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Introduction

Gastric cancer (GC) is one of the five common types of tumors and ranks the third leading cause of cancer-related mortality worldwide1. Despite new improvement to the standard of care therapy for GC patients, the prognosis of patients who were diagnosed in advanced stages remains poor, with a 5-year survival rate of less than 23%2,3. A better understanding of the regulatory mechani-sms underlying the development and progression of GC is critical for developing novel and effecti-ve therapeutic targets.

MicroRNAs (miRNAs) are noncoding RNAs of approximately 19-23 nucleotides that are tran-scribed by RNA polymerase II into pri-miRNAs4. Growing evidence has suggested that miRNAs play an important role in many physiological and pathological processes such as cell growth and differentiation, proliferation and apoptosis5,6. Re-cent studies indicated that aberrant expression of miRNAs can act as tumor suppressors or onco-genes, depending on the cellular function of their targets7. For instance, Zhou et al8 reported that miR-27a-3p promoted gastric cancer progression by directly targeting BTG2. Shan et al9 found that miR-448 acted as a tumor suppressor regulating lung cancer cells growth and metastasis by tar-geting DCLK1. Bao et al10 indicated that miR-194 suppressed proliferation of GC cell through targeting KDM5B. Previous reports have shown that miR-802 was involved in the carcinogene-sis of some tumors11,12. However, the expression and role of miR-802 in progression of GC has not been reported.

In the present work, we firstly detect the expres-sion levels of miR-802 in GC tissues and cells. In addition, the biological of forced miR-802 expres-sion in proliferation, migration, invasion and

Abstract. – OBJECTIVE: The aberrant expres-sion of microRNAs (miRNAs) has been observed in various types of cancer. Recently, miR-802 was found to play important role in tumor pro-gression. However, the function of miR-802 in gastric cancer (GC) remains unknown. The aim of the present study was to investigate biologi-cal effects and underlying mechanisms of miR-802 in GC.

PATIENTS AND METHODS: Quantitative RT-PCR was performed to evaluate the expression level of miR-802 in GC tissues and cell lines. The in vitro cell proliferation was measured using the MTT method. Cell invasion and migration assays were performed using the transwell assay. The effects of miR-802 on tumor growth were exam-ined using a GC xenograft model. Flow cytome-try method was used to detect the effect of miR-802 in apoptosis of GC cells. Targets of miR-802 were predicted using bioinformatics and validat-ed using luciferase reporter and Western blot analyses.

RESULTS: The results showed that miR-802 was significantly down-regulated in GC tissues and cell lines. The enforced expression of miR-802 induced growth suppression and apopto-sis of GC cells. Moreover, miR-802 overexpres-sion suppressed the migration and invasion of GC cells. Bioinformatics analysis predicted that the RAB23 was a potential target gene of miR-802. The results of luciferase reporter assay demonstrated that miR-802 could directly target RAB23. Additionally, in vivo analysis, the xeno-graft mouse model also confirmed the suppres-sive effects of miR-802 on tumor growth.

CONCLUSIONS: Our results are the first to demonstrate the tumor-suppressive role of miR-802 in GC. The identification of miR-802 and its novel target RAB23 will be valuable in develop-ing therapeutic applications for GC.

Key Words: miR-802, Gastric cancer, Proliferation, Metastasis,

RAB23.

European Review for Medical and Pharmacological Sciences 2017; 21: 4071-4078

X.-Y. ZHANG1, J.-H. MU1, L.-Y. LIU1, H.-Z. ZHANG2

1Department of General Surgery, The Third Affiliated Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China2Department of General Surgery, Mongolian Medicine Hospital of Bazhou, Hebei Province, China

Corresponding Author: Xian-Yi Zhang, MD; e-mail: hb691ww@yeah.net

Upregulation of miR-802 suppresses gastric cancer oncogenicity via targeting RAB23 expression

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apoptosis of GC cells was analyzed. Furthermore, we identified RAB23 as a miR-802 target in GC.

Patients and Methods

PatientsTotal 46 cases of GC tissues and the matched

adjacent normal tissues used in the study were collected from 46 patients between 2014 and 2015 in our hospital. All specimens were obtai-ned from patients through surgical resection and then histologically proven to be GC. All parti-cipants signed an informed consent for the use of their samples before recruitment. Written con-sent was received from all the patients and all the experiments have been approved by the Ethics Committee of our hospital.

Cell Lines and TransfectionFour GC cell lines (SGC-7901, MGC-803,

HGC-27, BGC-823), and normal gastric epithelial cell line (GES-1) were obtained from the Insti-tute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China). GC cells were cultured in Dulbecco’s modified Eagle’s me-dium (DMEM), Invitrogen (Carlsbad, CA, USA), supplemented with heat-inactivated 10% fetal bo-vine serum (HIFBS), Invitrogen (Carlsbad, CA, USA) in six-well plates. For transfection, miR-448 mimic and corresponding negative control (miR-NC) were synthesized by RiboBio (Guangzhou, China). Cell transfections were performed using Lipofectamine 2000 kit Invitrogen (Carlsbad, CA, USA) following the manufacturer’s instructions.

Real-time Quantitative Reverse Transcription-PCR(qRT-PCR)

TRIzol Reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from the GC cell lines and the patients’ samples according to the manufacturer’s instructions. cDNA was synthe-sized with the PrimeScript R Treagent Kit 128 (Promega, Madison, WI, USA). The expression levels of miR-802 and RAB23 mRNA were de-tected by qRT-PCRs. All qRT-PCRs were per-formed in triplicates on an ABI 7500 Real-ti-me PCR system (Applied Biosystems, Foster City, CA, USA). Primers for miR-802, RAB23 mRNA and the internal control GADPH gene were purchased from Ambion (Applied Biosy-stems, Foster City, CA, USA). The relative expression levels of RNA were quantified using 2-ΔΔCT method.

Cell Proliferation AssayHGC-27cells were seeded into 96-well plates

(6.0×103 cells per well). Cell viability was investi-gated by MTT assay (Beyotime Institute of Bio-technology, Shanghai, China). The absorbance at 450 nm was measured with a microplate reader. Each sample was assayed in triplicate.

Colony Forming AssayCells were seeded into 6-cm plates after the

cells were transfected with miR-802 mimic or miR-NC and then the cells were digested with trypsin for preparation of single cell suspension. After visible colonies formed in a fresh six-well plate, Cell colonies were fixed with cold metha-nol, stained with 0.1% crystal violet and counted.

Apoptosis DetectionApoptosis was assessed using Annexin V-FITC/

PI double staining kit (Beyotime, Jiangsu, China). Cell samples were analyzed by flow cytometry (FACScan, BD Biosciences, Franklin Lakes, NJ, USA). Each sample was assayed in triplicate.

In Citro Cell Transwell AssaysFor the invasion assay, 2x105 cells were plated

in the top chamber with a Matrigel-coated mem-brane (24-well insert; 8 μm pore size; BD Bio-sciences, Franklin Lakes, NJ, USA). The lower chamber was filled with 500 μl Roswell Park Me-morial Institute 1640 (RPMI-1640) medium sup-plemented with 10% fetal bovine serum (FBS) and the upper chamber was filled with cell suspen-sion. Following 24 h of incubation at 37°C, cells that invaded the matrigel to the lower membrane were stained with 0.1% crystal violet. Filtered cel-ls counted under a microscope (DP50; Olympus Corporation, Tokyo, Japan). The migration assay is the same with invasion assay. However, Matri-gel was not used and the permeating time for cells was 12 hours.

In vivo TumorigenicityXenograft model was used to detect the miR-

802 on the tumorigenicity. The procedure was de-scribed by previous study13.

Luciferase Reporter AssayA normal and a mutated 3’-UTR of RASA1

were constructed using PCR, and were then in-serted into the multiple cloning sites in the psi-CHECK-2 vector. GC cells were seeded into a 24-well plate (1×104 per well), and were co-tran-sfected with a luciferase reporter construct (200

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ng) and miRNA-802 mimics (50 nM) using Lipo-fectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA). After transfection for 48 h, luciferase activity was measured using the dual luciferase reporter assay system. All experiments were per-formed in triplicate.

Western Blot AnalysisThe cells were lysed on ice with RIPA lysis

buffer (Applygen Technologies Inc., Beijing, China) supplemented with protease inhibitors. Equal sample volumes of proteins (30 μg) were loaded for 10% sodium dodecyl sulphate-polya-crylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. The polyvinylidene fluoride (PVDF) membranes were blocked using 5% non-fat dry milk in a tris buf-fered sodium with tween-20 (TBST-20) solution at room temperature for 1 h, and incubated with monoclonal rabbit anti-RAB23 (1:1,000; Hai-ding, Beijing, China) overnight at 4˚C. Detection was performed with peroxidase-conjugated se-condary antibodies using an enhanced chemilu-minescence system (Applygen Technologies Inc., Beijing, China). The blot was photographed by FluorChem imaging system (Alpha Innotech. Corp., San Leandro, CA, USA). GADPH was used as a loading control.

Statistical AnalysisThe data were expressed as mean ± SD (stan-

dard deviation, SD), and were analyzed using Stu-dent’s t-test between two groups in vitro and in

vivo data. All data were analyzed using GraphPad Prism version 5.01 (GraphPad Software, San Die-go, CA, USA). p-values < 0.05 were considered to be statistically significant.

Results

Expression of miR-802 in GC Tissues and Cells

We firstly detected the expression levels of miR-802 in GC tissues and cells by RT-PCR. The results showed that the expression levels of miR-802 in GC tissues were lower than those in matched normal tissues (Figure 1A). Similarly, miR-26a was lower in all GC cells (SGC-7901, MGC-803, HGC-27, BGC-823) compared with normal gastric epithelial cell line (GES-1) (all p < 0.05, Figure 1B).

MiR-802 Overexpression Suppressed the GC Cell Proliferation, Colony Formation, and Induces Apoptosis

To discover the effect of miR-802 on GC cell proliferation, miR-802 mimics were tran-sfected into the human GC cell line HGC-27. The over-expression of miR-802 was confirmed by RT-PCR (Figure 2A). Then, colony formation assays and MTT were performed to detect the effect of miR-802 on GC cell proliferation. As shown in Figure 2B, we found that up-regulation of miR-802 in HGC-27 cells significantly inhibi-ted cell proliferation (p < 0.05), compared with

Figure 1. Up-regulation of expression of miR-802 in GC tissues and cell lines. (A) RT-PCT was used to detect expression of miR-802 in human GC tissues and normal gastric tissues. (B) RT-PCT was used to detect expression of miR-802 in GC cell lines (SGC-7901, MGC-803, HGC-27, BGC-823) and normal gastric epithelial cell line (GES-1). *p < 0.05 and **p < 0.01.

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the NC transfection. Furthermore, similar fin-dings were observed in colony formation assays (Figure 2C). Based on above results, we wonde-red whether miR-802 could influence HGC-27 apoptosis. Next, we performed flow cytometry assays and found that up-regulation of miR-802 inhibited apoptosis (p < 0.05, Figure 2D). These results suggested that miR-802 might be a tumor suppressor in GC.

MiR-802 Suppressed GC Cell Metastasis in vitro

In order to investigate whether the miR-802 played a critical role in GC cell metastasis, we performed transwell assays. As shown in Figure 3A, we observed that overexpression of miR-802 resulted in decreased migration of HGC-27 cells relative to controls. The similar findings were also observed in invasion assay (Figure 3B). These re-sults indicated that miR-802 plays a suppressive regulator in GC cell metastasis. Then, we tried to

explore the potential mechanism of miR-802 on regulating GC metastasis. Our attention focused on MMP-2 and -9, which play a critical role in the metastasis of GC. We detected the expression of MMP-2 and -9in HGC-27cells by Western blot-ting, our data showed that the expression level of MMP-2 and -9 were decreased in GC cells tran-sfected with miR-802 mimics (Figure 3C).

Identification of RAB23 as a Target Gene of miR-802

To explore how miR-802 affected malignant development of LSCC, we searched for potential regulatory targets of miR-802 by two prediction tools (miRanda and TargetScan). As shown in Fi-gure 4A, Sp1 may be a direct target gene of miR-802, and revealed that RAB23 mRNA contained a miR-802-nucleotide seed match at position of the RAB23 3’-UTR. We constructed luciferase reporter vectors to identify whether RAB23 is a direct target of miR-802 by luciferase reporter as-

Figure 2. Effects of miR-802 mimics transfection on cell proliferation, colony formation, apoptosis, of HGC-27 cells. (A) miR-802 levels in HGC-27cells transfected with miR-NC or miR-802 mimic. (B) The proliferation of HGC-27 cells was detected with MTT assay. (C) Effects of miR-802 overexpression on the colony formation of HGC-27 cells were determined. (D) Cell apoptosis was detected by flow cytometric analysis following transfection with miR-802 mimic or miR-NC. *p < 0.05 and **p < 0.01.

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says. The results showed that overexpression of miR-802 mimics led to a reduction of luciferase activity when the reporter construct contained the RAB23 3’-UTR (Figure 4B). However, muta-tion of the miR-802 binding site from the RAB23 3’-UTR abolished this effect of miR-802 (Figure 4B). Moreover, miR-802 over-expression signifi-cantly down-regulated RAB23 mRNA and pro-tein expression in HGC-27 cells (Figure 4C-D). Taken together, our results suggested that miR-802 modulates RAB23 expression by directly tar-geting its 3’-UTR.

The Effect of miR-802 in GC Xenograft Growth

The above experiment showed that miR-802 served as a tumor suppressor by inhibiting GC proliferation and metastasis. To confirm those results, we further examined the effects of miR-802 on tumor growth using a GC xenograft mo-del. Mice were transplanted with HGC-27 cells overexpressing miR-802 or miR-NC. The results showed that overexpression of miR-802 signifi-cantly reduced xenograft tumor volume (Figure 5A) and tumor weight (Figure 5B). Thus, it was also demonstrated that miR-802 interference inhibited the tumor growth in vivo.

Discussion

MiR-802, a miRNA located on chromosome 21, has been reported to be involved in some tu-mors progression14. For instance, Yuan et al15 found that miR-802 exhibits its antitumor effect in breast cancer cells by regulating FoxM1. Wu et al16 repor-ted that elevated expression of miR-802 inhibited tongue squamous cell carcinoma cell viability and invasion through inhibiting MAP2K4 expression. Wang et al17 indicated that overexpressed miR-802 significantly suppressed EMT, migration and inva-sion in prostate cancer cells by regulating Flot-2. All these studies suggested that miR-802 served as a tumor suppressor in various cancers. To our best knowledge, whether miR-802 played a similar role in GC remains unknown.

In the present work, we determined the expres-sion of miR-802 in GC tissues and cell lines. As expected, our results showed that miR-802 expression was significantly down-regulated in GC tissues and cell line, suggesting that miR-802 may exert a tumor-suppressive role in GC. To confirm our hypothesis, we used miR-802 mimic to up-regulate the expression levels of miR-802 in GC cells. Then, we performed in vitro and in vivo analysis to detect the role of miR-802 in GC

Figure 3. Effect of miR-802 on HGC-27 cell migration and invasion. (A-B) Transwell invasion and migration assays showed that the number of invaded/migrated cells was significantly lower in the miR-802-transfected group than in the other group. The MMP-2 and -9 protein expression in HGC-27 cells transfected with miR-802 mimic was determined by Western blotting. *p < 0.05 and **p < 0.01.

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cells proliferation, migration, invasion and apop-tosis. Our data also confirmed our suggestion that over-expression of miR-802 suppresses GC pro-gression. In order to explore how miR-802 regu-

lated GC cell invasion and migration, we focused on MMP-3 and -9; it is now widely accepted that the overexpression of MMPs, especially MMP3, may accelerate the process of invasion18,19. Our

Figure 5. miR-802 upregulation inhibited GC HGC-27 xenograft growth. (A) Growth curves of tumor volumes in xenografts of nude mice were established based on the tumor volume. (B) The weight of tumor tissues of different groups was measured. *p < 0.05 and **p < 0.01.

Figure 4. miR-802 targets the RAB23 gene. (A) The predicted binding sites of miR-802 in the WT and MUT 3’-UTR of RAB23. (B) Luciferase reporter assay of HGC-27 cells transfected with the wide-type or mutant-type RAB23 reporter plasmid and miR-802 mimic or miR-NC. (C) RT-qPCR detected RAB23 mRNA expression in HGC-27 cells transfected with the miR-802 mimics. (D) Western blot analysis of RAB23 protein expression in HGC-27 cells transfected with the miR-802 mimics. *p < 0.05 and **p < 0.01.

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results showed that over-expression of miR-802 resulted in down-regulation of MMP3- and -9.

RAB23 is located in the pericentromeric re-gion of chromosome 6. Increasing evidence sug-gests that RAB23 overexpression plays pivotal roles in malignancies. For example, Jiang et al20 reported that overexpression of RAB23 resulted in increased cell growth and invasion by promo-ting NF-κB signaling. Jian et al21 also reported that RAB23 promoted squamous cell carcinoma cell migration and invasion via integrin β1/Rac1 pathway. More importantly, Hou et al22 found that RAB23 served as a tumor promoter in GC. On the other hand, previous studies showed that RAB23 was a target of several tumors23,24. Importantly, Bin et al25 confirmed that over-expression of miR-367 suppressed the invasion and metastasis of gastric cancer by directly RAB23. All these evi-dence revealed the important value of miRNAs and RAB23. Thus, we searched the miRanda and TargetScan, and identified that RAB23 may be a target of miR-802. Furthermore, we demonstra-ted that RAB23 was a direct target of miR-802 in GC cells by luciferase reporter assay and Western blot analyses. We observed that overexpression of miR-802 repressed RAB23 expression at both mRNA and protein levels. Taken together, our results revealed an important role of miR-802 in regulating tumorigenesis possibly through regu-lating RAB23.

Conclusions

We demonstrated that miR-802 was frequent-ly downregulated in GC tissues and serves as a tumor suppressor by directly targeting RAB23. Therefore, this research may provide a therapeu-tic strategy for patients with GC.

Conflict of interestThe authors declare no conflicts of interest.

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