miR-335-5p inhibits GC cell proliferation in vitro
To investigate the role and function of miR-335-5p in GC cells, we analyzed its expression in 22 pairs of GC tissues and matched adjacent non-cancerous tissue samples by qRT-PCR. miR-335-5p levels were significantly lower in GC samples than in non-cancerous tissue samples (Figure 1A). These results were validated in five GC cell lines. miR-335-5p levels was lower in the BGC-823,SGC-7901, MKN-45,MKN-28, and AGS cell lines than in the GES-1 cell line (Figure 1B). To clarify the function of miR-335-5p in GCs, MKN-28 and SGC-7901 cells were selected for further analyses. As determined by qRT-PCR, miR-335-5p mimics successfully elevated miR-335-5p expression in two cell lines; the effect of the inhibitor was moderate due to the low expression of endogenous miR-335-5p in MKN-28 and SGC-7901 cells (Figure 1C). Thus, miR-335-5p may act as a tumor suppressor in GC.
Down-regulation of miR-335-5p in GC tissues and cells. (A) qRT-PCR analysis of miR-335-5p expression in 22 paired human gastric cancer and adjacent normal tissues. The expression of miR-335-5p was normalized to U6. (B) qRT-PCR analysis of miR-335-5p expression in normal gastric mucosal and gastric cancer cells and normalized against U6 RNA. (C) Expression levels of miR-335-5p were determined by qRT-PCR in GCs transfected with miR-335-5p mimics, inhibitor, or respective controls (*P < 0.05, **P < 0.01, and ***P < 0.005).
miR-335-5p induces cell cycle arrest and apoptosis in GC
Gain- and loss-of-function analyses were conducted by transfecting MKN-28 and SGC-7901 cells with miR-335-5p inhibitor-ctrl, inhibitor, miR-ctrl, and mimics. MTT and colony formation assays showed that the upregulation of miR-335-5p in MKN-28 and SGC-7901 cells inhibited cell growth and colony formation, while the inhibition of miR-335-5p exerted moderate adverse effects on GC cells, which may be explained by the low levels of miR-335-5p in MKN-28 and SGC-7901 cells (Figures 2A and 2B). Consistent with these results, a flow cytometry analysis revealed that the upregulation of miR-335-5p arrested cells in the G0/G1 phase and inhibited the transition to the G2/M phase; similar effects were not observed in the miR-ctrl-transfected cells (Figure 2C). Furthermore, flow cytometry confirmed that the upregulation of miR-335-5p induces apoptosis in GC cells. However, the miR-335-5p inhibitor resulted in a slight but non-significant difference in apoptosis compared to that in cells transfected with the negative control, which may be explained by the low expression level and low inhibitory efficiency in MKN-28 and SGC-7901 cells (Figure 2D). The inhibition of MiR-335-5p promoted proliferation and inhibited apoptosis in GC cells, while the inverse results were obtained in the miR-335-5p mimic group.
miR-335-5p inhibited proliferation and promoted apoptosis in gastric cancer cells. (A) The effects miR-335-5p on gastric cancer cell proliferation were determined by an MTT assay after the transfection of MKN-28/SGC-7901 cells with an miR-335-5p mimic or miR-335-5p inhibitor at 24, 48, and 72 h. (B) The growth of MKN-28/SGC-7901 cells was detected by colony formation after transfection with the miR-335-5p mimic or inhibitor. (C) Cell cycle progression was evaluated in MKN-28/SGC-7901 cells transfected with miR-335-5p inhibitor-ctrl, inhibitor miR-335-5p ctrl, and mimics. After 48 h, the cell cycle distribution was analyzed by flow cytometry. The histogram indicates the percentages of cells in G0/G1, S, and G2/M phases. (D) Apoptosis was detected by annexin-V/propidium iodide combined labeling flow cytometry in MKN-28/SGC-7901 cells 48 h after transfection with miR-335-5p inhibitor-ctrl, inhibitor, miR-335-5p ctrl, and mimics. Apoptosis was evaluated as the percentage of apoptotic cells (*P < 0.05, **P < 0.01, and ***P < 0.005).
Inhibition of miR-335-5p induces the migration and invasion of gastric cancer cells
To further confirm that miR-335-5p acts as a tumor suppressor, its effects on the invasion of MKN-28 and SGC-7901 cells were evaluated by a Transwell invasion assay and wound-healing assay. In the wound-healing assay, migration was slower in the miR-335-5p-transfected cells than in un-transfected cells. Over time, the difference in the metastasis rate between the two groups increased (Figure 3A). In the Transwell invasion assay, the transfection of cells with miR-335-5p mimics significantly impaired invasion compared to that in the miR-335-5p-ctrl group in MKN-28 and SGC-7901 cells. In contrast, the knockdown of miR-335-5p enhanced GC cell invasion. When transfected with the mir-335-5p inhibitor, the MKN-28 and SGC-7901 cell invasion rates increased significantly (Figure 3B). These results support the hypothesis that miR-335-5p contributes to the suppression of invasion and metastasis. To investigate the mechanisms underlying the roles of miR-335-5p in apoptosis and cell cycle progression, we measured the expression levels of apoptosis- and cell cycle-related proteins in GC cells. The transfection of MKN-28/SGC-7901 cells with MiR-335 downregulated CDK6, CDK4, CyclinD1, and BCL-2 and upregulated the expression of BAX. The overexpression of miR-335-5p reduced the expression levels of vimentin and β-catenin, and significantly increased E-cadherin levels in MKN-28 and SGC-7901 cells. Our results showed that the silencing of miR-355-5p significantly increased the relative expression levels of vimentin and β-catenin and decreased E-cadherin expression, comparable with the effects of miR-355-5p overexpression in MKN-28 and SGC-7901 cells (Figure 3C). These results suggest that mir-335-5p is involved in the progression, migration, and invasion of GCs.
miR-335-5p inhibited the migration and invasion of MKN-28 and SGC-7901 cells. (A) Scratch wound-healing assays of MKN-28 and SGC-7901 cells after treatment with miR-335-5p inhibitor-ctrl, inhibitor, miR-ctrl, or miR-335-5p mimics. (B) Transwell analysis of MKN-28 and SGC-7901 cells after transfection with miR-335-5p mimics, inhibitor, or their respective controls. (C) Western blot analysis of CDK6, CDK4, CyclinD1, BCL-2, BAX, E-cadherin, Vimentin, and β-catenin expression in MKN-28 and SGC-7901 cells transfected with miR-335-5p, inhibitor, or their respective controls.
MAPK10 is a direct functional target of miR-335-5p in GC cells
miRNA target prediction algorithms were used to search for potential miR-335-5p target genes. Levels of MAPK10 and mir-335-5p expression in GC based on TCGA data showed a negative correlation (P < 0.001; Figure 4A). MAPK10 had a potential miR-335-5p-binding site in the 3′-UTR and therefore was selected as a candidate target. To determine whether MAPK10 was directly targeted by miR-335-5p, we subcloned 3′-UTR MAPK10 fragments including wild-type (MAPK10-WT) and mutant (MAPK10-MUT) miR-335-5p-binding sites into the pmiRGLO dual-luciferase reporter vector (Figure 4B). pre-miR-335 and MAPK10-WT- or MUT-3′-UTR vectors were co-transfected into HEK293 cells. The relative luciferase activity of the MAPK10-WT pmirGLO-3-UTR vector was significantly reduced in miR-335-overexpressing HEK293 cells. As expected, miR-335-5p failed to inhibit the luciferase activity of the MAPK10-MUT pmirGLO-3′-UTR vector, indicating that miR-335-5p binds directly to the 3′-UTR of MAPK10 (Figure 4C). qRT-PCR was used to verify the relationship between miR-335-5p and MAPK10. The mRNA levels of MAPK10 decreased significantly by miR-335 mimics and increased by miR-335 inhibitors in MKN-28 and SGC-7901 cells. (Figure 4D) The protein levels of MAPK10 decreased significantly by miR-335 mimics and increased by miR-335 inhibitors in MKN-28 and SGC-7901 cells (Figure 4E). These findings demonstrated that miR-335-5p could directly target MAPK10 and suppress its expression in GC cells.
MAPK10 is a direct target of miR-335-5p in gastric cancer cell lines. (A) Correlation between MAPK10 expression and miR-335-5p expression in gastric cancer based on TCGA data. (B) A luciferase assay was performed using HEK293 cells in which miR-335 was co-transfected with the pGLO-MAPK10 wild-type or pGLO-MAPK10 mutant vector. (C) miR-335-5p is highly conserved across species and has binding sites within the 3′-UTR of human MAPK10. (D) mRNA expression levels of MAPK10 were measured by qRT-PCR after transfection with miR-335-5p mimics, miR-335-5pinhibitor, or their negative controls in MKN-28 and SGC-7901 cells. (E) Protein expression levels of MAPK10 were measured by western blotting after transfection with miR-335-5p mimics, miR-335-5pinhibitor, or their respectively negative controls in MKN-28 and SGC-7901 cells (*P < 0.05, **P < 0.01, and ***P < 0.005).
Bioinformatics analysis of MAPK10 in gastric cancer
The TCGA database was used to elucidate the effect of MAPK10 in GC tissues by a bioinformatics approach. The expression of MAPK10 was higher in GC tissues than in healthy counterparts, and its expression was associated with the histologic and pathologic stages of GC (Figure 5A–C). The expression of MAPK10 was associated with the DFI (disease-free interval, P = 0.033), PFI (progression-free interval, P = 0.013), DSS (disease-specific survival, P = 0.0068), and OS (overall survival, P = 0.017) in GC (Figure 5D–G), suggesting that MAPK10 plays a key role as an oncogene in GC.
(A–C) The expression of MAPK10 is associated with the pathologic and histologic grade of GC. (D–G) Bioinformatics analyses were used to elucidate the effect of MAPK10 in GC tissues. The expression of MAPK10 was related to the DFI (disease-free interval event), PFI (progression-free interval event), DSS (disease-specific survival event), and OS (overall survival).
Knockdown of MAPK10 reduces GC progression
We knocked down MAPK10 expression by RNA interference (small interfering RNA (siRNA)) to confirm that MAPK10 mediates the antitumor effects of miR-335-5p. MAPK10 expression levels were higher in GC cells than in GES-1 cells (Figure 6A) and were highest in MKN-28 and SGC-7901 cells. Western blotting indicated that the MAPK10 was obviously up-regulated in GC tissues than in their counterparts at the protein level (Figure 6B). MAPK10 was successfully knocked down by siRNA, as verified by analyses of both at the mRNA levels (Figure 6C). Similar to miR-335-5p-overexpressing cells, the downregulation of MAPK10 significantly inhibited proliferation and slightly inhibited colony formation in MKN-28 and SGC-7901 cells (Figure 6D and 6E). Moreover, the influence of MAPK10 siRNA on the cell cycle was similar to the effect of miR-335-5p upregulation (Figure 6F). Consistent with the effect of miR-335-5p on GC cell apoptosis, MAPK10 knockdown induced apoptosis in MKN28/SGC-7901 cells (Figure 6G), suggesting that MAPK10 is involved in the progression of GC.
Inhibition of MAPK10 suppressed GC progression. (A) mRNA and protein expression levels of MAPK10 in various GC and GES-1 cells. (B) MAPK10 protein expression in GC tissues vs counterparts' tissues was confirmed by using western blotting. (C) Expression levels of MAPK10 were measured by qRT-PCR in MKN-28 and SGC-7901 cells transfected with siMAPK10. (D) An MTT assay was performed to determine the growth of gastric cancer cells treated with siMAPK10 or a negative control (si-ctrl). (E) A colony formation assay was performed several days after the transfection of gastric cancer cells with siMAPK10 or a negative control (si-ctrl). (F) The cell cycle distribution was determined in gastric cancer cells 48 h after transfection with siMAPK10 by propidium iodide staining and flow cytometry. The histogram indicates the percentages of cells in G0/G1, S, and G2/M cell cycle phases. (G) Apoptosis was determined in gastric cancer cells at 48 h after transfection with siMAPK10 (*P < 0.05, **P < 0.01, and ***P < 0.005).
Knockdown of MAPK10 reduces the migration and invasion of GC cells
We silenced MAPK10 expression by RNA interference (RNAi) to evaluate whether it contributes to the effects of miR-335-5p on invasion and metastasis using MKN-28 and SGC-7901 cells. Based on a wound-healing assay, the group with low MAPK10 expression showed reduced rates of migration (Figure 7A). Transwell assays demonstrated that MAPK10 silencing inhibited the invasion and migration ability of GC cells (Figure 7B). Based on a western blot analysis, silencing MAPK10 significantly increased the relative expression levels of E-cadherin and decreased vimentin and β-catenin expression. These results were consistent with the effects of miR-355-5p overexpression in MKN-28 and SGC-7901 cells (Figure 7C), suggesting that MAPK10 functions as an oncogene in GC. We concluded that miR-335-5p suppresses GC progression by targeting MAPK10 (Figure 7D).
miR-335-5p inhibited cell invasion via MAPK10 knockdown. (A) Wound-healing assays of MKN-28 and SGC-7901 cells after treatment with si-ctrl and si-MAPK10. Representative images were captured at 0 h, 24 h, 48 h, and 72 h after transfection with si-ctrl and si-MAPK10. (B) The invasion viability of MKN-28 and SGC-7901 cells transfected with siMAPK10 was determined by a Transwell invasion assay. (C) The expression levels of MAPK10 were measured by western blotting in MKN-28/SGC-7901 cells transfected with siMAPK10. Protein expression levels of CDK6, CDK4, CyclinD1, BCL-2, BAX, E-cadherin, Vimentin, and β-catenin in gastric cancer cells transfected with siMAPK10 or si−ctrl were analyzed by western blotting. (D) Proposed model for the suppressive effect of miR-335-5p on gastric cancer progression via MAPK10 knockdown.