Effects of chitosan nanoparticle-mediated BRAF siRNA interference on invasion and metastasis of gastric cancer cells

Abstract

To observe the changes in invasion capacity of gastric cancer BGC823 cells after being treated with chitosan-encapsulated BRAF siRNA nanoparticles, and to evaluate the effects of the nanoparticle-mediated BRAF siRNA interference on cell invasion and metastasis, BRAF siRNA was encapsulated with chitosan into nanoparticles sized 350 nm to treat gastric cancer cells. Silencing of BRAF was detected by Western blot and PCR, and cell invasion was observed by the Transwell assay. The nanoparticles significantly downregulated BRAF expression in BGC823 cells (P < 0.01) and inhibited their invasion (P < 0.001). Chitosan nanoparticle-mediated BRAF siRNA interference evidently reduced the invasion capacity of gastric cancers.

Keywords::

• BRAF
• chitosan
• gastric cancer
• invasion
• metastasis
• nanoparticle

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Expression of Concern

Introduction

Gastric cancer is one of the most common malignant tumors, with the highest mortality rate. The therapeutic outcomes and survival rates of patients with gastric cancer are determined by invasion, metastasis, and recurrence (Kwak et al. 2005). Therefore, genes related to tumor invasion and metastasis have been employed as RNA interference (RNAi), to specifically block the expressions of corresponding genes (Elbashir et al. 2002). With the development of materials science, nanoparticles have attracted global attention as highly potential drug carriers. Besides, RNAi-carrying nanoparticles have been used to treat tumors.

As a member of the RAF gene family, BRAF mutates in several types of tumors and activates abnormal phosphorylation of downstream components, thus playing important roles in tumor onset and progression (Calipel et al. 2003). Recently, the BRAF gene has been associated with gastric cancer (Fu et al. 2007). To analyze the influence of RNAi-carrying nanoparticles on invasion and metastasis of gastric cancer cells, BRAF was herein selected as the target gene and chitosan nanoparticles were used as the carrier.

Experimental

Reagents

Chitosan was purchased from Sigma-Aldrich (USA). BRAF-specific and control siRNAs were designed with the templates in an on-line software provided by Ambion. The sense and antisense strands of BRAF siRNA were 5′-AAGAAGATGTAACGGTATCCACCTGTCTC-3′ and 5′-AATGGATACCGTTACATCTTCCCTGTCTC-3′ respectively. Lipofectamine 2000 Reagent was bought from Invitrogen (USA).

Cell culture

Highly invasive gastric cancer cell line BGC823 that highly expressed BRAF was preserved in our group, and cultured in 1640 culture medium (Invitrogen, USA) containing 10% fetal bovine serum (Invitrogen, USA) at 37°C in 5% CO₂.

Preparation of nanoparticles and particle size analysis

Chitosan was dissolved in 0.3 mol/L sodium acetate solution to the final concentration of 1 mg/ml, after pH adjustment to 5.5 with 0.01 mol/L NaOH. The solution was then filtrated by a 0.22 μm syringe filter for sterilization. siRNA was prepared to the final concentration of 100 μmol/L with diethyl pyrocarbonate (DEPC) solution, 60 μl of which was added into 500 μl of the above sodium acetate solution of chitosan. After 30 s of vortex and 1 h of standing at room temperature, siRNA-BRAF-chitosan nanoparticles were prepared. Afterwards, the suspension was diluted with DEPC solution to determine the average particle size, with the Malvern particle size analyzer. All the devices had been pretreated with DEPC solution.

Grouping and transfection

The BGC823 cells were divided into five groups. The untreated control group was cultured normally, the blank control group was cultured in chitosan solution, and the negative control group was cultured in siRNA-chitosan solution. For the siRNA-BRAF-liposome group, Lipofectamine 2000 was mixed with serum-free culture medium and left still at room temperature for 5 min, according to the manufacturer's instructions. BRAF siRNA was treated identically. Then they were mixed and left still at room temperature for another 15 min in complete culture medium until the final concentration of siRNA-BRAF was 100 nmol/L. For the siRNA-BRAF-chitosan group, siRNA-BRAF-chitosan was added into the culture medium until the final concentration of siRNA-BRAF was 100 nmol/L. The Transwell assay was performed after 24 h of treatment in culture medium containing 10% fetal bovine serum at 37°C in 5% CO₂, and Western blot and PCR were conducted for extracted proteins after another 24 h.

Validation of RNAi efficiency

The interference efficiency of BRAF siRNA-carrying nanoparticles was validated by real-time quantitative PCR and Western blot. Cultured cells were treated by Trizol (Invitrogen, USA) to extract total RNA, and reverse-transcribed into cDNA with RevertAid First Strand cDNA Synthesis kit (Thermo, USA). BRAF gene expression was quantitatively detected by the SYBR (Invitrogen, USA) method. PCR primers were designed by Invitrogen (USA). Sense strand of BRAF primer: 5′-TACCTGGCTCACTAACTAACGTG-3’; antisense strand: 5’-CACATGTCGTGTTTTCCTGAG-3’. Sense strand of internal control gene β-actin primer: 5’-CTGAGCAGATCATGAAGAC-3’; antisense strand: 5’-CTTGGTGGACGCATCCTGAG-3’. Real-time quantitative PCR was carried out on ABI 7500 FAST real-time PCR system. For Western blotting, cells in the logarithmic growth phase were collected, from which total protein was extracted to determine the concentration, with the Nano Drop 2000 spectrophotometer. After 5 × loading, buffer of the same volume as that of protein sample was added, the samples were denatured in a dry bath at 95°C for 10 min. Subsequently, the protein samples were subjected to 10% SDS-PAGE (amount of total protein in each lane: 30 μg). After being resolved, the proteins were transferred to a PVDF membrane by using the semi-dry transfer method for Western blotting. The membrane was then blocked with TBST solution containing 5% skimmed milk powder, and incubated with primary antibody for BRAF overnight at 4°C, and with secondary antibody at room temperature for 90 min. Afterwards, the membrane was incubated with ECL detection in dark, detected by sensitive X-ray film, allowed to develop color in a color development solution, and fixed in a fixing solution. Rabbit monoclonal and secondary antibodies for BRAF and β-actin were bought from Santa Cruz (USA).

Invasion assay

Invasion of gastric cancer cells was detected by the Transwell assay with a Matrigel-coated Transwell chamber (8 μmol/L per pore, Invitrogen, USA), according to instructions. The Transwell chamber was placed in a 24-well plate containing 0.7 ml of 1640 culture medium with 10% fetal bovine serum in each well. About 0.2 ml of serum-free culture medium was thereafter added in the upper chamber to suspend cells to the density of approximately 2 × 10⁴ in each well. Each experiment was performed in triplicate. After 24 h of culture, the cells were fixed by methanol and stained with crystal violet. Under a microscope with 20 × magnification, five visual fields were randomly selected to count the number of cells that penetrated the chamber, with mean and standard deviation used to represent invasion capacity.

Statistical analysis

All data were analyzed by SPSS 22.0. A value of P < 0.01 was considered statistically significant. The numerical data were expressed as mean ± standard deviation, and means were compared by the independent samples t-test. All experiments were performed in triplicate.

Results

Particle size analysis

BRAF siRNA was herein first encapsulated with chitosan into nanoparticles with the average size of 350 nm, which was suitable for experimental use (Figure 1).

Figure 1. Morphology of chitosan-encapsulated BRAF-siRNA nanoparticles under atomic force microscope (× 40000).

Interference efficiency

Different control and experimental groups were then divided and treated, with the aim of analyzing the interference effects of BRAF-siRNA-chitosan nanoparticles on BRAF expression. These nanoparticles managed to effectively interfere with the BRAF expression in BGC823 cells (Figures 2 and 3). The expression of BRAF mRNA (determined by the 2^−∆∆Ct method) in the siRNA-BRAF-chitosan group (0.57 ± 0.08) was significantly lower than the expression in the untreated control (1.03 ± 0.12), blank control (0.89 ± 0.05), and negative control (0.85 ± 0.11) groups (P < 0.01). However, the expression was similar to that of the siRNA-BRAF-liposome group (0.48 ± 0.12) (P = 0.137).

Figure 2. Chitosan-encapsulated BRAF siRNA significantly downregulated the level of BRAF mRNA (#P < 0.001).

Figure 3. Chitosan-encapsulated BRAF siRNA efficiently decreased the expression level of BRAF protein.

Reduction of invasion capacity by BRAF-siRNA nanoparticles

siRNA-BRAF-chitosan nanoparticles dramatically interfered with BRAF expression in BGC823 cells. Since BRAF is highly related with invasion and metastasis of gastric cancer, the invasion capacities of all groups were detected by the Transwell assay. The cells treated with siRNA-BRAF-chitosan nanoparticles had significantly lower invasion capacity (58.4 ± 1.42/visual field) than that in the untreated control (142.8 ± 4.12/visual field), blank control (133.6 ± 3.44/visual field), and negative control (129.6 ± 2.64/visual field) groups (P < 0.001, Figures 4 and 5). However, the siRNA-BRAF-chitosan and siRNA-BRAF-liposome (51.3 ± 2.43/visual field) groups showed similar invasion capacities (P = 0.203). Hence, siRNA-BRAF-chitosan nanoparticles were evidently able to inhibit the invasion of gastric cancer cells.

Figure 4. Transwell assay showing significantly decreased invasion of BGC823 cells transfected with BRAF siRNA (#P < 0.001).

Figure 5. Transwell assay showing significantly decreased invasion of BGC823 cells transfected with BRAF siRNA. The cells that migrated through the Matrigel were stained using crystal violet (Original magnification: × 200).

Discussion

The prepared chitosan-encapsulated BRAF-siRNA nanoparticles were spherical, uniform, and well dispersed. Nanocrystallization of chitosan can increase its solubility in aqueous solution, especially at physiological pH (Janes et al. 2001), and enhance the absorption by the gastrointestinal epithelium (Mooren et al. 1998). In addition, since tumor cells carry more negative charge than normal cells do and chitosan is a biomacromolecule carrying positive charge, it is selectively adsorbed by tumor cells through charge neutralization, thus exerting antitumor effects. Being selectively phagocytized by tumor cells, chitosan nanoparticles can both boost the therapeutic effects and reduce the peripheral side effects of drugs. Moreover, chitosan can cooperate with other antitumor agents by directly inhibiting tumor cells and by activating the immune system (Nagpal et al. 2010).

Gastric cancer is a prevalent malignant tumor with low survival rate, poor prognosis, and unsatisfactory therapeutic outcomes, during the progression of which prognosis is closely associated with invasion and metastasis of tumor cells. The aim of this study was to assess the influence of chitosan nanoparticle-mediated BRAF siRNA interference on invasion and metastasis of gastric cancer cells. BRAF-siRNA transfection effectively inhibited the expressions of the BRAF gene and protein in BGC823 cells, significantly suppressed cell growth, and reduced the migration and invasion ability of these cells, thereby demonstrating that the BRAF gene is related with both growth and invasion of gastric cancer cells. The chitosan-encapsulated BRAF-siRNA nanoparticles herein also evidently inhibited the invasion of gastric cancer cells, probably because silencing BRAF gene by using siRNA decreased the catalytic activity of BRAF and dephosphorylated downstream MEK, which thus reduced the activity of the RAF-MEK-ERK pathway or abnormally deactivated it.

The oncogene BRAF dominantly controls the onset and progression of tumors (Basto et al. 2005), and BRAF protein kinase is activated by binding RAS in the GTP state, thereby activating the phosphorylation of downstream MEK that further phosphorylates and activates ERK. Sustained activation of BRAF disrupts the RAF-MEK-ERK signaling transduction pathway and leads to malignant transformation by inducing excessive cell proliferation (Basto et al. 2005). Recent microarray and immunohistochemical studies have shown that BRAF, which is associated with gastric cancer (Fu et al. 2007), may be a potentially eligible target gene for its treatment. As to the biocarriers for targeted gene therapy, chitosan nanoparticles have been highlighted because they are non-toxic, stable, injectable, biodegradable, and immunoreaction-free (Lee et al. 2005). Although liposomes have high transfection efficiency, they are prone to degradation and thus limited in practical use. Chitosan nanoparticles, as novel materials, have been successfully applied in disease treatment, drug delivery, and ultrasound imaging (Chen et al. 2012, Shilpa and Paulose 2014). Such nanoparticles, when delivering siRNA, can prevent it from degradation, prolong its circulation time in blood, and augment its transfection efficiency. Moreover, chitosan nanoparticles can be chemically modified to realize targeted therapy, besides enhancing the effects of antitumor agents (Zhao et al. 2007, Zheng 2013).

Conclusions

Given the advantages of these nanoparticles and the positive results in this study, chitosan nanoparticle-mediated BRAF siRNA interference is promising in the targeted therapy of gastric cancer.

Declaration of interest

The author reports no declarations of interest. The author alone is responsible for the content and writing of the paper.