altered MFGE8 expression on the proliferation and migration of gastric cancer cell lines was investigated in vivo and in vitro. The CCK-8, colony formation, scratch healing and transwell assays showed that knockdown of MFGE8 expression significantly reduced the proliferation or migration capacity in two gastric cancer cell lines, MGC-803 and SGC-7901. Similarly, subcutaneous inoculation of nude mice or tail vein injection of mice with knockdown of MFGE8 in gastric cancer cell lines revealed slower tumor growth and fewer metastatic foci. Our results suggested that MFGE8 played an oncogene in gastric cancer. This result was consistent with previous studies, which have revealed the involvement of MFGE8 in malignant transformation and tumor progression.

LncRNA was a segment of nucleotide that did not encode a protein. It was initially considered a “Transcriptional Noise” [32]. However, more and more studies have recently shown that lncRNAs affected tumor progression by regulating protein expression [33]. Among them, lncRNA SNHG14, one of the most important members of the lncRNA SNHG family, has been shown to play an essential role in various cancers. Zhao et al. [34] found that lncRNA SNHG14/miR 5590 3p/ZEB1 formed a positive feedback loop to regulate the expression of immune checkpoint molecules PD 1/PD-L1. It promoted the progression and immune escape of diffuse large B cell lymphoma. And in hepatocellular carcinoma, lncRNA SNHG14 played an oncogenic role as a competing endogenous RNA that binds miRNA competitively with mRNAs, such as miR-4673 [35], miR-656 [36] and miR-876-5p [37]. Puzzlingly, Lu et al. revealed that silencing lncRNA SNHG14 inhibited glycolysis and proliferation of glioma cells while enhancing apoptosis [38]. However, Wang et al. [39] found the opposite phenomenon. Their study showed that lncRNA SNHG14 acted as a sponge to adsorb miR-92a-3p, inhibited cell proliferation and invasion and promoted apoptosis. The above results suggested that lncRNA SNHG14 could play different functions in different types of cancer. At present, there are few reports of lncRNA SNHG14 studies in gastric cancer. Therefore, in this study, we clarified the expression and biological functions of lncRNA SNHG14 in gastric cancer through in vivo and in vitro experiments. The expression of lncRNA SNHG14 was significantly reduced in cell lines with knockdown of MFGE8. Besides, lncRNA SNHG14 expression was found to be elevated in gastric cancer and combined with clinical information of STAD patients in TCGA database. The overall survival rate was lower in the lncRNA SNHG14 high expression group. Meanwhile, knockdown of lncRNA SNHG14 expression significantly reduced the proliferation and migration ability of gastric cancer. Overall, the diagnostic and therapeutic value of lncRNA SNHG14 in gastric cancer was revealed by our research. LncRNA SNHG14 has the promise as a new target molecule for the treatment of gastric cancer.

One of the characteristics of the malignant transformation of tumors is its stronger ability to invade and metastasize [40]. In recent years, the phenomenon of EMT is strongly associated with tumor metastasis [41]. The EMT caused epithelial tumor cells to acquire aggressive mesenchymal-like features. Epithelial cells will lose their epithelial characteristics after undergoing this process of EMT. For example, the epithelial gene expression was downregulated (E-cadherin). The expression of mesenchymal genes was activated (N-cadherin (CDH2) or vimentin) [41]. Previous studies showed that silencing lncRNA SNHG14 restored E-cadherin expression and decreased N-cadherin or vimentin expression in tumors in colorectal cancer cell lines [42]. Yu et al. [43] found that the EMT status of nasopharyngeal carcinoma is also regulated by lncRNA SNHG14. In gastric cancer, we came to the same conclusion. Overexpression of lncRNA SNHG14 activated the EMT state and promoted the expression of N-cadherin. However, the tumor-promoting effect mediated by lncRNA SNHG14 was partially alleviated after the knockdown of N-cadherin. It indicated that the expression of N-cadherin was influenced by the expression of lncRNA SNHG14. The specific molecular mechanism of N-cadherin regulation by lncRNA SNHG14 could be further elucidated in the future. In addition, in this study, we only preliminarily explored the relationship between MFGE8/lncRNA SNHG14/N-cadherin. More studies are needed to confirm whether MFGE8 mainly regulates the proliferation and migration of gastric cancer through this pathway.

In summary, we clarified the role of MFGE8 in regulating gastric cancer proliferation and migration through lncRNA SNHG14. Inhibition of MFGE8 or lncRNA SNHG14 expression significantly reduced the proliferation and migration ability of gastric cancer. These new findings help us to better understand the mechanisms behind the development and metastasis of gastric cancer. It provided the possibility to develop new therapeutic modalities.

Conclusion

In conclusion, our study has shown that MFGE8 expression was significantly elevated in gastric cancer tissues and is correlated with poor overall survival. We discovered that MFGE8 upregulates the expression of lncRNA SNHG14, which in turn upregulates CDH2 expression, thereby promoting the proliferation and migration of gastric carcinoma cells. These findings not only contribute to a deeper understanding of the mechanisms underlying gastric cancer proliferation and metastasis but also hold potential implications for the development of novel therapeutic strategies targeting MFGE8 and lncRNA SNHG14 in the treatment of gastric cancer.

Supplementary Material

Supplementary Materials.pdf
KCCY_A_2289745_SM2917.pdf (110.5KB, pdf)

Acknowledgments

We thank Meng-Ying Zhou for technical guidance and Chao Cheng for providing resource support.

Funding Statement

This study was funded by the Clinical Research Physician Program of Tongji Medical College, HUST (5001540018).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors Contributions

Zhou-Tong Dai: validation, investigation, conceptualization, methodology. Yong-Lin Wu: writing-original draft. Xing-Rui Li and Tao Xu: editing, formal analysis, and supervision.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Huazhong University of Science and Technology and Wuhan University of Science and Technology.

Data Availability Statement

The data presented in this study are available in this article and supplementary materials.

Supplementary materials

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2023.2289745.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials.pdf
KCCY_A_2289745_SM2917.pdf (110.5KB, pdf)

Data Availability Statement

The data presented in this study are available in this article and supplementary materials.

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