All clinical samples and information in this study were from the Department of Gastrointestinal Surgery, Provincial Hospital Affiliated to Shandong First Medical University. Fresh pathological specimens (GC and adjacent normal tissues) were from patients undergoing radical GC surgery at our hospital. All patients provided informed consent and were approved by the Medical Ethics Committee.

Human GC cell lines (MKN45, MKN28, HGC27, MGC803, N87, AGS and SGC7901) and the immortalized gastric mucosa cell line (GES-1) were provided by the Cell Center of the Chinese Academy of Medical Sciences. All cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, United States). Cells were cultured in incubator at 37 °C and 5% CO₂. Cells were passaged when they were 70% confluent.

The miR-143-5p mimics and inhibitor were purchased from RiboBio (Guangzhou, China). siRNAs targeting CASC20 (si-CASC20) and MEMO1 (si-MEMO1) were designed and synthesized by RiboBio (Guangzhou, China). Lipofectamine2000 (Invitrogen, United States) was used to transfect siRNA or miRNA mimics/inhibitor into cells following the manufacturer’s instructions. GC cells in logarithmic phase were collected and inoculated in a 6-well plate. Cells were transfected with siRNAs when cell confluence reached approximately 50%.

Trizol reagent (Invitrogen, United States) was used to extract total RNA from GC tissues and cells. RNA integrity was determined by evaluating the absorbance at OD₂₆₀/280 using a NanoDrop analyzer and confirmed at 1.8–2.0. Total RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara, Dalian, China) in accordance with the manufacturer’s instructions. The quantitative real-time polymerase chain reaction (qRT-PCR) three-step method was used, and the 2^-ΔΔCt method was used to calculate the relative expression levels of CASC20 and miR-143-5p. The primer sequences are as follows: CASC20, Forward 5′-ATAACCACCCTCCCCTCCTC-3′, Reverse 5′-GCTCCTCCCACTTTAACCCC-3′; miR-143-5p, Forward 5′-ATGGTTCGTGGGGTCCAGTTTTCCCAG-3′, Reverse 5′-GTGTCGTGGAGTCGGCAATTC-3′; MEMO1, Forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′, Reverse 5'-CGCTTCACGAATTTGCGTGTCAT-3′; and β-actin, Forward 5′-TGGCACCCAGCACAATGAA-3′, Reverse 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. U6, Forward 5′-CAGCACATATACTAAAATTGGAACG-3′ Reverse 5′-ACGAATTTGCGTGTCATCC-3′. β-actin (cytoplasm control) and U6 (nucleus control) were used for normalization.

GC cells were inoculated into 96-well plate at 5 × 10³ cells per well. At 24 h, 48 h, 72 h, 96 h and 120 h after transfection, 90 μL of fresh culture medium containing 10% FBS and 10 μL of CCK-8 reagent (Dojindo, Mashikimachi, Japan) were simultaneously added to each well. Cells were cultured for another 4 h. The OD value of each well at 450 nm was measured and proliferation curves were drawn.

GC cells were collected after trypsin digestion and centrifuged at 1000 rpm/min for 5 min. The cells were washed twice with phosphate buffer solution, and 500 μL binding buffer was added to adjust the cell concentration to 1 × 10⁶ cells/mL. Annexin V-FITC (5 μL) and propidium iodide solution (10 μL) (NeoBioscience, China) were added to the cell suspension, and cells were analyzed on a flow cytometer (BD Accuri C6).

After 48 h of transfection, cells were collected and the cell density was adjusted to 1 × 10⁵ cells/mL. The cell suspension was inoculated into a 6-well plate at 100 μL/well and the plates were incubated for 14 d. Cells were washed twice with phosphate buffered saline and fixed with 4% paraformaldehyde for 15 min; the cells were then stained with 0.1% crystal violet staining solution for 10 min. We counted the number of cell clones in five fields under the microscope and determined the clone formation rate as follows: Clone formation rate (%) = (number of clones/number of inoculated cells) × 100%. The experiment was repeated three times.

GC cells were cultured in a 6-well plate at 5 × 10⁵ cells per well. Linear scratch wounds were created in the cell monolayer with a sterile 100 μL pipette tip. Cells were cultured for 24 h, and the wound widths were photographed and measured.

Assays using a Transwell chamber (8 μm, Corning, United States) were used to evaluate cell migration and invasion abilities. GC cells were collected and resuspended in serum-free RPMI 1640 medium at 1.5 × 10⁴ cells/mL, and 200 μL of the cell suspension was inoculated into the upper chambers. Complete medium containing 10% FBS was added to the lower chamber. After culturing for 24 h, the cells were fixed with methanol. Cells in the upper chamber were removed with cotton swabs, and cells in the lower chambers were stained with 0.1% crystal violet solution for 30 min. The cells were observed under a microscope and photographed; five random fields were obtained to count the number of migrated cells. Each experimental group was performed in triplicate, and the average value was taken. For the invasion experiment, the upper chamber was coated with Matrigel; the assay was then conducted following the methods for the migration assay.

The online tool lncRNASNP2 was used to predict miRNAs that potentially bind to CASC20. We used miRDB, Targetscan, and miRTarbase to predict putative target genes of miR-143-5p.

Luciferase reporter pGL3 vectors with wild-type CASC20 (termed WT-CASC20), wild-type MEMO1 (MEMO1-WT), mutant CASC20 (MUT-CASC20) and mutant MEMO1 (MEMO1-MUT) were constructed (Promega, Madison, United States). The reporter plasmids were transfected into MGC803 cells along with miR-143-5p mimics. At 48 h after transfection, the luciferase activity was measured using the Dual Luciferase Reporter Assay system (Promega, United States) in accordance with the manufacturer’s instructions.

Cells were collected and lysed with RIPA lysis buffer (Beyotime, Shanghai, China). After lysates were centrifuged, the supernatant was collected and protein concentration was evaluated by the BCA method. The samples (60 μg/well) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro-transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, United States). After blocking in 5% skim milk at room temperature for 2 h, the membrane was incubated with primary antibody overnight at 4 °C. The membrane was then washed three times with Tris buffered saline tween and incubated with the secondary antibody at room temperature for 2 h. ECL luminescent solution was used for chemiluminescence and Image J software was used to analyze the protein expression level. MEMO1 and β-actin monoclonal antibodies were obtained from Sigma-Aldrich (St Louis, MO, United States). The other antibodies were purchased from Abcam (Cambridge, England).

Statistical analyses were performed using GraphPad Prism (version 7.0; GraphPad Prism software, San Diego, CA, United States). Data are shown as mean ± SD. For measurement data, the difference between two or more groups was calculated using the Student’s t-test or one-way ANOVA analysis. Chi-square test was used to analyze the correlation between CASC20 and clinicopathological parameters of GC patients. The survival curve was determined by the Kaplan–Meier method and log-rank test. P < 0.05 was considered statistically significant.

Analysis of TCGA database revealed that CASC20 expression was significantly higher in 408 GC tumor tissues than that in 211 normal gastric tissues (P < 0.01, Figure 1A and B). We performed qRT-PCR to assess the expression of CASC20 in 50 cases of GC tissues and paired normal gastric mucosa tissues adjacent to tumors. The results showed that the expression of CASC20 in GC tissues was significantly higher than that in normal adjacent gastric mucosa tissues (P < 0.01, Figure 1C). We further found that the expression of CASC20 in seven GC cell lines was significantly higher than that of GES-1 normal gastric mucosal epithelial cells (Figure 1D). We next analyzed the relationship between CASC20 expression and the survival rate of GC patients in TCGA database. The results indicated that the survival rate of GC patients with high expression of CASC20 was significantly lower than that of patients with low expression (Figure 1E). Together these data indicate that CASC20 is highly expressed in GC and may play an important role in the development of GC.

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Figure 1 High level of cancer susceptibility 20 in gastric cancer tissues and cell lines. A and B: Cancer susceptibility 20 (CASC20) expression in gastric cancer (GC) samples and normal samples from TCGA GC database; C: CASC20 expression in 50 pairs of clinically collected gastric cancer tissues and normal tissue samples; D: CASC20 expression in GC cell lines and GES-1 cell line was analyzed by quantitative real-time polymerase chain reaction; E: Overall survival curves of TCGA GC patients with different CASC20 expression. ^aP < 0.05 and ^cP < 0.001 vs control.

Next, we examined the correlation between CASC20 expression and the pathological characteristics of 50 GC patients. GC patients were divided into high and low CASC20 expression groups based on the median ratio of relative CASC20 expression. High CASC20 expression in GC patients was significantly correlated with a higher risk of lymphatic metastasis (P = 0.011), but did not show associations with age, gender, TNM stage and differentiation degree (Table 1). These results suggest that CASC20 is closely related to the malignant biological behavior of GC.

We next evaluated the role and effects of CASC20 in GC cells. We transfected siRNA targeting CASC20 into GC cells and qRT-PCR confirmed that the expression of CASC20 was significantly down-regulated after siRNA transfection (Figure 2A). Flow cytometry results showed that knockdown of CASC20 significantly increased the apoptosis rate of SGC7901 and MGC803 GC cells compared with the control cells (Figure 2B and C). CCK8 assays demonstrated that knockdown of CASC20 significantly inhibited the proliferation of SGC7901 and MGC803 cells (Figure 2D). Colony formation experiments revealed that inhibiting CASC20 expression resulted in decreased clonogenic survival (Figure 2E and F). Transwell experiments showed that knocking down CASC20 significantly reduced the numbers of invading and migrating GC cells (Figure 2G and H). Together, these findings show that knockdown of CASC20 inhibits the malignant phenotype of GC cells, suggesting that CASC20 exhibits oncogenic functions in GC.

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Figure 2 Konckdown of cancer susceptibility 20 inhibits the apoptosis, proliferation and metastasis of gastric cancer cells. A: Efficacy assessment of cancer susceptibility 20 (CASC20) silence through quantitative real-time polymerase chain reaction method in MGC803 and SGC7901 cells; B and C: Cell apoptosis evaluation in transfected cells through flow cytometry; D: Cell viability was verified by CCK-8 experiments after transfection with si-CASC20; E and F: Colony formation assay was used to detect the cell proliferation ability after transfection with si-CASC20; G and H: Transwell assay was employed for examining the effect of CASC20 low-expression on gastric cancer cell migration and invasion. ^aP < 0.05 vs control.

To gain better insights into the function of CASC20, we first determined the subcellular location of CASC20 by GeneCards software (https://www.genecards.org). The results showed that CASC20 is mainly located in the cytoplasm. The results of nuclear cytoplasmic experiments further confirmed that CASC20 mostly localizes in the cytoplasm (Figure 3A and B), suggesting that CASC20 may function as a ceRNA to “sponge” miRNA.

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Figure 3 miR-143-5p served as a target of cancer susceptibility 20 in gastric cancer cells. A and B: Relative cancer susceptibility 20 (CASC20) expression levels in nucleus and cytoplasm of SGC7901 and MGC803 cells; C: The relative expressions of miRNAs in gastric cancer (GC) cells after downregulation of CASC20; D: The expression of miR-143-5p was detected by quantitative real-time polymerase chain reaction in GC cells after transfection with si-CASC20; E: The expression levels of miR-143-5p in GC from TCGA database; F: The detection of CASC20 expression after transfection of miR-143-5p mimics; G: The correlation analysis between miR-143-5p expression and CASC20 expression in 50 GC tissues; H: Bioinformatics presentation of CASC20 and miR-143-5p binding sites; I: The verification of CASC20 and miR-143-5p interaction by luciferase report assay. ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001 vs control.

We next used bioinformatics tools (lncRNASNP2) to predict miRNAs with potential binding sites for CASC20 and the results identified miR-143-5p, miR-515-3p, miR-543 and miR-616-5p. We evaluated the expressions of these miRNAs in cells transfected with si-CASC20 and found that the expression of miR-143-5p was up-regulated when CASC20 was knocked down, while miR-515-3p, miR-543 and miR-616-5p showed no significant changes (Figure 3C and D). We thus selected miR-143-5p for further analyses.

TCGA database analysis showed that miR-143-5p was expressed at low levels in GC tissues (Figure 3E), which was consistent with previous studies on miR-143-5p[15]. We transfected miR-143-5p mimics into GC cells and observed significantly decreased expression of CASC20 (Figure 3F). In addition, we identified a negative correlation between CASC20 and miR-143-5p in 50 GC tissues (Figure 3G).

To further determine the regulatory mechanism between CASC20 and miR-143-5p, we performed dual luciferase reporter assays. The results showed that transfection of miR-143-5p mimics decreased the luciferase activity of the WT-CASC20 reporter in MGC803 cells, while miR-143-5p mimics had no impact on the MUT-CASC20 reporter activity (Figure 3H and I). This result suggests that the regulatory relationship of CASC20 and miR-143-5p in GC cells involves interactions between CASC20 and miR-143-5p.

To determine whether miR-143-5p played a role in the effects of CASC20 in GC cells, we co-transfected si-CASC20 and miR-143-5p inhibitor into SGC7901 and MGC803 cells. CCK8 assays showed that si-CASC20 transfection inhibited GC cell proliferation, and co-transfection of miR-143-5p inhibitor restored proliferation (Figure 4A and B). Transwell experiments showed while transfection of si-CASC20 inhibited the invasion and migration of GC cells, co-transfection of miR-143-5p inhibitor restored the invasion and migration abilities (Figure 4C–E). Wound healing assays also confirmed that co-transfection of miR-143-5p inhibitor partly rescued the inhibitory effect of knockdown of CASC20 on GC cells (Figure 4F–H). These results indicate that CASC20 regulates GC cell activities through miR-143-5p.

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Figure 4 The regulation of cancer susceptibility 20 on gastric cancer cells is mediated by miR-143-5p. A and B: Gastric cancer (GC) cells were treated with si-NC, si-cancer susceptibility 20 (CASC20), si-CASC20 + inhibitor NC, si-CASC20 + miR-143-5p inhibitor, CCK-8 assays evaluated the proliferation; C-E: Transwell assays tested invasion and migration abilities of transfected cells; F-H: GC cells were treated with si-NC, si-CASC20 + inhibitor NC, si-NC + miR-143-5p inhibitor, si-CASC20 + miR-143-5p inhibitor, wound healing assay was used to detect migration ability. ^aP < 0.05 vs control.

To further explore the role of miR-143-5p in GC, we screened and identified the potential target mRNAs of miR-143-5p. Bioinformatics tools miRDB, Targetscan and miRTarbase predicted several mRNAs (HDAC7, ZNF85, ZNF347, MEMO1, ESCO1, MLXIP, MAP3K2 and DYRK1A mRNAs) that potentially interact with miR-143-5p (Figure 5A). Among them, MEMO1 was selected as the target gene of miR-143-5p because it had the highest score in the prediction analysis.

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Figure 5 Cancer susceptibility 20 regulates the miR-143-5p target MEMO1. A: Bioinformatic tools were used to predict the potential target mRNA of miR-143-5p; B: The expression levels of MEMO1 in gastric cancer (GC) from TCGA STAD database; C: MEMO1 expression in 50 pairs of gastric cancer tissues and normal tissue samples; D: Putative miR-143-5p binding sequence and mutation sequence of MEMO1 mRNA were as shown; E: Dual luciferase reporter assays were used to confirm the direct target between miR-143-5p and MEMO1; F: MEMO1 expression was decreased in GC cells transfected by miR-143-5p mimics; G: The correlation analysis between cancer susceptibility 20 (CASC20) and MEMO1 expression in 50 GC tissues; H: MEMO1 expression was decreased after knockdown of CASC20. ^aP < 0.05, ^bP < 0.01 vs control.

TCGA database analysis showed that MEMO1 was highly expressed in GC tissues (Figure 5B). In addition, the mRNA expression of MEMO1 was significantly increased in 50 GC tissues compared with its expression in adjacent tissues (Figure 5C).

We next designed luciferase reporter vectors containing the wild-type 3′UTR of MEMO1 mRNA or the 3′UTR with mutated binding sites (Figure 5D). Dual luciferase reporter assay showed that miR-143-5p significantly reduced the luciferase activity of the reporter vector with the wild-type 3′UTR of MEMO1 mRNA but had no effects on the mutated reporter (Figure 5E), suggesting that MEMO1 mRNA may be a target gene of miR-143-5p. We then transfected miR-143-5p mimics into MGC803 cells and found that the expression of MEMO1 was significantly reduced at both gene and protein levels (Figure 5F). These data indicate that miR-143-5p regulates MEMO1 mRNA expression.

We also found that the expression of CASC20 was positively correlated to MEMO1 expression in GC tissues (Figure 5G). In cells with CASC20 knockdown, MEMO1 expression was downregulated, suggesting a positive regulatory relationship between CASC20 and MEMO1 (Figure 5H).

To explore the role of MEMO1 in GC, we transfected siRNA targeting MEMO1 into GC cells. Scratch experiments and Transwell experiments revealed that knockdown of MEMO1 inhibited the invasion and migration of GC cells. The migration and invasion abilities were partially rescued by co-transfection with miR-143-5p inhibitor (Figure 6A–G), indicating that miR-143-5p inhibition reversed the effects of MEMO1 knockdown.

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Figure 6 Inhibition of miR-143-5p blocked the function of MEMO1 knockdown in gastric cancer cells. A-D: MGC803 and SGC7901 cells were transfected with si-NC, si-MEMO1, si-MEMO1 + inhibitor NC, si-MEMO1 + miR-143-5p inhibitor, respectively. Transwell assay was employed for the detection of cell invasion and migration ability; E-G: MGC803 and SGC7901 cells were transfected with si-NC, si-MEMO1 + inhibitor NC, si-NC + miR-143-5p inhibitor, si-MEMO1 + miR-143-5p inhibitor, respectively. Wound healing assay was used to detect migration ability; H: The protein expression of MEMO1 was detected by western blot assay after transfecting with miR-143-5p mimics or si-cancer susceptibility 20; I: Western blot was utilized to detect the expressions of EMT-related proteins (ZEB1, Vimentin and E-cadherin). ^aP < 0.05 vs control.

To investigate whether CASC20 regulates MEMO1 through miR-143-5p, we transfected si-CASC20 and miR-143-5p inhibitor into MGC803 cells. Western blot results indicated that MEMO1 expression decreased in GC cells after transfection with si-CASC20, while MEMO1 expression was restored after co-transfection with si-CASC20 and miR-143-5p inhibitor (Figure 6H).

We next evaluated the effects of CASC20 on EMT. In MGC803 cells with CASC20 knockdown, the expression level of the epithelial protein marker E-cadherin was up-regulated, while the expression levels of the mesenchymal protein markers Vimentin and ZEB1 were down-regulated. After transfection with miR-143-5p inhibitor, the expressions of MEMO1, Vimentin and ZEB1 were up-regulated, and the expression of E-cadherin was down-regulated (Figure 6H and I).

Together these results indicated that CASC20 may sequester miR-143-5p to increase the MEMO1 expression, leading to the induction of EMT and metastasis of GC.

LncRNAs have been indicated to play critical roles in gastric cancer (GC) tumorigenesis and progression. However, their roles in GC remain to be further elucidated.

To discover promising diagnostic and therapeutic targets for GC.

To investigate the underlying mechanisms of the lncRNA cancer susceptibility 20 (CASC20) in GC.

The expression of CASC20, miR-143-5p and MEMO1 genes were detected by quantitative real-time polymerase chain reaction. The oncogenic functions of CASC20 was investigated by experiments including cell counting kit-8, colony formation, wound-healing and Transwell assays in GC cells in vitro. Bioinformatics and dual-luciferase report assay were performed for determining the regulatory relationship of lncRNA-miRNA-mRNA.

We found that the expression of lncRNA CASC20 was significantly upregulated in GC tissues and cell lines, and increased expression of CASC20 was positively correlated with lymph node metastasis in patients with GC. Additionally, knockdown of CASC20 could inhibit GC cells growth, migration and invasion, whereas its overexpression could reverse these effects. Mechanistically, CASC20 specifically inhibited miR-143-5p expression, thus up-regulating MEMO1 expression.

CASC20 promotes GC cell metastasis via adsorbing miR-143-5p and up-regulating MEMO1, it plays an important oncogenic function in GC.

This study identified the lncRNA CASC20/miR-143-5p/MEMO1 axis in GC and provided insights into the mechanistic relationships among CASC20, miR-143-5p and MEMO. The results of our research suggest that CASC20 may act as a promising diagnostic marker for GC.