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. 2017 Dec 12;21(1):151–160. doi: 10.3233/CBM-170462

Enhanced expression of the long non-coding RNA SNHG16 contributes to gastric cancer progression and metastasis

Dongbo Lian 1,*, Buhe Amin 1, Dexiao Du 1, Wei Yan 1
PMCID: PMC13075754  PMID: 29081409

Abstract

This paper aimed to probe into the expression of long non-coding RNA (lncRNA) SNHG16 in human gastric cancer (GC) and its potential tumor biological functions. The expression of lncRNA SNHG16 was detected in GC and adjacent tissues and GC cell lines using qRT-PCR. GC MGC-803 cells were transfected with siRNA of lncRNA SNHG16, as well as blank and negative control. A series of experiments including CCK-8, flow cytometry, transwell, and wound healing assay were adopted to evaluate the effects of lncRNA SNHG16 on cell growth and metastasis. Besides, the nude mouse xenograft tumor model was established to draw tumor growth curve and measure tumor volume during treatments. TUNEL staining was used to determine the apoptosis rate of tissues. The expression of lncRNA SNHG16 in GC tissue, significantly associated with invasion depth, lymph node metastasis, TNM stage and histological differentiation (all P< 0.05), was upregulated compared with adjacent tissues. Transfected with siRNA of lncRNA SNHG16 inhibited GC MGC-803 cell proliferation, and arrested cells in the G0/G1 phase, and then promoted apoptosis rate with reduced cell invasion and shortened migration distance. Additionally, the nude mice xenograft presented lower tumor growth rate and weight loss alongside elevated apoptosis rate of tumor tissues. LncRNA SNHG16 is highly expressed in GC, while suppression of SNHG16 expression can inhibit proliferation, weaken invasion and migration of GC cells, and enhance apoptosis, to be a novel target for GC clinical treatment.

Keywords: Long non-coding RNA SNHG16, gastric cancer, clinicopathological features, proliferation, migration, invasion, apoptosis, cell cycle

1. Introduction

Gastric cancer (GC) is one of the most frequently diagnosed therioma, ranking the second in cancer-causing death in global [1]. The rate of newly diagnosed cases and deaths increases by over 70% annually worldwide [2]. Surgery accompanied with effective adjuvant chemoradiotherapy still remains the primary curative treatment for GC, whereas it left poor clinical efficacy for the patients with GC metastasis [3]. Thus, seeking for a new biomarker for early diagnosis of GC and comprehensively exploring the mechanisms of GC occurrence and progression is required for improving the survival rate and quality of life of GC patients [4, 5].

Long non-coding RNA (lncRNA) is a RNA that comprises a class of transcripts with more than 200 nucleotides in length but do not encode proteins [6], which could not only regulate the normal physiological activities such as organism development, differentiation and metabolism, but also participant in diverse diseases including tumorigeneses [7, 8, 9]. For example, previous studies demonstrated that lncRNA is closely linked to biological activities of multiple tumors, such as breast cancer [10], colorectal cancer [11], lung cancer [12], renal carcinoma [13] and liver cancer [14], and it also plays a potential role in GC incidence and development [15, 16]. Moreover, Yang et al. found that lncRNA H19 expression was upregulated in GC tissue, while tumor cell proliferation got suppressed and apoptosis was enhanced when H19 levels in GC cells were silenced by a small interfering H19 RNA [17]. Saito et al. also revealed that lncRNA ATB activated by TGF-β may lead to GC epithelial-mesenchymal transition (EMT) changes to facilitate invasion and metastasis of GC [18], indicating that lncRNA is promising to be a desired target for GC diagnosis. Small nucleolar RNA host gene 16 (SNHG16, or ncRAN), as a lncRNA being rare studied, was firstly discovered in neuroblastoma with an upregulation level, markedly associated with poor prognosis [19]. Further, as shown by Qi’s group, SNHG16 was widely expressed in colorectal cancer cells and inhibited the metastasis and invasion of tumor cells [20]. However, whether SNHG16 could be expressed in GC, as well as its function or mechanism in GC is still unclear.

Therefore, we discussed the expression of lncRNA SNHG16 in GC and the potential molecular mechanism involved in the progression of GC to provide new insights into the pathogenesis and treatment of GC in this paper.

2. Materials and methods

2.1. Ethics statement

According to the declaration of Helsinki, all the subjects were informed and signed the informed consent before entering the investigation, which was approved by the ethics committee of the Beijing Shijitan Hospital of Capital Medical University. And all the animal experiments were carried out in strict accordance with the animal protocols and guidance approved by the ethics committee concerning animal research at our hospital. Every effort designed to minimize impact on the animals under study.

2.2. Subjects

A number of 122 cases of patients received GC radical treatment in the General Surgery Department of Beijing Shijitan Hospital of Capital Medical University were involved in the following experiments from June 2014 to June 2016. Inclusion criteria: (a) primary GC cases without recurrence and metastasis; (b) no previous history of other malignancies; (c) not treated with chemotherapy, radiotherapy or biotherapy before surgery; (d) patients’ pathologic biopsy reports after surgery reviewed by a pathologist conformed to GC pathological diagnosis guidelines; (e) with complete clinical and pathological data. The tumor tissue (non-necrotic parts) and the corresponding adjacent tissues (over 5 cm away from the tumor tissue) of each patient were collected, respectively, and immediately stored in frozen tubes in liquid nitrogen. Of the participants, 83 cases were males and 39 cases were females, and the age of the patients ranged from 30 to 81 with an average of 58.14 ± 16.57 years.

2.3. Cell culture

The GC cell lines (AGS, BGC-823, MGC-803, SGC-7901, and MKN451) and normal gastric epithelial cell line GES-1 were all purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were inoculated in RPMI-1640 (Gibico, USA) containing 10% FBS (Gibico, USA), 100 U/mL penicillin and 100 μg/mL streptomycin at 37∘C in 5% CO2 atmosphere. Cell passage was conducted nearly every 2 ∼ 3 days when cell fusion reached about 80% ∼ 90%, and cells in the logarithmic phase were collected to seeded into in 6-well plates with 5 × 103 cells each well for subsequent trials.

2.4. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA from GC, adjacent tissues and all GC cell lines was extracted following the kits instructions (Promega, USA). Reverse transcription PCR was performed on a Reverse Transcription System A3500 (Promega, USA) according to manufacturer’s procedures. PCR was achieved using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR amplification was carried out with cycling conditions: 95∘C for denaturation for 2 min, followed by 28 cycles of 95∘C for 15 s, 55∘C for 15 s and 72∘C for 20 s. The GAPDH gene was used as the reference gene. Relative expression of the target gene (fold changes) was calculated using the formula 2-Δ⁢Δ⁢Ct, where Ct is the inflection point of amplification curve representing a cycle value. The relative expression was calculated using the equation ΔCt = Ct-target gene CtGAPDH; folds = 2-(Δ⁢Ct (tumor)-Δ⁢Ct (normal)).

2.5. Cell grouping and transfection

The MGC-803 cells were utilized for transfection and grouped into Blank group (MGC-803 cell without any treatment), negative control (NC) group (MGC-803 cells transfected with negative plasmid), si-SNHG16-1 group (MGC-803 cells transfected with SNHG16 siRNA-1 plasmid), and si-SNHG16-2 group (MGC-803 cells transfected with SNHG16 siRNA-2 plasmid). By the way, two sequences of si-SNHG16 (si-SNHG16-1/-2) were designed according to the previous study [21]. Cells in the logarithmic phase were obtained in each group and inoculated in 6-well plates with a density of 4 × 105 per well. Lipofectamine 3000 (L3000015, Invitrogen, California, USA) was introduced for cell transfection.

2.6. Cell proliferation by CCK-8 assay

The harvested cells of each group were washed with PBS and then digested with trypsin. The uniform cell culture was seeded with inoculation of 100 μL per well and 3 replicates in parallel. Being incubated at 37∘C containing CO2, the culture plates were added with 10 μL CCK-8 (CK04, Tongren, Japan) per well at 24 h, 48 h and 72 h, respectively. With incubation for one hour, microplate reader (Thermo Fisher, Waltham, MA) was introduced to determine optical density (OD) at 450 nm. It was repeated 3 times and average OD value was calculated.

2.7. Cell cycle by flow cytometry

After transfection for 48 h, the cells in each group were fixed with ice cold anhydrous ethanol at 4∘C overnight. After rinsed with PBS, the cell suspension was centrifuged at 1000 rpm for 5 min to discard supernatant and then resuspended in 100 μL PBS, and stained using 300 μL propidium iodide (5 μg/mL) at 4∘C and then incubated in the dark for 15 min. Cell cycle was assayed with the aid of flow cytometry (BD, San Diego, CA, USA).

2.8. Apoptosis rate analysis by flow cytometry

Annexin V Kit (C1063, Beijing, China) was adopted to evaluate cell apoptosis. With 0.25% trypsin digestion and gently triturating, the digestive solution was mixed with the previously collected medium after transfection for 48 h. Afterwards, the mixtures were centrifuged at 12000 × g for 5 min at 4∘C, removing the supernatant, and cell precipitation was resuspended in PBS containing 50 ∼ 100 thousand cells. With another centrifugation, cellular sediment of each group was treated with 5 μL Annexin V-FITC (20 μg/mL) and 10 μL propidium iodide (50 μg/mL) for 30 min in the dark, and apoptosis was detected by a flow cytometry (BD Pharmingen, San Diego, CA, USA) after ice-bath. The early apoptosis was the right lower quadrant, while the late apoptosis was the right upper quadrant, and the survival cells were the left lower quadrant. Apoptosis rate = the early apoptosis rate + the late apoptosis rate.

2.9. Transwell assay

The prepared matrigel was mixed with serum-free DMEM medium as a ratio of 1:3, and then was coated onto the upper surface of Transwell chamber for air-drying at room temperature. The trypsin digested cells were diluted into a density of 1 × 105 cells using DMEM free of serum for starvation cultivation for 24 h, and pre-treated with mitomycin C at 10 μg/mL for 2 h to inhibit cell proliferation [22]. The upper Transwell chamber was inoculated with cell culture for 200 μL per chamber. Transwell chambers were seeded into each well in 24-well plates prepared with 500 μL DMEM containing 10% FBS. After 48 hours’ incubation, the chambers were taken out and rinsed with PBS to remove the medium in the upper chamber. Cells and matrigel present at the microporous membrane was collected using cotton swab and fixed with 95% ethanol for 15 min, and then stained with crystal violet. The average number of cells observed to migrate through the basement membrane was recorded under an inverted microscope.

2.10. Wound healing assay

The cells in each group were inoculated in 6-well plates with 5 × 104 cells each well, and pre-treated with mitomycin C at 10 μg/mL for 2 h to inhibit cell proliferation. Then, wound scratch was performed using a spatula with a width of 2 mm as the cells grew with adherence, and then cultured for 24 h. The cells of all groups experienced incubation for another 24 h based on above grouping treatments. It was observed and photographed under a microscope at 0 h and at 24 h after scratch, respectively, and Image-Pro Plus software was used for distance measurement between the scratches. Cell migration distance (mm) = scratch distance at 0 h - scratch distance at 24 h.

2.11. Establishment of human GC xenografts model in nude mice

A number of 30 female BALB/C nude mice (4 ∼ 6 weeks old and weighed 18 ± 2 g) were purchased from China Scientific Research Institute of Animal, and raised in the experimental animal center of our hospital. The nude mice and experimental conditions involved all met the specific-pathogen-free (SPF) standard. Adaptive feeding for one week, the mice were divided into 3 groups similar with the cell grouping, 10 mice per each group. Meanwhile, the right backs of the nude mice in each group were injected with 0.2 mL of cells from the corresponding group (Blank group, NC group and si-SNHG16 group), which were digested using trypsin and suspended to 1 × 108 cells/mL. The tumor volume (V = a × b ×0.5; a represents the long diameter and b represents the short diameter) was measured every week. The xenograft was isolated and weighed 35 days later.

2.12. TUNEL staining

The isolated xenografts were fixed using TUNEL detection kit (Roche, Switzerland) at room temperature 30 min, washing with PBS, and then they were immersed in 0.2% Triton X-100 for 5 min to strengthen the permeability. An addition of 50 μL mixtures (the concentrated enzyme solution and marking liquid was mixed as a ratio of 1:9) was followed after the tumor issue was rinsed with PBS and dried, and the tumor system was cultured at 37∘C for 60 min in a wet container. When PBS washing finished, 5 field were randomly selected and analyzed for each slice using fluorescence microscope, and apoptotic rate = apoptotic positive cells/total cells × 100%.

2.13. Statistical analysis

All statistical data were analyzed by the SPSS 19.0 software (SPSS, Inc, Chicago, IL, USA). The measurement data were expressed as means ± standard deviation (SD). Predictive value of SNHG16 was evaluated by the receiver operating characteristic curve (ROC) analysis. The t-test was used for the pair comparisons with normal distribution. Differences between groups were evaluated using One-Way ANOVA. P< 0.05 was considered statistically significant.

3. Results

3.1. Expression of lncRNA SNHG16 in GC tissues and cell lines

QRT-PCR was performed for lncRNA SNHG16 expression analysis in GC tissues and cell lines. The lncRNA SNHG16 expression in GC tissue was higher than in adjacent tissues (P< 0.05, Fig. 1A). In addition, the optimal cutoff value of SNHG16 expression was 1.38-fold with the largest Youden’s index according to the relative expression levels of SNHG16 (Fig. 1B). Based on the cutoff score, 110 cases (90.16%) were up-regulated (cutoff score ⩾ 1.38), while the resting 12 cases (9.84%) were down-regulated (cutoff score < 1.38). Collectively, these findings strongly suggested the increased SNHG16 in GC. Similarly, the expression of lncRNA SNHG16 in GC cell lines (AGS, BGC-823, MGC-803, SGC-7901 and MKN45) were all highly upregulated when compared with the normal gastric epithelial cell line GES-1(P< 0.05, Fig. 1C). Then the MGC-803 cells were the preferred subjects for follow-up trials because they had the most significant increased expression of lncRNA SNHG16 (P< 0.01).

Figure 1.

Figure 1.

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Expression of lncRNA SNHG16 in GC tissues and cell lines detected by qRT-PCR. Note: A, Expression of lncRNA SNHG16 in 122 pairs of GC tissues and matched adjacent tissues; B, Receiver operating characteristic (ROC) curve analysis was used to determine whether SNHG16 is really a good candidate to discriminate tumor tissues from non-tumor tissues; C, Expression of lncRNA SNHG16 in GC cells, *refers to P< 0.05 compared with the GES1 cell. Data were presented as 2-Δ⁢Δ⁢Ct.

3.2. Relationship between lncRNA SNHG16 expression and clinicopathological features of GC

The lncRNA SNHG16 expression was observed to have no clearly relevance to clinical pathological features in age, gender, tumor size and tumor site (all P> 0.05), but was closely connected to the GC invasion depth, TNM staging, differentiation and lymph node metastasis, indicating the severer invasion, the higher staging, the lower differentiation alongside with lymph node metastasis all resulted in higher SNHG16 expression level in GC patients (P< 0.05, Fig. 2).

Figure 2.

Figure 2.

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Relationship between lncRNA SNHG16 expression and GC clinicopathological features. Data were presented as 2-Δ⁢Δ⁢Ct.

3.3. Effects of siRNA of lncRNA SNHG16 on cell proliferation

In contrast with Blank group and NC group, the expressions of lncRNA SNHG16 in si-SNHG16-1 and si-SNHG16-2 groups were markedly downregulated (P< 0.05), but no significant differences were shown in Blank group and NC group (P> 0.05), indicating remarkable efficiency through knockdown of si-SNHG16-1 and si-SNHG16-2 (Fig. 3A). Cell proliferation in si-SNHG16-1 and si-SNHG16-2 groups were declined obviously at 48 h, 72 h and 96 h as compared with Blank group and NC group (P< 0.05), which presented no significant differences between Blank group and NC group, as shown in Fig. 3B.

Figure 3.

Figure 3.

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Comparison of SNHG16 expression and cell proliferation in each group when transfected with siRNA of lncRNA SNHG16. Note: A, lncRNA SNHG16 expression in each transfected group detected by qRT-PCR, *refers to P< 0.05 compared with Blank group; B, Cell proliferation in each transfected group analyzed by CCK8.

3.4. Effects of siRNA of lncRNA SNHG16 on cell cycle and apoptosis in each group

Cell cycle observed by flow cytometry showed that the si-SNHG16-1 and si-SNHG16-2 groups performed elevated number of cells in G0/G1 phase (all P< 0.05) and reduced cell ratios in S and G2/M phase (all P< 0.05) when compared with Blank group and NC group, while it showed no significant differences between Blank group and NC group in G0/G1, S and G2/M phase (all P> 0.05, Fig. 4A). Apoptosis rate in si-SNHG16-1 and si-SNHG16-2 groups were turned out to be conspicuous higher than Blank group and NC group (all P< 0.05, Fig. 4B).

Figure 4.

Figure 4.

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Effects of siRNA of lncRNA SNHG16 on cell cycle and apoptosis of GC cells. Note: A, Cell cycle examined by flow cytometry; B, Apoptosis analyzed using flow cytometry. *refers to P< 0.05 compared with the Blank group.

3.5. Effects of siRNA of lncRNA SNHG16 on migration and invasion of cells in each group

Transwell assays confirmed that the si-SNHG16-1 and si-SNHG16-2 groups showed markedly reduction in invasive cells in comparison with Blank group and NC group (P< 0.05), with no obvious differences in cell invasion for the Blank group and NC group (P> 0.05, Fig. 5A). The scratch activity was observed at 0 h and 24 h to exhibit poorer wound healing and shorter migration distance in si-SNHG16-1 and si-SNHG16-2 groups than Blank group and NC group (all P< 0.05). Similarly, the migration distances of the Blank group and NC group showed no significantly difference (all P> 0.05, Fig. 5B).

Figure 5.

Figure 5.

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Transwell and wounding healing assay for cell invasion and migration analysis in each transfected group. Note: A, Comparisons of cell invasion in each group; B, Comparisons of cell migration in each group. *refers to P< 0.05 compared with the Blank group.

3.6. Effects of siRNA of lncRNA SNHG16 on the growth of human GC xenografts in nude mice

The xenografts in si-SNHG16-1 and si-SNHG16-2 groups grew slower than that in Blank group and NC group (all P< 0.05), and Blank group and NC group exhibited no markedly differences (P> 0.05). The si-SNHG16-1 and si-SNHG16-2 groups also appeared distinctly tumor weight loss when compared with Blank group and NC group (all P< 0.05), which was no different from each other (P> 0.05). It was proved by TUNEL staining that normal nucleus was visible to be uniform blue and apoptotic cell nucleus was stained with pale brown or dark brown. Statistical analysis made it known that the apoptosis rate of si-SNHG16-1 and si-SNHG16-2 groups were extremely higher than Blank group and NC group (all P< 0.05), while the Blank group and NC group showed no markedly differences when compared with each other (P> 0.05). It was shown in Fig. 6.

Figure 6.

Figure 6.

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Subcutaneous xenograft trials in nude mice. Note: A, Xenograft growth curve in nude mice; B: Comparison of xenograft weight in nude mice; C: TUNEL staining for xenograft apoptosis analysis. *refers to P< 0.05 compared with the Blank group.

4. Discussion

Despite being absent of protein-coding capacity, lncRNA performed active roles in regulating gene expression at the epigenetic, transcription and post-transcription level [23]. To our knowledge, lncRNA is not only widely involved in diverse physiological processes including growing development, and its specific expression may also facilitate the occurrence and development of many diseases, particularly the tumorigenesis [24]. In recent years, more and more lncRNAs have been identified in extensive observations to be obviously overexpressed in various tumors. For instance, the LSINCT5 was proved to be highly expressed in both breast and ovary cancer cells and tissues [25], and LncRNA HULC was substantially upregulated in liver and pancreatic cancers, which had a closely relationship with tumor size, lymphatic metastasis and vascular invasion [26, 27]. Moreover, HIF1A-AS2 was validated to be associated with advanced tumor stage, lymph node metastasis, severe invasion, or poorer prognosis as it presented elevated expression, indicative of a novel marker for GC diagnosis and prognosis [28]. Besides, Yang et al. also discovered that c-Myc can directly bind to the E-box element in the promoter region of long non-encoding CCAT1, and c-Myc ectopic expression can activate CCAT1 to be highly upregulated in GC, and thus promoting GC metastasis [29]. Additionally, it is important to emphasize that SNHG16 expression in highly aggressive neuroblastoma specimens and neuroblastoma cell lines was greatly upregulated, implying SNHG16 to be related to cancer progression and nervous system development [19]. Not surprisingly, SNHG16 observed in this study was significantly upregulated in GC tissue and GC cell lines, which was consistent with previous findings, especially under conditions of poorer degree of differentiation, more advanced tumor stage, and increased tumor invasion and metastasis, suggesting that SNHG16 may function as an oncogene in GC incidence and development, more importantly, its overexpression even aggravated cell malignant transformation and tumor progression through expanding GC cell invasion and metastasis.

Another important findings in our experiment demonstrated that migration distance and invasive cell number were clearly decreased in the SNHG16 siRNA-transfected group. In the past studies, evidence revealed that lncRNA HOTAIR appeared high levels in GC tissue and cells, whereas knockdown of HOTAIR could control GC cell proliferation and induce apoptosis, so as to weaken tumor cell invasion and reduce cell migration [30]. As authenticated by Zhao’s group, silencing of HULC, another lncRNA, could effectively reverse the EMT phenotype in GC cells [31]. Similarly, silencing the expression of Linc00152 could suppress GC cell proliferation and metastasis, arrest cell cycle at G1 phase, and then increase apoptosis by regulating the EMT procedure [32]. Therefore, we used siRNA to interfere with SNHG16 expression for further elucidating the associations between SNHG16 expression and GC occurrence, and our results turned out that the expression of SNHG16 was dramatically lowered after transfected with SNHG16 siRNA in GC cells, which could markedly reduce GC cell proliferation and invasion and migration, promoted cells arrest at G0/G1 phase and enhanced apoptosis rate, showing that SNHG16 expression could be served as a strong prognostic indicator of GC cell growth and metastasis. Similar finding was also obtained in bladder cancer that high SNHG16 expression was confirmed to promote cell proliferation, invasion and migration, while silencing of SNHG16 improved the chemosensitivity of bladder cancer [33], supporting the findings we did in this research. As we know, disruption of a proper regulation of cell cycle contributing to cancer development resulting from the oncogenes activation or tumor suppressor genes inactivation, leads to uncontrolled cellular proliferation [34], providing an explanation that silencing of SNHG16 expression mitigated cell growth and suppressed cell invasion and migration, possibly by functioning on the cell cycle control program to affect the progression of tumor cell cycle. Considering the above, SNHG16 was similar to many other lncRNAs in GC, as well as its biological characteristics to influence GC, so as to accelerate tumorigenesis and progression, which may support the notion that SNHG16 may become a potential target for GC therapy.

In addition, we established a model of human GC xenografts in nude mice, which is a good model to offer valuable approaches for further studying its biological and malignant behavior of SNHG16 in GC, because the histological form or tumor biological features of transplant tumor is the same as original tumor [35, 36]. The present study showed that the xenograft in si-SNHG16 group presented inhibited tumor growth, decreased tumor volume and improved apoptosis rate, demonstrating that SNHG16 plays an important role in regulation of the growth of GC xenografts in vivo, which was in accordance with the discoveries of the cellular tests in vitro [24]. More interestingly, according to Christensen LL reports, SNHG16 may act as the ceRNA (competing endogenous RNA) to achieve functionality by interacting with miRNA to implicate in GC pathogenesis [21]. However, further and comprehensive studies about whether SNHG16 combines with miRNA by competitive sponge adsorption to effect on GC cell growth and metastasis are still to be explored.

In conclusion, up-regulation of SNHG16 exhibits a tumorigenic effect in GC, and suppression of SNHG16 could restricted cell proliferation and metastasis, and promoted apoptosis in GC cells in this paper, providing a promising target for GC diagnosis and treatment.

Acknowledgments

The authors appreciate the reviewers for their meaningful comments in the current study.

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Articles from Cancer Biomarkers: Section A of Disease Markers are provided here courtesy of SAGE Publications

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