Effect of Silver Chloride Nanoparticles on Expression of LncRNA GAS5 Gene in Liver Cancer Cells (HepG2)

Liver cancer is one of the deadliest cancers and the most common cancers worldwide. Nanoparticles due to their wide applications in diagnostics, imaging and drug delivery, have attracted significant attentions (6). Upon a previous studies, the green-route mediated production of metal nanoparticles inhibits the progression of cancer cell lines (9, 10). Inprevios study the inhibition of the breast cancer cells were conducted by silver chloride nanoparticles synthesized by Onopordum acanthium L. extract through inducing apoptosis pathway (11).Lnc RNA GAS5 is a tumor suppressor, it’s an expression in some malignant tumors decrease. In liver Cancer Cells (HCC) The GAS5 gene Expression is reduced, which identifies the poor prognosis of this gene (5). However, a limited number of reports have been investigated on the cytotoxic effect of silver chloride nanoparticles on cancer cells, so in this study, we aimed to investigate it. In our study, we investigated the silver chloride nanoparticles synthesized by O. acanthium L. extract, as well as its anticancer effects on liver cancer and normal cell lines. The aim of this study was investigating the cytotoxicity effect of silver chloride nanoparticles and evaluation of LncRNA GAS 5 gene expression in liver cancer cells.

Brifly, 100 mL of a solution of 1 mM silver nitrate was prepared in distilled water. Then, 5 mL of solution of O. acanthium L. extract (4 mg/mL) and KCl salt (1 mM) were drop wise added to the stirred silver nitrate solution. Subsequentely, the resulted solution was stirred at room temperature for 24 hours. This solution was then centrifuged at 13,000 rpm for 10 minutes; the supernatant was discarded and then centrifuged again with distilled water (11). In this study, HepG2 and HEK293 cells were obtained from the Pasteur Institute Cell Bank of Tehran and treated with silver chloride nanoparticles after 24 hours. Cell viability percentage of nanoparticles were determined by MTT (3-(4, 5-Dimethyltetrazollium Bromide) assay toward HepG2 and HEK293 cells. The HepG2 and HEK293 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Scotland) medium with 10% FBS serum and 1% streptomycin antibiotic (Gibco, Scotland). The cells were then stored at 37 ° C with 5% CO2 in a humidified cell culture. To evaluate the in vitro cell viability, the colorimetricMTT method was assessed for investigating the appropriate lethal dose of silver chloride nanoparticles. Afterward, they were treated with silver chloride nanoparticles in various concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 mg/mL) for 24 hours. Nanoparticles were added to the cells (1 × 104 cells per well) cultured on 96-well plate, which were incubated an overnight at 37 °C. Thereafter, MTT solution (100 μl of 0.05 mg/well) was added to the plate. Subsequently, dimethyl sulfoxide (DMSO) solvent we added of to each well to solubilize the formazan crystals. Finally, the optical density of the treated and untreated cells was evaluated with the ELISA reader (Eppendorf, Germany) at the wavelength of 570 nm. Finally, the viability of the cells was calculated according to the following formula: percentage of cell viability (%) = OD value of sample / OD value of control × 100.Levels of LncRNA GAS5 gene expression in HepG2 liver cancer cell were quantified using quantitative real-time reverse transcription-PCR (qRT-PCR) technique. To assess the qRT-PCR method, total cellular RNA extraction was carried out using the RNA extraction kit based on to the instructions in the Qiagen kit (Qiagen, Germany). To synthesis of transcriptional complementary DNA (cDNA), PrimeScriptTM RT Kit (Takara, Japan) was carried out (12). GAS5 and GAPDH primers were then conducted using NCBI primer blast in this study (Table 1). Subsequently, qRT-PCR method was evaluated by 100 ng of cDNA, SYBER Green PCR Master Mix (2X), followed by 2mM each of reverse and forward primers in final volume to 20 mL with nuclease free water. Also, the data was investigated by ABI StepOne using the Applied Biosystems qRT-PCR (ABI 7300 system, Applied Biosystems). Statistical significance between treatment and untreated groups were evaluated by One-way analysis of variance (ANOVA) with SPSS software version 22 and the Tukey post hoc. Data were expressed as mean ± the standard deviation of three replicates from three independent experiments. and P ˂0.05 was considered significance level.

The results showed that silver chloride nanoparticles decrease significantly the cell viability of cancerous HepG2 cells as compared to normal HEK293 cell line. In this regard, silver chloride nanoparticles were able to increase cell toxicity in a dose-dependent manner, as shown in Figure 1 and 2. Also, IC50 concentration of silver chloride nanoparticles toward HepG2 and HEK293 cells was 49 and 160 μg/mL, respectively. To conduct the effect of IC50 dose of nanoparticles on cell lines, we investigated HepG2 and HEK293 cells treated with 0, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 mg/mL of nanoparticles for overnight using MTT assay. At concentration of 12.5, 25, 50, 100, 200, and 400 µg/mL of silver chloride nanoparticles, the viability of HepG2 was reduced to (79.85±6.44 )%, (63.68±7.05) % ,(46.87 ± 4.7)% , (31.83 ± 4.44)% , (18.63± 6.1) %, and (18.63± 6.1) (P < 0.001), respectively (Figure 1). At concentration of 50, 100, 200, and 400 µg/mL of silver chloride nanoparticles, the viability of HEK293 was reduced to (80.28±6.1 )%, (69.91±6.13) % ,(43.67 ± 7.76)% , and (24.99 ± 4.42)% (P < 0.001), respectively (Figure 2). In order to determine the effect of silver chloride nanoparticles on cancerous cells, alterations in the mRNA expression levels of LncRNA GAS5 gene in HepG2 cells were carried out using qRT-PCR technique followed by the exposure to nanoparticle. According to our study, the results of real time PCR displayed that the expression of LncRNA GAS5 was up-regulated by a 4.6±0.34 (p˂0.001) fold as compared to control gene.

In summary, the effect of silver chloride nanoparticles against HepG2 and HEK293 cells showed that these nanoparticles have high cytotoxicity. Also, increase in LncRNA GAS5 gene expression level reveals the triggering apoptosis in HepG2 cells. silver chloride nanoparticles can be considered as a promising strategy for the treatment of liver cancer. Thus, for the liver cancer therapy, further in vivo investigations are required to determine the efficacy of the silver chloride nanoparticles.