Up-regulation of Rb by small activating RNA inhibits cell invasion and migration in gastric cancer cells

Background: Gastric cancer (GC) is a lethal disease that needs further investigation. Recent studies have reported that small activating RNA (saRNA), involved in a process called RNA activation, plays an important role in the development of cancer. Methods: The expression of retinoblastoma (Rb) was detected in human GC tissues and cell lines. We designed three different saRNAs targeting the Rb gene promoter in GC cell lines. Upregulated Rb expression after transfection of the saRNAs was conrmed by PCR and western blotting. And GC cell proliferation, migration, and invasion were detected using CCK8 assays and Transwell assays. Results: Here, we found that Rb displayed lower expression in GC tissues and cells. saRNA-3 signicantly increased the expression of Rb in both SGC-7901 and MKN-28 cells. saRNA-3 signicantly inhibited the proliferation of both cell types by day 3 after transfection. There was no signicant difference between the negative control saRNA and mock transfection groups. saRNA-3 decreased the tumor volume and weight compared with the mock and dsControl groups. In addition, saRNA-3 transfection decreased the migratory ability of SGC-701 and MKN-28 cells. Conclusions: Overall, Rb is a promising novel prognostic biomarker of GC and, due to its role in metastasis, a novel therapeutic target for the clinical management of invasive and metastatic GC.


Background
Gastric cancer (GC) is the fth most common tumor and third leading cause of cancer-related mortality in the world [1]. However, in China, GC has the highest morbidity and mortality rates of all cancers [2].
Although the incidence of GC is decreasing by nearly 2% per year worldwide, the overall treatment effect has not fundamentally improved due to population growth [3]. The highly invasive behavior of GCs often leads to recurrence and metastasis, which are the main causes of death in patients [4]. GC-related genes, including oncogenes and tumor suppressor genes, are closely related to the malignant biological behavior of GC [5]. Therefore, in-depth investigation of the mechanisms of GC-related genes can provide the necessary theoretical basis for identifying new targets for GC treatment.
Small activating RNAs (saRNAs) act mainly on tumor suppressor genes, including those that negatively regulate the cell cycle, promote cell death, and inhibit tumor invasion and metastasis [6]. saRNAs upregulate the expression of such genes to promote tumor cell differentiation, increase apoptosis, and inhibit tumor cells [7]. In the process of RNA activation (RNAa), the expression of saRNA target genes is upregulated after the saRNA binds to a speci c region of the promoter region or against the recognition of a tumor suppressor gene by non-coding small RNAs (microRNAs) [8]. In addition, saRNAs promote post-transcriptional upregulation of target genes via different pathways [9]. Compared with the traditional methods of increasing target gene expression, RNAa has unique advantages involving simple operation, low cost, very speci c activation, and physiological regulation [10]. Furthermore, its effects persist in the cell but can be terminated by stopping the drug, and the activation effect is easily controlled.
At present, the mechanism of RNAa is unclear, but different studies have shown that saRNAs activate target genes at the transcriptional level by binding to certain regions of the promoter or to the antisense transcript of the target gene [11,12]. Because saRNAs act primarily on oncogenes and have achieved important clinical results, we believe that saRNAs targeting tumor suppressor genes may play an important role in the treatment of GC.

Materials And Methods
Collection of gastric cancer tissues and normal tissues 106 GC tissues and corresponding adjacent non-tumorous gastric samples were obtained from Total cellular proteins were extracted using 1% NP-40 and 1 mM phenylmethylsulfonyl uoride and were quanti ed using the bicinchoninic acid assay (Sigma-Aldrich, St. Louis, MO, USA). The proteins were then separated by SDS-PAGE and electroblotted onto polyvinylidene di uoride membranes. After sequential incubations with primary and secondary antibodies, the bands were visualized by enhanced chemiluminescence using the Immobilon TM Western Kit (Millipore, Burlington, MA, USA). GAPDH served as a loading control.

Cell proliferation assay
The proliferation of the GC cell lines SGC-7901 and MKN-28 was assessed using the Cell Counting Kit-8 (CCK8: Beyotime Biotechnology, Shanghai, China). All procedures were performed according to the manufacturer's instructions.

Cell migration and invasion assays
After 72 h, the cells were harvested following treatment with the Rb-targeting saRNA, dsControl, mock transfection, or saRNA/short-hairpin RNA combinations. Cell invasion and migration were measured via Transwell assays using chambers with membranes containing 8 μm pores (Corning, Corning, NY, USA).
The cells were suspended in 100 μL serum-free medium and seeded in the upper chamber, and the lower chamber was lled with 500 μL complete media. After incubation at 37°C for 24 h, the Transwell membranes were xed and stained with 0.5% Crystal Violet/methanol solution for 30 min. The cells that did not migrate were removed using cotton swabs, and the membranes were photographed using a digital camera. In the cell invasion assay, the Transwell membranes were coated with Matrigel (BD Biosciences, San Jose, CA, USA), and the cell incubation period was 48 h.

In vivo model of tumor growth
All animal experiments were approved by the Animal Care and Ethics Committee of Shandong Provincial Hospital a liated to Shandong University and conducted in the Animal Center of Shandong Provincial Hospital a liated to Shandong University. Animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press). The Rb-targeting saRNA and dsControl saRNA were transfected into GC cells, and the mock control group consisted of GC cells treated with the transfection reagent only. Nude mice were randomly divided into three groups (n = 6) consisting of mice injected with the following: (1) GC cells transfected with the Rb-targeting saRNA (saRNA group), (2) GC cells transfected with the dsControl saRNA (dsControl group), and (3) mock transfected GC cells (mock group). The cells (5 × 10 6 ) were subcutaneously injected into the right side of nude mice. After 21 days, the mice were sacri ced, and solid tumors were isolated for further analysis.

Statistical analysis
All experimental results are expressed as the mean ± standard deviation, and one-way analysis of variance followed by the Tukey-Kramer test for multiple comparisons were conducted using Prism statistical software (GraphPad, San Diego, CA, USA). Differences with a value of P < 0.05 were considered statistically signi cant.

Results
Rb was downregulated expressed in GC tissue and cells In the rst, we detected the expression of Rb in GC tissues and cells. As shown in Fig. 1A, Rb level was signi cantly decreased in GC tissues compared with the adjacent normal tissues. Besides, Rb expression in GC cells (MKN-28 and SGC-7901) was also signi cantly downregulated compared with the human gastric mucosal epithelial cell line (GES-1) (Fig. 1B).
saRNA transfection e ciency First, we determined the transfection e ciency of the saRNAs targeting Rb. According to PCR, of the three saRNAs tested, saRNA-3 signi cantly increased the expression of Rb in both SGC-7901 and MKN-28 cells (P < 0.01). However, there was no signi cant difference in Rb expression between the control groups (P > 0.05) and the cells transfected with saRNA-1 or saRNA-2 (Fig. 2).

Signi cant activation of Rb expression by saRNA in GC cells
Next, our date showed that saRNA-3 notably increase the Rb expression levels in mRNA and protein (Fig.3). The Rb mRNA (4.96 folds or 4.53 folds) and protein (2.87 folds or 2.64 folds) levels after transfected with saRNA-3 were increased compared with the mock group (P < 0.01).

Effective inhibition of in vitro proliferation by saRNA in GC cells
The CCK-8 assay was used to detect the effect of saRNA-3 on SGC-7901 and MKN-28 cell proliferation.
saRNA-3 signi cantly inhibited the proliferation of both cell types by day 3 after transfection. In addition, there was no signi cant difference in proliferation between the mock and dsControl groups (Fig. 4).

Effective inhibition of in vivo proliferation by saRNA in GC cells
For in vivo validation, we established a GC model by subcutaneously inoculating cells into BALB/c nude mice. saRNA-3 decreased the tumor volume and weight compared with the mock and dsControl groups, consistent with the in vitro results (Fig. 5).
Effective inhibition of migration and invasion by saRNA in GC cells saRNA-3 transfection decreased the migratory ability of SGC-701 and MKN-28 cells compared with the control groups (P < 0.01; Fig. 6). Conversely, there was no signi cant difference between the dsControl and mock groups (P > 0.05). Moreover, the invasion assay showed that saRNA-3-transfected SGC-701 and MKN-28 cells exhibited a lower invasion rate compared with the dsControl and mock groups (P < 0.01; Fig. 7).

Discussion
Globally, GC is the third most lethal tumor, with a mortality rate of approximately 723,000 patients per year. Despite advances in medical technologies, the clinical manifestations of GC are not obvious, and it is usually not detected until an advanced stage. Therefore, the mortality of GC patients therefore has remained high. GC is a heterogeneous disease associated with a variety of genetic backgrounds and epigenetic changes, including genetic mutations, somatic cell copy number changes, structural variations, and epigenetic variations. Characterizing the molecular mechanisms involved in GC progression is therefore a di cult challenge. However, identifying new molecular targets in GC may lead to novel approaches to determine disease prognosis and suitable clinical treatment strategies.
Since the discovery of saRNAs, different RNAa models have been developed, and numerous studies have been conducted on this class of molecules. Janowski and Li et al. con rmed that different saRNAs activate E-cadherin, p21, VEGF, and PR and inhibit tumor cell growth [13,14]. Junxia et al. reported that saRNAs inhibit breast and bladder cancers by activating E-cadherin [15]. Kang et al. established an orthotopic tumor model in mice and treated bladder cancer cells with relevant saRNA liquid particles to successfully inhibit tumor growth [16]. Ren also con rmed this mouse model using a similar experiment.
Furthermore, saRNAs inhibited prostate cancer cell growth and prolonged survival in mice [17]. In our previous study, we designed an saRNA targeting the VEZT gene and found it inhibited the proliferation, invasion, and migration of GC cells via upregulated expression of VEZT [18].
Kundson et al. evaluated the genetic basis of childhood retinoblastoma in 1971 and found that tumor formation requires simultaneous deletion of 13q14 or inactivation of a pair of alleles of the Rb gene. Lee et al. sequenced and predicted that the protein corresponded to this RNA, and the gene was identi ed and isolated in 1989. Because the gene is closely related to the occurrence of retinoblastoma, it was named the Rb gene, which was the rst successfully cloned tumor suppressor gene [19]. Recently, it was reported that the Rb gene is closely related to the development of many malignant tumors such as retinoblastomas, osteosarcomas [20], breast cancer [21], bladder cancer [22], and lung cancer [23]. Furthermore, inactivation of Rb contributes to tumor formation.
In the present study, we rst used three saRNAs to reactivate Rb expression in GC cells (SGC-7901 and MKN-28) and selected saRNA-3 because it most effectively upregulated Rb expression. saRNA-3 inhibited GC cell proliferation both in vivo and in vitro.

Conclusion
Our ndings also con rmed that saRNA-3 signi cantly inhibited GC cell invasion and migration. These results indicate that saRNA-mediated upregulation of Rb inhibited the tumorigenicity of GC cells.
WGC conceived the study. PLP, ZSJ and KS designed the experiments; TSB, XT and PLP conducted the experiments. PLP, WGC and ZSJ wrote the manuscript. All authors have read and approved the nal manuscript.