A total of 79 human GC tissues and matched adjacent non-cancerous tissues (ANTs) (at a distance of 5 cm from the tumor margin) were obtained at the time of surgery from April 2015 to May 2017 at The Affiliated Hospital of North Sichuan Medical College (Sichuan, China). None of the patients received radiotherapy, chemotherapy, or other anti-tumor treatments before surgery. Following excision, the GC and non-cancerous tissues were immediately frozen in liquid nitrogen and preserved at -80 °C until use. The clinicopathological parameters of patients with GC were collected. All patients provided written informed consent, and the entire study protocol was approved by The Ethics Committee of the Affiliated Hospital of North Sichuan Medical College, Nanchong, China.

Five human GC cell lines, AGS, MGC-803, BGC-823, SNU-1, and SGC-7901, and the normal gastric mucosal cell line GES-1 were purchased from Shanghai Cell Bank (Shanghai, China). The results of short tandem repeat (STR) analysis showed that there was no tri-allelic phenomenon in any locus, and no cross-contamination of other human cells was found. STR analysis found a 100% match to the reference data in the ATCC cell bank. Cells were cultured in DMEM or RPMI-1640 (Gibco BRL, Gaithersburg, MD, United States) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, United States) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere with 5% CO₂.

The pc-HOXD-AS2 plasmid vector was purchased from Beijing Syngentech (Beijing, China). The plasmid was transfected into SGC-7901 and SNU-1 cells using Lipofectamine 3000 (Invitrogen, United States). After 48 h, the efficiency of each transfection group was compared with regard to the expression of HOXD-AS2.

The PI3K inhibitor LY294002 was purchased from MedChemExpress (United States). LY294002 was dissolved in DMSO (100 mmol/L) and stored at -20 °C for 1 wk. Before use, LY294002 was quickly diluted into culture medium at a final concentration of 10 µmol/L.

Total RNA was extracted from GC tissues, ANTs, and GC cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), and reverse transcription reactions were performed using the GoScript Reverse Transcription System (Promega, Madison, United States) according to the manufacturer’s instructions. Real-time PCR was performed according to the instructions using standard Roche fluorescence quantitative PCR reagents (Roche, United States) and Roche fluorescent PCR instruments. β-actin or GAPDH was used as an internal reference. Each sample was detected three times. After the end of PCR, the dissolution profile of each specimen was stringently checked to ensure that there was only a single amplification product. HOXD-AS2, HOXD8, GAPDH, β-actin, PI3K, Akt, MAM2, and p53 primers were purchased from Shanghai Biotech (Shanghai, China). The primer sequences are shown in Supplementary Table 1. Analysis of the relative quantification of gene expression was performed using the classical 2^-ΔΔCt method.

The invasive ability of GC cells (SGC-7901 and SNU-1) was detected by Matrigel Transwell assay. A 24-well Transwell plate was used with membranes separated by 8 μm pores (Costar, Cambridge, MA, United States), and 50 mg/L Matrigel (BD Pharmingen, San Jose, CA, United States) was added to the upper chamber. Then, the 24-well plate was incubated at 37 °C in a humidified incubator containing 5% CO₂ for 2 h. SGC-7901 and SNU-1 cells transfected for 48 h were inoculated into the upper chamber at 5 × 10⁵ cells per well, and complete medium containing 10% FBS was added to the lower chamber. After 48 h of incubation, the cells that did not invade the Matrigel were wiped off with a cotton swab, and the invaded cells attached to the lower surface of the membrane were fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, United States), and stained with 1% crystal violet (Beyotime, Shanghai, China). The migration assay was similar to the invasion assay except that Matrigel was not added. Finally, three fields of view were randomly selected under a microscope for invasive and migratory cell counting. Three replicate wells were set in all experiments.

Cell migration ability was measured using a scratch wound healing assay. A straight line was marked with a marker in the middle on the back of a 3.5 cm dish. The SGC-7901 and SNU-1 GC cells transfected 24 h after each group were inoculated into a culture dish. After 24 h, a scratch was made on the bottom of the culture dish with a 200 μL tip, and the cell fragments were washed with sterile phosphate buffered saline (PBS). After incubation at 37 °C and 5% CO₂ for 48 h, the distance between the two edges of the scratch was observed under a microscope.

Cell counting kit-8 assay (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China) was used to detect the cell viability. Cells were seeded at 5000 cells per well in a 96-well plate. After the cells were transfected with plasmids for 24 h, 10 μL of CCK-8 was added to each well at 0 h, 24 h, 48 h, and 72 h. The absorbance at 450 nm of each well was measured with a spectrophotometer. All experiments were performed in triplicate.

Cell apoptosis was analyzed with Annexin V-APC/7-AAD Apoptosis Detection kit (KeyGEN BioTECH, China) and a NovoCyte flow cytometer (ACEA, China). Forty-eight hours after cell transfection, the cells were digested with trypsin without EDTA and washed twice with cold PBS. Then the digested cells were put into the flow sampling tube, and 500 μL of Binding Buffer was added to the tube according to the ratio of 100:1, followed by the addition of 5 µL Annexin V-APC and 5 µL 7-AAD staining solution. After 15 min of reaction at room temperature and protection from light, samples were taken on the machine for apoptosis analysis. Each experiment was repeated three times.

For cell cycle detection, the cells were collected into a brown EP tube. Add pre-cooled 70% alcohol was added, the tube was shaken gently, and put in a refrigerator at 4 °C for 4 h. After the cell membrane was broken, 0.5 mL of propidium iodide staining solution was added to the tube for staining at room temperature in the dark for 30 min, and then the sample was subject to cell cycle analysis. This experiment was performed in triplicate.

Total protein was extracted, and the concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States). Sample lysates (10 μg of protein) were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with specific antibodies against HOXD8 (1:1000), PI3K (1:1000), Akt (1:2000), MDM2 (1:500), or p53 (1:1000) (Abcam, Cambridge, MA, United States) at 4 °C overnight, followed by incubation with secondary antibody. Protein levels were normalized to those of total GAPDH, which were detected using a monoclonal anti-GAPDH antibody (1:10000; Sigma-Aldrich Corporation, St. Louis, MO, United States). Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad).

All experimental data were analyzed using SPSS20.0 (SPSS, Chicago, IL, United States) and GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, United States), and the measurement data are expressed as the mean ± SD. Two-tailed Student’s t-test was used to compare two groups of measurement data. The relationship between lncRNA expression levels and clinical indicators of patients was calculated by Chi-square test or Fisher's exact probability test (two-sided). Survival analysis for patients with GC was performed by the Kaplan–Meier method, and the differences between survival curves were estimated by the log-rank test. Spearman correlation analyses were performed to investigate correlations between gene expression levels. All tests were two-tailed, and P < 0.05 was considered statistically significant.

As shown in Figure 1A, the relative expression level of lncRNA HOXD-AS2 in 79 GC tissues was significantly decreased in comparison with that in the corresponding adjacent tissues (P = 0.030). As shown in Figure 1B, decreased expression of HOXD-AS2 was observed in 49 (62.03%) out of 79 cases, while high expression was observed in the 30 remaining cases (37.97%). The relationship between the expression level of HOXD-AS2 and the clinicopathological features of GC patients was analyzed. As shown in Table 1, low expression of HOXD-AS2 was associated with lymph node metastasis (P = 0.009) and TNM stage (P = 0.040). Data from the Kaplan-Meier Plotter database (http://kmplot.com/) was used to analyze the relationship between HOXD-AS2 expression and the overall survival rate of 348 patients with GC (109 patients with low HOXD-AS2 expression and 239 with high HOXD-AS2 expression). The prognosis of the HOXD-AS2 low expression group was worse than that of the HOXD-AS2 high expression group, and the difference was statistically significant (P = 0.012, Figure 1C).

Open in New Tab   Full Size Figure   Download Figure

Figure 1 HOXD-AS2 is downregulated in gastric cancer tissues and is associated with a poor prognosis in gastric cancer patients. A: The relative expression level of HOXD-AS2 in gastric cancer (GC) tissues was significantly lower than that in adjacent non-cancerous tissues (ANTs; P = 0.030, n = 79); B: HOXD-AS2 expression levels were decreased in human GC tissues and ANTs. Bars represent the ratio between the expression levels in GC tissues and ANTs (C/N, log scale) from the 79 patients. GC tissues expressed significantly lower levels of HOXD-AS2 than ANTs in the majority of patients (62.03%); C: Kaplan-Meier survival analysis showed that patients with low HOXD-AS2 expression had a poorer prognosis than those with high expression. Low: The expression level of HOXD-AS2 is lower than that of ANTs. High: The expression level of HOXD-AS2 is higher than that of ANTs. Expression levels were normalized to β-actin levels. The results are shown as the mean ± SD. ^aP < 0.05, two-tailed Student’s t-test. GC: Gastric cancer; ANTs: Adjacent non-cancerous tissues.

As shown in Figure 2A, the relative expression level of lncRNA HOXD-AS2 in the five GC cell lines SGC-7901, MGC-803, BGC-823, SNU-1, and AGS was lower than that in the normal gastric mucosal cell line GES-1 (P = 0.005, = 0.006, < 0.001, = 0.004, and < 0.001, respectively). The expression of HOXD-AS2 was the lowest in SGC-7901 and SNU-1 cells, so we selected these two cell lines for subsequent experimental studies. As shown in Figure 2B, after transfection of SGC-7901 and SNU-1 cells, the expression of HOXD-AS2 in the pcHOXD-AS2 group was significantly increased compared with that in the control group (P = 0.002 and 0.002, respectively).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 HOXD-AS2 is downregulated in gastric cancer cells. A: The expression of HOXD-AS2 was downregulated in gastric cancer AGS, MGC-803, SGC-7901, BGC-823, and SNU-1 cells compared with gastric epithelial GES-1 cells; B: The expression of HOXD-AS2 in SGC-7901 and SNU-1 cells after transfections. The results are shown as the mean ± SD. ^bP < 0.01, ^eP < 0.001.

The effects of HOXD-AS2 on the invasion and migration of SGC-7901 and SNU-1 cells were detected by wound healing assay and Transwell assay. It was found that overexpression of HOXD-AS2 inhibited the invasion and migration of SGC-7901 and SNU-1 cells (Figure 3A). We calculated the number of invasive and migratory cells in the pc-HOXD-AS2 group and control group, and cell migration and invasion in the pc-HOXD-AS2 group were significantly decreased compared with those in the control group (Figure 3A). The wound healing assay also confirmed the ability to inhibit the migration of SGC-7901 and SNU-1 cells after overexpression of HOXD-AS2 (Figure 3B). These results show that HOXD-AS2 is involved in the regulation of invasion and migration of GC cells.

Open in New Tab   Full Size Figure   Download Figure

Figure 3 Overexpression of HOXD-AS2 inhibits gastric cancer cell migration and invasion in vitro. A: Transwell assays were performed to detect the effect of HOXD-AS2 on the migration and invasion of SGC-7901 and SNU-1 cells. The results revealed that overexpression of HOXD-AS2 inhibited the migration and invasion of SGC-7901 and SNU-1 cells; B: Wound healing assay further showed that overexpression of HOXD-AS2 inhibited gastric cancer cell migration and invasion. ^aP < 0.05, ^bP < 0.01. Data are shown as the mean ± SD. All the experiments were performed in triplicate.

The cell proliferation activity was detected by the CCK8 experiment, and it was found that overexpression of HOXD-AS2 can inhibit the proliferation ability of SGC-7901 and SNU-1 cells (Figure 4A and B). We examined the effect of HOXD-AS2 on the apoptosis of SGC-7901 and SNU-1 cells by flow cytometry. The results showed that the apoptosis rate of the HOXD-AS2 overexpression group was higher than that of the control group (Figure 4C). Cell cycle detection results showed that in SGC-7901 and SNU-1 cells, overexpression of HOXD-AS2 can increase the number of G0/G1 phase cells, reduce the number of S phase cells, and block the cell cycle in G0/G1 phase (Figure 4D).

Open in New Tab   Full Size Figure   Download Figure

Figure 4 Overexpression of HOXD-AS2 inhibits gastric cancer cell proliferation and cell cycle progression and promotes apoptosis in vitro. A and B: The effect of HOXD-AS2 on cell proliferation was determined by cell counting kit-8 assay. Cell proliferation was decreased after overexpression of HOXD-AS2 in SGC-7901 (P = 0.0019) and SNU-1 (P < 0.001) cells; C: Flow cytometry was used to detect the percentage of apoptotic cells in the pc-HOXD-AS2 and the control groups. The percentage of apoptotic cells was increased after overexpression of HOXD-AS2 in SGC-7901 (P = 0.007) and SNU-1 (P = 0.021) cells; D: The effect of HOXD-AS2 on cell cycle was measured by flow cytometry after 7-AAD staining in SGC-7901 and SNU-1 cells. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001.

To explore the regulatory mechanism of HOXD-AS2 in GC, we predicted the co-expressed genes associated with lncRNA HOXD-AS2 via circlncRNAnet (http://app.cgu.edu.tw/circlnc/) and found that HOXD-AS2 was positively correlated with its nearby gene HOXD8 (r = 0.785, P < 0.05) (Figure 5A). Then, we included our qPCR data in the analysis and confirmed that HOXD-AS2 had a positive correlation with HOXD8 (r = 0.9157, P < 0.05) (Figure 5B). As shown in Figure 5C, G, and H, the relative mRNA expression level of HOXD8 in 79 GC tissues was significantly decreased in comparison with that in the corresponding adjacent tissues (P = 0.048), and the protein expression levels of HOXD8 in six GC tissues was significantly decreased in comparison with that in the corresponding adjacent tissues (P < 0.001). As shown in Figure 5D, E, and F, the relative mRNA and protein expression levels of HOXD8 in the five GC cell lines SGC-7901, MGC-803, BGC-823, SNU-1, and AGS were lower than those in the normal gastric mucosal cell line GES-1 (P < 0.001). In SGC-7901 and SNU-1 cells, we overexpressed HOXD-AS2 and found that HOXD8 protein levels also increased (Figure 6A, D, and E). According to these results, we speculate that HOXD-AS2 is likely to participate in the progression of GC cells via interaction with HOXD8.

Open in New Tab   Full Size Figure   Download Figure

Figure 5 HOXD8 is downregulated in human gastric cancer tissues and positively correlated with HOXD-AS2 expression. A: CirclncRNAnet database analysis results show that HOXD-AS2 is positively correlated with HOXD8 (r = 0.785, P < 0.05); B: Bivariate correlation analysis of the relationship between HOXD-AS2 and HOXD8 expression levels, and the resulting Spearman correlation coefficient was calculated as 0.9157 with P < 0.0001 (n = 79); C and D: The relative mRNA expression level of HOXD8 was decreased in human gastric cancer (GC) tissues (P = 0.048); E and F: The relative protein expression levels of HOXD8 were decreased in human GC cells (P < 0.001); G and H: The relative protein expression level of HOXD8 was decreased in human GC tissues (P < 0.001). The results are shown as the mean ± SD. ^aP < 0.05, ^eP < 0.001. GC: Gastric cancer; ANTs: Adjacent non-cancerous tissues.

Open in New Tab   Full Size Figure   Download Figure

Figure 6 Overexpression of HOXD-AS2 inhibits the PI3K/Akt signaling pathway. A, D, and E: Overexpression of HOXD-AS2 reduced p-PI3K, p-Akt, and p-MDM2 protein levels, and increased p53 and HOXD8 protein levels; B: HOXD-AS2 mRNA levels were significantly increased following treatment with LY294002 in SGC-7901 (P = 0.009) and SNU-1 (P = 0.001) cells for 48 h. Expression levels were normalized to GAPDH levels. The results are shown as the mean ± SD; C, F, and G: HOXD8 and p53 protein levels were significantly increased but p-PI3K, p-Akt, and p-MDM2 protein levels were significantly reduced following treatment with LY294002 in SGC-790 and SNU-1 cells for 48 h. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001.

In our previous research, through Gene Ontology (GO) analysis and KEGG signaling pathway enrichment analysis of GC-related signaling pathways, we identified the top 10 most enriched signal pathways in GC[14]. In this study, we found that lncRNA HOXD-AS2 may play an important role in regulating the progression of GC cells through the PI3K/Akt signaling pathway. The results showed that in SGC-7901 and SNU-1 cells, the protein levels of p-PI3K, p-Akt, and p-MDM2 in the pc-HOXD-AS2 group were lower than those in the control group, while the level of p53 was higher than that in the control group (Figure 6A, D, and E). To further demonstrate whether HOXD-AS2 regulates the progression of GC cells via the PI3K/Akt signaling pathway, we added 10 μmol/L PI3K inhibitor to SGC-7901 and SNU-1 cells. After the use of the PI3K-specific inhibitor LY294002, a similar effect was obtained as with transfection of the HOXD-AS2 plasmid. The results also showed that the expression of HOXD-AS2 increased and the expression of HOXD8 protein increased, which further demonstrated that HOXD-AS2 regulates the progression of GC cells by inhibiting the activation of the PI3K/Akt signaling pathway (Figure 6B, C, F, and G).

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with a length of more than 200 nucleotides and lack an open reading frame. They mainly regulate RNA transcription and mRNA splicing and play an important role in regulating the stability of RNA in the cytoplasm and the activity of microRNAs. More and more studies have shown that abnormally expressed lncRNAs are involved in all aspects of tumor occurrence and development, and are closely related to tumor proliferation, apoptosis, invasion, metastasis, drug resistance, and poor prognosis. In recent years, more and more studies have found that abnormally expressed lncRNAs are involved in the occurrence and development of gastric cancer (GC), and they are expected to become new biomarkers for the diagnosis and treatment of GC.

GC is one of the most common malignant tumors in the world. The incidence and mortality of GC are in the forefront of all malignant tumors. Although a major breakthrough has been achieved in the study of lncRNAs in the pathogenesis of GC, the specific mechanism of lncRNAs in the occurrence and development of GC has not yet been fully elucidated. Exploring new lncRNAs can help to understand the molecular mechanism of GC more deeply.

The main purpose of this study was to explore the effect of downregulation of HOXD-AS2 on the biological behavior of GC cells SGC-7901 and SNU-1 and the underlying mechanism. Studies have found that the downregulation of HOXD-AS2 can regulate the expression of its neighboring gene HOXD8, and can also activate the PI3K/Akt signaling pathway, thereby promoting the progression of GC cells. The results of this study may provide a new idea for the treatment of GC.

The pcHOXD-AS2 plasmid vector was constructed and transfected into SGC-7901 and SNU-1 GC cells. Matrigel Transwell and wound healing assays were used to confirm the effect of HOXD-AS2 on invasion and migration of GC cells. Cell counting kit-8 assay and flow cytometry were used to verify the effect of HOXD-AS2 on proliferation, cell cycle, and apoptosis of GC cells. The relevant regulatory mechanism between HOXD-AS2 and HOXD8 and PI3K/Akt signaling pathway was verified by Western blot analysis.

In this study, we found that the low expression of lncRNA HOXD-AS2 was associated with lymph node metastasis and tumor-node-metastasis stage in GC. In vitro functional experiments demonstrated that overexpression of HOXD-AS2 inhibited GC cell progression. Mechanistic studies revealed that HOXD-AS2 regulated the expression of its nearby gene HOXD8 and inhibited the activity of the PI3K/Akt signaling pathway.

These results indicate that downregulation of HOXD-AS2 significantly promotes the progression of GC cells by regulating HOXD8 expression and activating the PI3K/Akt signaling pathway. HOXD-AS2 may be a novel diagnostic biomarker and effective therapeutic target for GC.

This study combines basic experimental research and bioinformatics results to reach a relatively novel conclusion. To further confirm the results of this study, siRNA and animal experiments may be better.