miR-182-5p improves the viability, mitosis, migration, and invasion ability of human gastric cancer cells by down-regulating RAB27A

We investigated the effect of miR-182-5p on the viability, proliferation, invasion, and migration ability of human gastric cells by regulating the expression of RAB27A. Real-time PCR assay was used to detect the expression of miR-182-5 and RAB27A in human gastric carcinoma tissues, para-carcinoma tissues, and different cell lines. Western blotting was also used to determine the RAB27A expression in both tissues and cell lines. We chose the HGC-27 cell line as experiment subject as it demonstrated the highest miR-182-5p level. HGC-27 cells were transfected with different vectors and the cell viability, mitosis, invasion, and migration ability were measured through MTT assay, flow cytometry (FCM) analysis, Transwell assay, and wound healing assay. In comparison with the normal tissues, miR-182-5p is expressed at a higher level in gastric cancer (GC) tissues, while RAB27A is expressed at a lower level in cancerous tissues. The down-regulation of miR-182-5p and up-regulation of RAB27A can significantly decrease the viability, migration, invasion, and mitosis of HGC-27 cells. The target relationship between miR-182-5p and RAb27A was confirmed through a dual-luciferase reporter gene assay and Western blot assay. miR-182-5p enhances the viability, mitosis, migration, and invasion of human GC cells by down-regulating RAB27A.


Introduction
Gastric cancer (GC) is one of the most common malignant tumors of the digestive system, has an estimated 951,600 new cases and causes 723,100 deaths every year [1]. Although there has been a steady decline in the incidence and mortality rate of GC in developed countries, in less developed areas, it still has a threatening third rank in both cancer incidence and mortality [1]. The causes for GC are complex and may include factors such as an unbalanced diet, smoking, alcohol, and infection of the Helicobacter pylori [2]. Furthermore, GC pathogenesis is also reported to be related with genetic factors such as DNA methylation, the epigenetic inactivation of several genes, gene amplifications and deletions, and aberrant somatic mutations [2].
The absence of specific clinical symptoms sets obstacles for the early diagnosis of GC [3]. Therefore, patients with GC are always diagnosed at advanced stages leading to serious metastasis and poor prognosis. The 5-year survival rate is less than 30% [4][5][6]. Surgery has been the primary treatment option for GC during the past few decades, with an auxiliary treatment of chemoradiation and chemotherapy [7,8].
Gene-based drug therapy is a potential approach for GC treatment [9]. However, due to the lack of understanding of the molecular mechanisms behind GC development, there is currently no effective therapy for GC [10]. RAB27A is an isoform of RAB27 and a member of the small GTPase Rab family. RAB27A is unique as its dysfunction is related to human hereditary diseases such as type 2 Griscelli syndrome [11]. Previous studies have reported that the deregulation of RAB27A is related with carcinogenesis and progressions such as colorectal carcinoma [12,13], pancreatic cancer [14], and lung cancer [15]. In breast cancer, the overexpression of RAB27A was found to promote various cell activities such as growth, invasion, and metastasis [16,17]. Additionally, scientists have found in vivo that RAB27A can serve as a prognostic biomarker in gliomas [18,19] and hepatocellular cancer [20]. All studies mentioned above jointly indicate that a close relationship exists between RAB27A and cancer. However, the role of RAB27A in GC has not been thoroughly explored. miRNAs are a collection of small non-coding RNAs with a length of 21-25 nt. Through binding to the 3 -UTR regions, miRNA can suppress gene expression at both the mRNA and translational levels [21,22]. Previous studies have suggested that miRNAs such as miR-29c, miR-135b, miR-193b, and miR-532-5p are key regulators of tumor proliferation, apoptosis, and migration. They can also serve as potential biomarkers and therapeutic targets in GC [23][24][25]. The aberrant expression of miR-182-5p has been proven to play an oncogenic role in variant malignant tumors such as bladder [26] and prostate cancer [27]. However, a study performed by Xu et al. [28] indicated that the down-regulation of miR-182-5p promotes the proliferation in renal cell carcinoma by targeting the AKT/FOXO3a signaling pathway.
Data from the TargetScan database suggest that a targeting relationship exists between miR-182-5p and RAB27A. We made the assumption that miR-182-5p regulates activity in GC cells by targeting RAB27A and conducted a series of experiments to test this hypothesis. We investigated the role of miR-182-5p and evaluated its regulatory mechanism in gastric tumorigenesis and progressions.

Human tissues
Thirty human GC and the para-carcinoma tissues (the distance from the gastric carcinoma was 2 cm) were obtained from the Affiliated Yantai Yuhuangding Hospital of Qingdao University from March 20, 2015 to May, 20 2016. Samples were subsequently frozen in liquid nitrogen for further study. Para-carcinoma tissues were identified by three physicians in the hospital and confirmed to be cancer-free. All patients gave their informed consent and the ethical approval was obtained from the Human Ethics Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University.

RNA extraction and quantitative real-time PCR
Total RNA of tissues and cells were extracted using the TRIzol reagent according to the manufacturer's instructions. Total RNAs (5 μg) and miRNAs (5 μg) were then reverse transcribed into complementary DNAs (cDNAs). The SYBR Green PCR mix was used for quantitative analysis in the quantitative real-time PCR (qRT-PCR) assay. The primers for miR-182-5p, RAB27A, and other genes were purchased from RiboBio Co., Guangzhou, China ( Table 1). PCMV-Sport6 vectors (Invitrogen, Carlsbad, CA, U.S.A.) were used to recombine the RAB27A cDNAs, and DNA sequencing technique (contracted with Sangon Biotech, Shanghai, China) was used to confirm the successful recombination. U6 (RNU6B) and GAPDH were used as internal normalization standards for miR-182-5p and RAB27A mRNA respectively. Statistics were analyzed using the 2 − C t method.

Cell transfection and grouping
To investigate the role of miR-182-5p on GC cell activities, HGC-27 cells in the logarithmic growth phase were transfected with 50 nM miR-182-5p mimics or 8 ng mimics control (GenePharma, Shanghai, China) using 1 μl of the Lipofectamine 2000 reagent. The groups were designed as follows: control group, miR-mimics group, anti-miR group, anti-negative control (NC) group, and miR-NC group. To study the role of RAB27A on GC cell activities, HGC-27 cells were transfected with lentiviral vectors recombined with human RAB27A gene sequence (the constructed vector was plenti-GIII-Ubc-RAB27A). These were assigned to the plenty-RAB27A group and cells transfected with the empty vector plasmid were designated as the NC group (named plenti-Null group).

Flow cytometry (FCM)
Cell cycle and cell apoptosis were assessed using flow cytometry (FCM) method. Forty eight hours after transfection, HGC-27 cells were washed with PBS and blended in a −20 • C freezer with 70% alcohol at a concentration of 10 6 cells/ml [29]. Subsequently, cells were left at 4 • C overnight. On the next day, samples were centrifuged, washed with PBS, and then the supernatant was discarded. Cells were then resuspended in a liquor containing PI (50 mg/l) and RNase (50 mg/l) and incubated for approximately 25 min. In addition, other transfected cells were blended with the liquor Annexin V-FITC/PI according to the manufacturer's instructions. After the cells were incubated for 20 min, FCM was used to assess cell apoptosis.

Transwell assay
The invasion ability of cells was assessed using Transwell assay. The upper side of membranes was coated with Matrigel. RPMI 1640 (500 μl) containing 10% FBS was added to the lower chambers. Serum-free medium and the HGC-27 cells (3 × 10 5 ) were placed in the upper chambers. After culturing for 48 h, 4% methanol and Crystal Violet were used to fix and stain the cells that invaded the membrane respectively. Subsequently, a microscope (100×) was utilized to count the cells in ten random fields in order to estimate the relative invasiveness of cells in different groups. Cell optical absorption was measured under 570 nm wavelength in each group.

Wound healing assay
Wound healing assay was conducted to examine the migratory ability of HGC-27 cells. The cell suspension was seeded into a six-well plate with 2 ml of suspension in each well. After the confluence reached 80%, a sterile pipette was used to scratch the cell surface. Cells were then cultured in an incubator at 37 • C and 5% CO 2 . The cell surface was photographed using an inverted microscope and the wound closure was measured at 0 and 24 h after scratching.

Dual-luciferase reporter gene assay
The mutated RAB27A 3 -UTR was constructed using site mutation technology. The wild-type RAB27A 3 -UTR and mutated RAB27A 3 -UTR sequences were amplified using PCR. The amplified sequences were recombined with plasmids that contained the firefly luciferase gene. A total of 3 × 10 5 cells were seeded into a 12-well plate and left overnight. When cells reached approximately 80% confluency, the [30] recombinant vectors and the Rellina Luciferase internal control pRL-CMV were co-transfected with either miR-mimics or miR-NC respectively into the HGC-27 cells using Lipofectamine 2000. The relative activity of luciferase was detected 48 h after transfection using the dual-luciferase reporter gene assay system according the manufacturer's instructions.

Western blotting
A total of 5 × 10 5 cells were seeded into a 6-well plate, cultured overnight, and then the [30] total protein of tissues and cells was extracted using RIPA buffer. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE; 12.5% gel) was conducted to isolate the proteins within the lysates. The proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane and 5% defatted milk was used to block the membrane at room temperature for 1 h. The membranes were incubated with primary antibodies overnight at 4 • C. The next day, membranes were washed with tris-buffered saline with Tween (TBST) and incubated with horseradish peroxidase-coupled secondary antibodies. Signals were observed using chemiluminescence and the Western blotting detection system. ImageJ software was used to analyze the protein density.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 and GraphPad Prism 6.0 software. The statistical significance between two groups was determined using the Student's test, and the mean calculated after each experiment was repeated three times. Measurement data are presented as the mean + − standard deviation (x + − s), and ANOVA was used to compare differences between multiple groups. A P-value < 0.05 is considered to be statistically significant.

Overexpression of miR-182-5p in both GC tissues and cell lines
Quantitative real-time PCR analysis was utilized to investigate the expression of miR-182-5p in tissues and cells. The results demonstrated that GC tissues had a significantly higher expression of miR-182-5p than adjacent tissues (P<0.05) ( Figure 1A). Moreover, the GC cell lines HGC-27, MKN-45, SGC-7901, and MGC-803 had higher levels of miR-182-5p than GES cell lines ( Figure 1B). HGC-27 cells that had the highest level of miR-182-5p expression among the four GC cell lines were chosen for later studies.

Under expression of RAB27A in GC tissues
The RAB27A mRNA levels in GC and adjacent tissues were detected using qRT-PCR. We found that the expression of RAB27A mRNA in GC tissues was much lower than that in adjacent tissues (P<0.05) ( Figure 1C). Western blotting assay was performed to determine the expression of RAB27A protein in GC and adjacent tissues. A lower level of RAB27A protein was seen in GC tissues in comparison with adjacent tissues ( Figure 1D).

Validation of RAB27A 3 -UTR as a direct target of miR-182-5p
The complimentary sequences of miR-182-5p and 3 -UTR of RAB27A were obtained from the TargetScan database and are shown in Figure 2(A). Luciferase assay was used to investigate the binding relationship between miR-182-5p and RAB27A. The wild-type and mutated RAB27A 3 -UTR segments were inserted into the plasmids and transformed into the pGLO-wt and pGLO-mut recombinant vectors. The recombinant vectors and the control plasmid pGLO-NULL were co-transfected with either miR-mimics or miR-NC in HGC-27 cells. The results showed a dramatic reduction in the luciferase activity of the miR-182-5p mimics + pGLO-RAB27A-3 -UTR (wt) group compared with the control group. This validates that RAB27A is negatively regulated by miR-182-5p ( Figure 2B).
Western blotting was used to determine the impact of anti-miR-182-5p on the expression of RAB27A ( Figure 2C). RAB27A expression in the anti-miR, anti-NC, and control groups was detected and the results displayed that the inhibition of miR-182-5p could increase the expression of RAB27A.  The apoptosis rates of cells in different groups are shown in Figure 3(D) and (E). There was no significant difference found between the control and all NC groups. Apoptosis cells in the anti-miR and plenti-RAB27A groups significantly

The low expression of miR-182-5p or the overexpression of RAB27A suppresses the invasive and migratory ability of HGC-27 cells
No significant difference was seen between the control and other NC groups in regard to the invasive and migratory cell numbers. In order to explore the effect of miR-182-5p and RAB27A on cell invasiveness, we manipulated the expression of miR-182-5p and RAB27A in HGC-27 cells. The results are shown in Figure 4(A) and (B). We found that the number of cells that invaded the Transwell polycarbonate filter was significantly less in the anti-miR and plenti-RAB27A groups than in the anti-miR-NC and plenti-NULL groups (P<0.01). Moreover, there was little difference between the plenti-RAB27A+miR-mimics, control, plenti-NULL, and anti-miR-NC groups. These results demonstrate that the under expression of miR-182-5p or the overexpression of RAB27A can negatively regulate the invasiveness of HGC-27 cells.
The migratory ability of HGC-27 cells was measured through the use of wound healing assay ( Figure 4C). The figures show that the migration distance of cells in the anti-miR and plenti-RAB27A groups is significantly lower than that in the corresponding NC groups. The under expression of miR-182-5p or the overexpression of RAB27A can negatively regulate the migration of HGC-27 cells. Compared with the corresponding NC groups, the migration of cells in the anti-miR group and plenti-RAB27A group significantly decreased. ***P<0.01 were considered statistically significant.

Discussion
miR-182-5p was found to be overexpressed in GC tissues than in corresponding adjacent tissues. Similarly, miR-182-5p was found up-regulated in the four GC cell lines (HGC-27, MKN-45, SGC-7901, and MGC-803) in comparison with normal gastric epithelial cells (GES). These results suggest that miR-182-5p might be an oncogene of GC. Also, studies have demonstrated that miR-182-5p functions as an oncogene in different types of cancers such as melanoma [31] and breast cancer [32]. On the other hand, RAB27A was found to be down-regulated in GC tissues and cell lines, indicating that RAB27A may function as a tumor suppressor gene in GC. The Rab family reported by Grosshans et al. [33] was shown to play an essential role in membrane traffic by binding to their effectors. Scientists also found that Rab genes could regulates the activation of signal transduction, cell metabolism, membrane receptor, and the pathophysiology of diseases such as obesity, immune deficiency, diabetes, infection, and cancer [34]. This indicates a close relationship between Rab genes and diverse biological processes. RAB27A tends to be up-regulated in cancers such as hepatocellular carcinoma [20], breast cancer [16], and colorectal carcinoma [12]. In 2013, Wu et al. [35] found that RAB27A can enhance the glioma cell ability, accelerate proliferation and invasion, and inhibit cell apoptosis. We hypothesize that RAB27A may serve as a tumor suppressor by regulating signal transduction, cell metabolism, and membrane receptors in GC.
To see whether miR-182-5p function by regulating one of its effector gene RAB27A, we conducted dual-luciferase gene assay. miR-182 has been reported to target various tumor suppressors such as close homolog of LI (CHL1) and special AT-rich sequence-binding protein 2 (SATB2) in different cancers [36,37]. We confirmed that a targeting relationship exists between miR-182-5p and RAB27A. This was previously predicted by the TargetScan software. We speculate that the miR-182-5p/RAB27A axis has a close relationship with the formation and progression of GC.
It has been illustrated in previous studies that miR-182 and RAB27A are involved in regulating the biological process such as proliferation, apoptosis, migration, and invasion in various cancer cells [35,38,39]. In the present study, we observed that when miR-182-5p was inhibited or RAB27A was overexpressed, cell viability of GC cells line became inhibited, a large proportion of cells were held up in the G0/G1 phase, the apoptosis rate increased, and the invasive and migratory ability of GC cells were weakened. We demonstrated that miR-182-5p promotes the progression of GC by suppressing RAB27A. These may help understand how miR-182-5p/RAB27A influences GC progression at the cellular level.
In conclusion, the present study verifies that a negative regulatory relationship exists between miR-182-5p and RAB27A. This may be a novel target for the treatment and prognosis of GC and a possible diagnostic biomarker in clinical practice.

Author Contribution
Conception and design: Yuling Li, Shudong Chen and Zhengfei Shan; Analysis and interpretation of data for the work: Liyan Bi, Shengqiang Yu and Yongwei Li; Drafting the work and revising: Sen Xu; Final approval: All authors.