Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying both Ligand and siRNA

Gastric cancer is the second leading cause of cancer-related death worldwide. RNA nanotechnology has recently emerged as an important field due to recent finding of its high thermodynamic stability, favorable and distinctive in vivo attributes. Here we reported the use of the thermostable three-way junction (3WJ) of bacteriophage phi29 motor pRNA to escort folic acid, a fluorescent image marker and BRCAA1 siRNA for targeting, imaging, delivery, gene silencing and regression of gastric cancer in animal models. In vitro assay revealed that the RNA nanoparticles specifically bind to gastric cancer cells, and knock-down the BRCAA1 gene. Apoptosis of gastric cancer cells was observed. Animal trials confirmed that these RNA nanoparticles could be used to image gastric cancer in vivo, while showing little accumulation in crucial organs and tissues. The volume of gastric tumors noticeably decreased during the course of treatment. No damage to important organs by RNA nanoparticles was detectible. All the results indicated that this novel RNA nanotechnology can overcome conventional cancer therapeutic limitations and opens new opportunities for specific delivery of therapeutics to stomach cancer without damaging normal cells and tissues, reduce the toxicity and side effect, improve the therapeutic effect, and exhibit great potential in clinical tumor therapy.

fluorescent magnetic nanoparticles 12 , Her2 monoclonal antibody-conjugated RNase-A-associated CdTe quantum dots 13 , folic acid conjugated upper conversion nanoparticles 14 , Folate conjugated gold nanorods 15 , ce6-conjugated carbon dots 16 , ce6-conjugated Au nanoclusters(Au NCs) 17,18 . However, clinical translation of these prepared nanoparticles still presents great challenge because all these prepared nanoparticles are not only distributed to the site of gastric cancer, but also partially accumulated in other organs. The development of safe and effective nanoparticles for in vivo targeted delivery, imaging and simultaneous therapy of early gastric cancer have become our major concerns.
In recent years, several new nano-delivery systems with different materials and physic-chemical properties have been developed 19 . However, effective strategies to block tumor progression and prevent metastasis are lacking, there are several challenges including specific cancer targeting, tissue penetration, intracellular delivery, toxicities and side effects due to organ accumulation, nonspecific cell entry, particle heterogeneity, aggregation, dissociation due to dilution after systemic injection, and unfavorable pharmacological profiles [20][21][22][23][24] . In recent years, RNA nanotechnology has shown great advances as a new theranostic platform for medical applications 25,26 . RNA nanoparticles can be fabricated with precise control of shape, size and stoichiometry, as demonstrated by the packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor, which forms dimmers, trimers, and hexamers via hand-in-hand interactions of the interlocking loops [27][28][29] . The pRNA contains an ultra-stable three-way junction (3WJ) motif [30][31][32] , which can be assembled from three short fragments with extremely high affinity. Recently we have obtained the crystal structure of the pRNA-3WJ motif 33 and a variety of therapeutic RNA nanoparticles using the pRNA-3WJ and pRNA-X motifs as scaffolds have been constructed 34,35 . The pRNA-3WJ nanoparticles display thermodynamically stable properties, including high melting temperature with low free energy, resistance to denaturation in 8 M urea, and resistance to dissociation at very low concentrations in the blood 31 . Boiling resistant RNA nanoparticles with controllable shapes and defined stoichiometry have recently been reported 36 . Various imaging groups, such as fluorophores; targeting ligands, such as receptor binding aptamers; and therapeutic modules, such as siRNA, miRNA or ribozymes can be integrated into the 3WJ scaffold without affecting the folding and functionality of the core motif and incorporated functional moieties 27,30,31,35 . Upon 2'-Fluoro (2'-F) modifications of Uracil (U) and Cytosine (C) nucleotides, the RNA nanoparticles become resistant to RNase degradation with enhanced in vivo half-life while retaining authentic functions of the incorporated modules 32,37 . Furthermore, the pRNA nanoparticles are non-toxic, non-immunogenic, and display favorable biodistribution and pharmacokinetic profiles in mice 32 . These favorable findings prompted the use of this novel platform for the treatment of stomach cancer, which is one of the challenging tasks in clinical oncology.
Such targeted delivery systems call for a ligand-receptor pair that is specifically found in cancer cells. Many, but not all, cancer cells, including stomach, ovarian, lung, breast, kidney, endometrium, colon and hematopoietic cells, over-expressed folate receptors (FRs) than normal cells for high uptake of folate 38 , since folate is essential component during DNA replication and methylation in highly proliferating cells 39 . Folic acid (FA), a synthetic oxidized form of folate, has been widely used as a ligand conjugate in various cancer targeting materials [40][41][42][43][44][45][46][47][48] . BRCAA1 (breast cancer-associated antigen 1,AF208045) has been confirmed to exhibit over-expression in breast cancer and gastric cancer, and no or lower expression in normal gastric mucosa and normal breast tissues 49 . Our previous studies have demonstrated that gastric cancer MGC803 cells were transfected with constructed plasmids of shRNA-BRCAA1, the cell growth was greatly inhibited and the rate of cell apoptosis was significantly higher than those of untransfected group and mock plasmid transfected group 50 . We also screened out a new antigen epitope SSKKQKRSHK 49 , and also screened out matched two monoclonal antibody cell lines, and successfully prepared monoclonal antibody conjugated fluorescent magnetic nanoparticles, and realized the targeted imaging and hyperthermal therapy of in vivo gastric cancer 12,[51][52][53][54] . Therefore, the BRCAA1 gene is a potential therapeutic target for gastric cancer. We also confirmed that folic acid receptor exhibited overexpression in gastric cancer MGC803 cells, prepared folic acid-conjugated silica-modified gold nanorods were successfully used for X-ray/CT imaging-guided dual-mode radiation and photothermal therapy of gastric cancer 15 .
Herein, we adopted an innovative RNA nanotechnology approach to overcome some of the aforementioned challenges, and report for the first time a new strategy to target and deliver therapeutic BRCAA1 siRNA to in vivo stomach cancer tissues using FA-conjugated pRNA-3WJ nanoparticles. Our objective is to construct multi-functional, thermodynamically and chemically stable RNA nanoparticles that allow specific binding to stomach cancer specific cell surface antigens or receptors resulting in the internalization of RNA nanoparticles into target cells and delivery of the siRNA, miRNA, and drugs for attaining synergistic effects for the treatment of stomach cancer, we also investigated the effects of prepared RNA nanoparticles on the regression of gastric cancer tissues in vivo, and potential molecular mechanism, with the aim of laying foundation for further clinical application in near future.

Materials and Methods
Construction and characterization of FA conjugated BRCAA1-siRNA pRNA-3WJ nanoparticles. The pRNA-3WJ nanoparticle consisted of three fragments, a 3WJ , b 3WJ and c 3WJ , was functionalized with folate, as targeting ligand; Alexa 647 , as imaging module; and BRCAA1 siRNA (or scrambled control), as therapeutic module. The RNA fragments were then synthesized chemically (TriLink), self-assembled into RNA nanoparticles, and characterized by 1.2% agarose gel shift assays and Atomic Force Microscopy (AFM) imaging as well as Zeta potential/Particle Sizer, as described previously 52 .
In order to evaluate the effects of a wide pH range on the stability of RNA nanoparticles, the prepared RNA nanoparticles were dispersed in varied pH buffers for 12 h, RNA nanoparticles/ buffer = 1:1(v/v), and pH ranged from 2 to 13 (Table s1 in supporting data: details of preparation of a series of buffer solutions), then 1.2% agarose gel electrophoresis was used to characterize the stability of prepared RNA nanoparticles. Effects of pH on the fluorescent intensity of RNA nanoparticles were investigated by measuring the fluorescent intensity of RNA samples with different pH via the photoluminescence (PL) spectra (Perkin Elmer LS55 spectrofluorimeter).
As we previously reported 25,26 , the RNA nanoparticles contained 2'-F modified U and C nucleotides to make them resistant to RNase degradation. However, Effects of RNAase A on the stability of RNA nanoparticles was still investigated. RNase A-free purified water was used to dilute RNAse A (Sigma Company), the resulting solutions were respectively exhibited different concentration of RNAse A (10 U, 50 U, 100 U, 500 U, 1000 U, 10000 U) , then, each tube was respectively added into 1μ g RNA nanoparticles, incubated at 37 °C for 12 h, then we used 10% SDS-PAGE(sodium dodecylsulfate-polyacrylamide gel electrophoresis) gel electrophoresis to observe effects of RNAse A on the stability of RNA nanoparticles. The pRNA-3WJ was prepared by diluting 100 μ M of the complexes in diethylpyrocarbonate (DEPC) treated water with PBS at 1:1 (v/v) right before the experiments.
Effects of prepared RNA nanoparticles on cell binding efficiency and specificity. The human gastric cancer MGC803 cells and human gastric epithelial GES-1 cells (Cell Bank of Type Culture Collection of Chinese Academy of Sciences) were maintained at 37 °C (5% CO 2 ) in Dulbecco's Modified Eagle's Medium (DMEM, HyClone) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 U/mL penicillin, and 1 mg/mL streptomycin. Cell culture products and reagents were purchased from GIBCO. 200 nM AlexaFluor647 labeled 3WJ-FA-A647 was incubated with 1 × 10 5 MGC803 and GES-1 cells at 37 °C for 1 h, after washing with PBS for three times, the cells were collected and resuspended in PBS buffer, followed by analyzed with a FACS Calibur (BD Biosciences).
In order to investigate the specificity of RNA nanoparticles binding to MGC803 cells, MGC 803 cells were cultured in a humidified 5% CO2 balanced air incubator at 37 °C for 2 days. All the cells were collected and implanted onto 18 mm glass coverslips in a 12-well tissue culture plate, and culturing was continued for 3 days. After the cells were rinsed 3 times, 500 μ L of medium containing prepared RNA nanoparticles was added into each dish and incubated for 30 min. Three dishes of all dishes were first incubated with free folic acids for 30 min, then incubated with RNA nanoparticles, then washed with PBS buffer, and then examined under the dark field microscopy. Dark-field images were obtained on an upright Olympus IX71 optical mi8croscope integrated with a CRi Nuance multispectral imaging system(Cambridge Research & Instrumentation, Inc., Woburn, MA, USA).

Effects of RNA nanoparticles on the silencing of BRCAA1 gene in MGC803 cells. MGC803
cells were transfected with a positive BRCAA1 siRNA control using Lipofectamine 2000 (Invitrogen) as the carrier. Two 3WJ-RNA constructs were assayed for the subsequent BRCAA1 gene silencing effects: one harboring folate and BRCAA1 siRNA; and, the other harboring folate and BRCAA1 siRNA scramble control. After 48 h of treatment, total RNAs from MGC803 cells were isolated by using Trizol (Invitrogen) and Direct-zol™ RNA MiniPrep (Zymo Research) according to manufacturer's instructions. First-strand cDNA was obtained by using 1 μ g of total RNA and random primers and M-MLV reverse transcriptase (Promega). All reactions were carried out in a final volume of 25 μ l and assayed in triplicate. qRT-PCR was performed using a BioRad iQ5 iCycler Detection System with a three-step PCR protocol (95 °C for 10 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 30 s) with HieffTM qPCR SYBR® Green Master Mix (Yeasen). The data was analyzed by the Δ Δ CT method. The primers for BRCAA1 and GAPDH are as follows: BRCAA1: forward: 5'-ACCAAATCTCCCGCAAGG-3'; reverse: 5'-CATATTTTCCAGGTCCGACA-3' . GAPDH: forward: 5'-GAAGGTGAAGGTCGGAGTC-3'; reverse: 5'-GAAGATGGTGATGGGATTTC--3' . The qRT-PCR data were treated by using comparative Ct method, the calculation formation is as follows: 2-Δ Δ Ct; Δ Δ Ct = (treated group Ct-treated group GAPDH Ct)-(control group Ct-control group GAPDH Ct). The results obtained indicate the relative ratio is calculated that target gene mRNA expression levels in the treated group are divided by mRNA expression level in the control group.

Effects of RNA nanoparticles on growth and apoptosis of MGC803 cells. Effects of prepared
RNA nanoprobes on viability of MGC803 cells and GES-1 cells were analyzed using Cell Counting Kit-8 (CCK8) assay 23 . MGC803 cells and GES-1 cells were cultured in the 96-well microplate at the concentration of 5000 cells per well and incubated in a humidified 5% CO2 balanced air incubator at 37 °C for 24 h. Except for control wells, the remaining wells were added into medium with prepared RNA nanoparticles, final concentrations were, respectively, 10, 20,40 and 80 μ g/ml, then those cells were continued to culture for 24 h, 48 h and 72 h, respectively, then, the ODs were measured using the thermomultiskan MK3 ELISA plate reader according to the protocol of CCK8 assay kit, and calculated the survival rate of cells. The survival rate of cells can be calculated by the following equation: Cell RNA nanoparticles for targeted therapy of in vivo gastric cancer. Nude mice loaded with gastric cancer MGC803 cells were prepared according to our previous reports [12][13][14][15] , and were randomly divided into three groups: test group (10 mice) (FA-pRNA-3WJ-BRCAA1siRNA, 1 mg/kg body weight); control group (10 mice) (FA-pRNA-3WJ-Scram siRNA, 1 mg/kg body weight) and blank control (10 mice) (untreated). When the tumor sizes reached about 5 mm in diameter, the nude mice were injected with prepared RNA nanoparticles in PBS via tail vein (1 mg/kg body weight). Every two days, the tumor volume was measured, up to 15 days. Then, these mice were sacrificed.
Effects of RNA nanoparticles on important organs. The mice in testing group were sacrificed after being raised for 15 days. For histological evaluation, excised important organs including heart, liver, spleen, lung and kidney were frozen and embedded by medium at − 20 °C, and then were sectioned into 8 μ m slices, then were stained by hematoxylin and eosin (HE) stain method, and were observed by microscopy to confirm whether there is pathological lesion in important organs existed. Statistical analysis. Each experiment was repeated three times in duplicate. The results were presented as mean ± SD. Statistical differences were evaluated using the t-test and considered significance at P< 0.05.

Results
Construction and characterization of triple-functional pRNA-3WJ nanoparticles. The pRNA-3WJ nanoparticles were prepared by mixing the three strands a 3WJ , b 3WJ , and c 3WJ respectively, at equal molar ratio (Fig. 1a). The dynamic light scattering (DLS) experiments showed that the size of the nanoparticle is 5.20 ± 0.83 nm in diameter, and the zeta potential is − 16.57 ± 0.75 mv, as shown in Figure  S1A and S1B (supporting data). The effects of pH on the fluorescent intensity and stability of RNA nanoparticles were also investigated. As shown in Figure S2A (supporting data), in the range of pH 2 to 13, RNA nanoparticles exhibited different fluorescent intensity, in the range of pH 5-9, RNA nanoparticles displayed more than 90% strong fluorescent signals. As shown in Figure S2B (supporting data), prepared RNA nanoparticles displayed the identical position on the gel, similar brightness, no degradation, which highly suggest that prepared RNA nanoparticles are very stable in the range of pH 2 to 13.
The melting temperature of the 3WJ-BRCAA1 siRNA nanoparticle was determined as 69.2 ± 0.9 °C by real-time PCR, as shown in Figure S3 (supporting data). Effects of RNAase A on the stability of RNA nanoparticles were also investigated. As shown in Figure S4 (supporting data), RNA nanoparticles on different lanes exhibited identical position, similar brightness, no obvious degradation, which highly suggests that prepared RNA nanoparticles own good stability against RNase A (less than 10000 U) degradation.
The resultant pRNA-3WJ nanoparticles are thermodynamically and chemically stable, which makes them an attractive candidate for in vivo nano-delivery for the purpose of cancer detection or treatment. In our study, we incorporated folate, as targeting ligand; Alexa 647 , as imaging module; and BRCAA1 siRNA (or scrambled control) into the pRNA-3WJ scaffold.
Binding efficiency of pRNA nanoparticles to gastric cancer cell. Flow cytometry data in Fig. 2 showed that the prepared 3WJ-FA-A647 nanoparticles can bind with the MGC803 cells with almost 100% binding efficiency, while the GES-1 cells display a weak signal, which highly suggested that the prepared RNA nanoparticles did not bind with GES-1 cells. Our results also demonstrate that folate receptor exhibits over-expression on the surface of MGC803 cells, no expression on the surface of GES-1 cells, similar to our previous report 15 . Fig. 3a showed that, prepared FA-pRNA-3WJ-BRCAA1 siRNA nanoparticles could knockdown the expression of BRCAA1 gene in MGC803 cells after incubating with MGC803 cells for 48 h, in contrast, prepared FA-pRNA-3WJ-Scram siRNA nanoparticles could not knockdown the expression of BRCAA1 gene in MGC803 cells after incubation for 48 h, between two groups, there existed statistical difference (P < 0.01). Compared with BRCAA1 siRNA, prepared FA-pRNA-3WJ-BRCAA1 siRNA nanoparticles achieved similar silencing efficiency of BRCAA1 gene in MGC803 cells. The Ct, ΔCt, and ΔΔCt values for the qRT-PCR assay are shown in Table S2 (supporting data). Additionally, as shown in Fig. 3b, Western blotting results further confirmed that prepared FA-pRNA-3WJ-BRCAA1 siRNA   siRNA group, inhibition rate is 12.5 ± 1.9%, there existed statistical difference between two groups, P < 0.01.

Effects of RNA nanoparticles on the silence of BRCAA1 gene in MGC803 cells. The qRT-PCR results in
Our previous study shows that BRCAA1 can inhibit MGC803 cell apoptosis and improve the proliferation of MGC803 cells, we hypothesized that the performed RNA interference (RNAi) by RNA nanoparticles could induce MGC803 cell apoptosis. As shown in Fig. 5, the transfection with 25 nM FA-pRNA-3WJ-BRCAA1 siRNA in MGC803 cells induced 2.51% of early apoptotic cells and 15.0% of late apoptotic cells, respectively, in the normal control, MGC803 cells exhibited 0.085% of early apoptotic cells, there existed statistical difference between treated group with FA-pRNA-3WJ-BRCAA1 siRNA and control group, P < 0.05. The light scattering plot of MGC 803 cells treated with FA-pRNA-3WJ-BRCAA1 siRNA nanoparticles for 48 h is shown in Figure S5. These results show that prepared FA-pRNA-3WJ-BRCAA1 siRNA nanoparticles can induce apoptosis of MGC803 cells.
Fluorescent RNA nanoparticles for in vivo imaging of gastric cancer. It has been reported that unmodified siRNA ribonucleic acid sequences have extremely poor pharmacokinetic properties due to short in vivo half-life and fast kidney clearance caused by their small size (hydrodynamic diameters, HDs; typically < 5 nm, which is smaller than the kidney filtration threshold (KFT) of 5.5 nm). Tumor targeting efficiency by RNA nanoparticles was investigated by collecting and analyzing in situ fluorescence images of MGC803 xenografts in nude mice at different post-injection (p.i.) time points (Fig. 6a,c). Tumor area  was hardly distinguished in the mouse in the first 30 min p.i. because of the strong fluorescence background in normal tissues. However, as the time increased, the decrease in the fluorescence background of normal tissues and the accumulations at the tumor site caused the tumor area became readily defined 5 h p.i.. Ex vivo images of normal tissues, organs, and tumors taken from the RNA nanoparticles-injected mice showed that the tumors taken at 5 and 24 h p.i. exhibited the strongest signal (Fig. 6b). In terms of tumor accumulation kinetics, RNA nanoparticles reached their highest accumulation within 5 h and remained in the tumor site 96 h p.i., which indicted the high tumor targeting efficiency and tumor retention capability of the constructed RNA nanoparticles.
RNA nanoparticles for in vivo targeted therapy of siRNA to gastric cancer. As shown in Fig. 7 and 8, the tumor in the mouse without treatment grew very rapidly, the size of tumor enlarged as a control. In contrast, the tumor in mice with treatment showed regressed growth and the size of tumor is smaller comparing to controls. The difference between FA-pRNA-3WJ-BRCAA1siRNA treated group and FA-pRNA-3WJ-Scram-siRNA treated group was statistically different (P< 0.01). The result fully demonstrated that prepared FA-pRNA-3WJ-BRCAA1 siRNA nanoparticles can specifically inhibit the growth of gastric cancer cells in vivo.

Undetectable of organ damage by RNA nanoparticles after systemic injection. We used
Harris Hematoxylin and Eosin (HE) staining to check the potential damage to important organs including the heart, liver, spleen, lung and kidney by the RNA nanoparticles. As shown in Fig. 9, no obvious tissue damages were observed, which indirectly suggested that the prepared RNA nanoparticles displayed good biocompatibility and no negative effects on important organs in the body was observed.

Discussion
In recent years, RNA nanotechnology has made great advance. RNA has been used as nanomaterials to construct varieties of nanostructures for targeted imaging and cancer therapy in vivo with the advantages of high delivery efficient, high accumulation in the site of tumor, low toxicity, no damaging of normal cells and tissues, and integration of targeting imaging, nucleic acid drug, and therapy into one nanostructure [30][31][32][33][34][35][36][55][56][57][58][59][60] , which displays great potential for applications in clinical imaging and therapy in the near future 25,26 .
Gastric cancer is the second most common cancer in China. How to achieve simultaneous diagnosis and therapy of early gastric cancer has become a great challenge. Although RNA nanoparticles have been constructed and in vitro studies exhibited great potential of using RNA nanoparticles for cancer theranostic applications, up to date, no report demonstrated RNA nanoparticles can be used for targeted imaging and therapy of gastric cancer in vivo. In order to investigate the feasibility of applying RNA nanoparticles as theranostic agents for gastric cancer diagnosis and therapy, we designed the pRNA-3WJ nanoparticle consisting of three fragments, a 3WJ , b 3WJ and c 3WJ , and functionalized with folate, as targeting ligand; Alexa 647 , as imaging module; and BRCAA1 siRNA (or scrambled control), as therapeutic module,  respectively. We successfully prepared FA-pRNA-3WJ-BRCAA1 siRNA nanoparticles and the resulting RNA nanoparticles showed good pH and thermodynamic stability, good stability against RNase A (less than 10000 U) degradation, and exhibited stability of fluorescent intensity. These results demonstrated that the prepared RNA nanoparticles should be very stable in the blood circulation and can act as high efficient theranostic agent for targeted imaging and siRNA therapy of gastric cancer in vivo, which lay foundation for RNA nanoparticles' further clinical application.
Nanotoxicity of nanotheranostic agents has caused broad attention. In this study, prepared RNA nanoparticles did not exhibit obvious toxicity. After being injected into in vivo blood circulation via tail vain, RNA nanoparticles gradually accumulated in the site of in vivo tumor within 6 h p.i., clearly displayed the imaging of tumor tissues, and exhibited specific targeting ability. The RNA nanoparticles were also proved to be able to retention in the tumor for long time and generate tumor regression effects. In addition, the alteration of biochemical parameters in the mice after treating with FA-pRNA-3WJ-BRCAA1 siRNA nanoparticles was investigated as shown in Table S3 (supporting data) and no obvious tissue damages were observed for liver and kidneys. Further HE staining results also confirmed that prepared RNA nanoparticles did not damage important organs such as brain, heart, lungs, liver and kidneys. Therefore, we can confirm that the prepared RNA nanoparticles should be safe for in vivo application.
In this study, the results of in vivo evaluation of therapeutic efficacy also showed that the prepared RNA nanoparticles can actively target in vivo gastric cancer tissues and inhibited tumor growth significantly. However, the concrete molecular mechanism is not well understood. In order to investigate the potential molecular mechanism, we used Western Blot to detect the expression level of BRCAA1, Bcl-2, Rb and Bax in MGC803 cells treated with prepared RNA nanoparticles for 24 h and 48 h. As shown in Fig. 10, RNA nanoparticles can down-regulate or silence the expression of BRCAA1 gene, down-regulate the expression of Bcl-2 gene, adversely up-regulate the expression of Rb and Bax genes in MGC803 cells. Based on these results, we proposed a molecular mechanism of RNA nanoparticle induced MGC803 growth inhibition: the prepared RNA nanoparticles (FA-pRNA-3WJ-BRCAA1 siRNA) actively bind to the folic acid receptor on the surface of MGC803 cells via folic acids conjugated on the RNA nanoparticles, and then induce the endocytosis of RNA nanoparticles into tumor cytoplasm. The double-stranded BRCAA1 siRNA region on the RNA nanoparticle can be recognized by RNA-induced silencing complex (RISC) in the cytoplasm and processed. The released siRNA antisense strand can further recognize target BRCAA1 mRNA, degrade it, and result in silence of BRCAA1 gene in MGC80 3 cells. The down regulate or silencing of the BRCAA1 gene will cause subsequent down-regulation of Bcl-2 gene, and further up-regulation of Rb and Bax gene, which will end up with inducing cell apoptosis and inhibiting the cell growth. The proposed mechanism is summarized in Fig. 11 and the concrete study of the regulation signal pathway is under way.
In recent years, BRCAA1 gene, as an important member of ARID family, called as ARID4B, has been found to involve in the regulation of the male fertility and stem cells, ARID4B protein can regulate Rb binding protein 1, which highly suggest that ARID4B may be a tumor suppressor. Up to date, our experiment data confirm that BRCAA1 exhibit over-expression in the gastric cancer MGC 803 cells, therefore, we predict that BRCAA1 (ARID4B) may exist in gastric cancer MGC 803 cells with gene mutation or other way, further investigation is still under way.

Conclusions
Folate-conjugated 3WJ-BRCAA1 siRNA-pRNA nanoparticles were successfully developed, and resulted in specific fluorescent targeted imaging, high efficient siRNA delivery, significantly inhibiting the growth of gastric cancer MGC803 cells, and reducing the size of gastric cancer xenografts in vivo, which exhibiting potential clinical applications. More importantly, the prepared RNA nanoparticles exhibited remarked accumulation in tumor as well as little accumulation in crucial organs such as liver, spleen, kidneys, etc. and no damage to non-tumor tissues. The potential molecular mechanism is: the prepared RNA nanoparticles can enter into the cytoplasm specifically via folic acid receptor mediated endocytosis and inhibit BRCAA1 expression in gastric cancer cells by uploaded BRCAA1 siRNA, resulting in the up-regulation of Rb and Bax, down-regulate the expression of BCl-2, and inducing of gastric cancer cell apoptosis. These actions finally regress the tumor growth in the studied mice. Our results also provide a new paradigm for the applications of RNA nanoparticles to specific tumor cells to maximize therapeutic effects while minimizing the toxicity of the drug delivery system. The prepared RNA nanoparticles showed great potential in applications such as gastric cancer targeted imaging, drug delivery, and siRNA therapy in near future. Figure 11. The potential mechanism of RNA nanoparticles for gastric cancer therapy.