miR-335-5p suppresses gastric cancer progression by targeting MAPK10

Recent studies have established the roles of microRNAs (miRNAs) in cancer progression. The aberrant expression of miR-335-5p has been reported in many cancers, including gastric cancer (GC). In this study, the precise roles of miR-335-5p in GC as well as the molecular mechanisms underlying its effects, including the role of its target MAPK10, were evaluated. Quantitative real-time PCR was used to evaluate miR-335-5p levels in GC cell lines and tissues. MTT and colony formation assays were used to detect cell proliferation, and Transwell and wound-healing assays were used to evaluate the invasion and migration of GC cells. The correlation between levels of miR-335-5p and the cell cycle-related target gene mitogen-activated protein kinase 10 (MAPK10) in GC was analyzed. In addition, the candidate target was evaluated by a luciferase reporter assay, qRT-PCR, and western blotting. The levels of miR-335-5p were downregulated in GC tissues and cell lines. Furthermore, miR-335-5p inhibited the proliferation and migration of GC cells and induced apoptosis. Additionally, miR-335-5p arrested the cell cycle at the G1/S phase in GC cells in vitro. Levels of miR-335-5p and the cell cycle-related target gene MAPK10 in GC were correlated, and MAPK10 was directly targeted by miR-335-5p. These data suggest that miR-335-5p is a tumor suppressor and acts via MAPK10 to inhibit GC progression.


Background
Gastric cancer (GC) is still a significant public health problem worldwide [1,2]. Over 1,000,000 new cases and an estimated 783,000 deaths were reported in 2018, making it the fifth most frequently diagnosed cancer and the third leading cause of cancer deaths [3]. A wide range of factors, such as lifestyle, Helicobacter pylori infection, polyps, gastric ulcers, genetic factors, and gastric residual tissue, may be involved in gastric tumorigenesis [4]. Although there are many methods for the diagnosis and treatment of GC, 30% of patients are diagnosed at an advanced stage [5]. Thus, useful biomarkers for early screening or detection are essential for improving survival rates [6]. miRNAs of about 22 nucleotides contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors [10]. For example, miR-181d [11], miR-99a [12], miR-105 [13], and others have suppressive effects on GC development, whereas other miRNAs, including miR-188-5p [14] and miR-221 [15], promote GC growth.
MiR-335-5p is abnormally expressed in many cancers. For instance, miR-335-5p is significantly downregulated and has a vital role in the metastasis of non-small cell lung cancer [16]. miR-335-5p is downregulated in breast cancer cells and is a promising biomarker for breast cancer treatment [17]. Additionally, miR-335-5p is downregulated in renal cell carcinoma and is a candidate therapeutic target [18]. The downregulation of miR-335 in GC has been reported [19]; however, its precise roles in GC cells are not fully understood.
MiRNAs function by binding to the 3′UTR of target mRNAs in a complementary base-pairing manner, thereby contributing to cell apoptosis, proliferation, and differentiation [20][21][22][23]. They are thought to regulate more than 50% of protein-coding genes. Based on a literature review and gene target prediction algorithms, including TargetScan, miRanda, and miRBase, we hypothesized that mitogen-activated protein kinase 10 (MAPK10) is a potential target of miR-335-5p. MAPK10 is a member of the Jun N-terminal kinase subgroup of mitogen-activated protein kinases. MAP kinases act as integration points for multiple biochemical signals and are involved in a wide variety of cellular processes, such as proliferation, differentiation, transcription regulation, and development [24,25]. Expression patterns of MAP kinases differ depending on the tumor type. Zhang et al. showed that MAPK10 is expressed at low levels in cervical cancer tissues and cells [26]. However, the percentage of MAPK10 protein-positive cells is significantly higher in ovarian serous, mucinous, and clear cell carcinomas than in normal tissues [27].
In this study, the effect of miR-335-5p on GC progression via MAPK10 was evaluated. In particular, we compared the expression levels of miR-335 in GC tissues and cells with those in matched normal tissues and MKN-28 and SGC-7901 cell lines. In addition, we used bioinformatics approaches, luciferase assays, qRT-PCR, and western blotting to evaluate its relationship with MAPK10 and further evaluated the functions of miR-335 and MAPK10 in GC cell proliferation, metastasis, and apoptosis. Overall, our results demonstrated that miR-335 suppresses the progression of GCs by targeting MAPK10. Human GC cell lines (AGS, BGC-823, MKN-45, MKN-28, and SGC-7901), a normal gastric epithelial cell line  (GES-1), and model cells (HEK-293) were provided by the Biomedical Experiment Center of Xi'an Jiaotong University (China). The use of these cell lines was approved by the Ethics Committee of Yan'an University College of Medicine (China). Human GC cells were cultured in DMEM (PAA Laboratories, Pasching, Australia) containing 10% fetal bovine serum and RPMI1 640 medium (PAA Laboratories) at 37 °C in a 5% CO 2 incubator. The culture medium was changed once every 2-3 days. MKN-28 and SGC-7901 cells in the logarithmic growth phase were collected and subjected to the following experiments.

Cell transfection
GC cells in the logarithmic growth phase were digested and inoculated onto a 6-well culture plate. After cells reached 60-80% confluence, the miR-335-5p-mimics and miR-335-5p-inhibitor (GenePharma, Shanghai, China) were added to the corresponding wells for further culture for 24-48 h.

MTT assay
Cell proliferation was assessed using the MTT Kit (Sigma, St Louis, MO, USA). Cells in the logarithmic growth phase were harvested and seeded on a 96-well plate. At 24, 48, and 72 h after seeding, 10 μL of MTT was added to each well and the cells were incubated for 4 h. Each well was supplemented with 150 μL of DMSO, and the optical density (OD) was recorded at 490 nm.

Colony formation detection
Transfected cells in the logarithmic growth phase were seeded onto a 6-well plate. After 2 weeks of culture, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. Images were obtained and cells were counted.

Flow cytometry
Transfected cells in the logarithmic growth phase were inoculated onto a 6-well plate and cultured for 1 day. Cells were fixed in 70% ethanol for 24 h and treated with propidium iodide and RNase provided with the kit. The cell cycle distribution was detected by flow cytometry.

Cell invasion assay
Transwell chambers (8-μm pore size; Millipore, Billerica, MA, USA) were coated with Matrigel (15 μg/filter; BD Biosciences, Franklin Lakes, NJ, USA). Cells (2.0 × 10 4 ) in serum-free medium were added to the upper chamber, and the bottom wells were filled with complete medium. The cells were allowed to cross the Matrigel-coated membrane for 48 h.

Wound-healing assay
A wound-healing assay was performed to examine metastasis. Briefly, after cells reached 90% confluence in 12-well plates, a single scratch wound was generated with a 200-μL disposable pipette tip. The extent of wound closure was measured after 48 h.

Statistical analysis
Results are shown as means ± SEM of at least three different experiments. The SPSS22.0 was used for statistical analyses. Bioinformatics analyses were performed using the ggstatsplot package in R. Experimental data were processed using GraphPad Prism7.0. Comparisons were conducted with the independent t-test. P < 0.05 was considered statistically significant.

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 (Fig. 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 (Fig. 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 (Fig. 1c). Thus, miR-335-5p may act as a tumor suppressor in GC.

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 (Fig. 2a, b). 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 (Fig. 2c). 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) 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 (Fig. 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.

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 (Fig. 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 (Fig. 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 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) 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 (Fig. 3c). These results suggest that mir-335-5p is involved in the progression, migration, and invasion of GCs.

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 (Fig. 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 (Fig. 5d-g), suggesting that MAPK10 plays a key role as an oncogene in GC.

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 (Fig. 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 (Fig. 6b). MAPK10 was successfully knocked down by siRNA, as verified by analyses of both at the mRNA levels (Fig. 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 (Fig. 6d, e). Moreover, the influence of MAPK10 siRNA on the cell cycle was similar to the effect of miR-335-5p upregulation (Fig. 6f ). Consistent with the effect of miR-335-5p on GC cell apoptosis, MAPK10 knockdown induced apoptosis in MKN28/ SGC-7901 cells (Fig. 6g), suggesting that MAPK10 is involved in the progression of GC.

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 (Fig. 7a). Transwell assays demonstrated that MAPK10 silencing inhibited the invasion and migration ability of GC cells (Fig. 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 (Fig. 7c), suggesting that MAPK10 functions as an oncogene in GC. We concluded that miR-335-5p suppresses GC progression by targeting MAPK10 (Fig. 7d).

Discussion
The occurrence of GC is a complex process involving multiple factors, including genetic and epigenetic events. Many miRNAs have been demonstrated to contribute to GC tumorigenesis and development, and these serve as a valid diagnostic and therapeutic targets [28]. In addition, some specific microRNAs able to regulate the expression of genes in GC cells at the post-transcriptional level have been identified as diagnostic biomarkers for GC [29][30][31]. Therefore, studies of potential miRNAs associated with the development of GC may provide opportunities for improvements in diagnosis, treatment, and prognosis. In this study, the roles of mir-335-5p in GC were evaluated. Recent studies have shown that miR-335-5p acts as a tumor suppressor and inducer in several cancer types [32][33][34][35]. For example, miR-335-5p is downregulated in thyroid cancer cells and inhibits proliferation [36]. miR-335-5p functions via lactate dehydrogenase B to exert tumor inhibitory effects in colorectal cancer [37]. In the present study, based on expression analyses of miR-335-5p in tissues, we found that miR-335-5p acts as a tumor suppressor in GC, and the overexpression of miR-335-5p inhibits proliferation, invasion, and metastasis and induces apoptosis in vitro. Additionally, miR-335-5p could induce cell cycle arrest at the G1 phase and downregulated the G0 and G1 phase cycle-associated proteins Cyclin D1, CDK 6, and CDK4. The abnormal activation of CDK and its modulators has been reported in many tumors [38,39]. Furthermore, miRNAs participate in the regulation of the cell cycle [40,41]. For example, the miR-15a/16 family regulates G0/G1 cell cycle progression by targeting cyclin D1 (CCND1) [42] addition, miR-16 regulates various mRNA targets, including CDK6, CDC27, and G1-related cyclins, which jointly control cell cycle progression [43]. These studies strongly support our observation that miR-335-5p plays a key role in cell proliferation in GC. Migration and invasion are closely related to the occurrence and development of tumors. Moreover, many migration-and invasion-related proteins, including E-cadherin, vimentin, and β-catenin, are involved. For instance, p0071 interacts with E-cadherin in the cytoplasm and promotes invasion and metastasis in non-small cell lung cancer [44]. Furthermore, ubiquitin specific peptidase 20 (USP20) regulates the deubiquitination of β-catenin to control the invasion and migration of cancer cells [45]. Our results showed that the overexpression of mir-335-5p decreases the expression of vimentin and β-catenin and increases E-cadherin in MKN-28 and SGC-7901 cells. Our data suggest that mir-335-5p is involved in the migration and invasion of GCs.
In addition, our results showed that the upregulation of miR-335-5p inhibits the expression of MAPK10 in MKN-28/SGC-7901 cancer cells at the RNA and protein levels. Using bioinformatic analyses and a dual-luciferase reporter assay, we demonstrated that miR-335-5p directly targets MAPK10 by binding to its 3′-UTR and inhibiting translation. To further clarify the tumor suppressive effect of miR-335-5p via MAPK10, siRNA was used. MAPK10 silencing inhibited cell proliferation and migration and induced cell apoptosis, similar to the observed effects of miR-335-5p overexpression in GC cells in vitro. Accordingly, the expression levels of related proteins, including CDK6, CDK4, CyclinD1, BCL-2, BAX, E-cadherin, vimentin, and β-catenin were also altered by siMAPK10. MAPK10 is a member of the Jun N-terminal kinase subgroup of the mitogen-activated protein kinases, which are implicated in important physiological processes [46]. MAPK10 regulates the occurrence and development of several types of cancer. The downregulation of MAPK10 contributes to the suppression of ovarian cancer [47]. miR-27a-3p promotes the growth and invasion of NPC cells by targeting Mapk10 [48]. These 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) results robustly suggested that the downregulation of MAPK10 induced by miR-335-5p could inhibit GC progression. Our findings highlight that mir-335-5p or MAPK10 may be considered as potential targets for GC therapy in the near future.

Conclusion
We obtained the following new findings.