shRNA mediated inhibition of Cdc42 gene expression in Calu-6, lung cancer cells

Background Information RNAi technique as a new strategy in gene therapy is the effective gene silencing method. Cdc42 is a member of Rho GTPases involving in lung cancer cells migration and proliferation. In the present study, targeting and inhibiting the Cdc42 expression in Calu-6 cells was investigated. Recombinant lentiviral particles were produced by co-transfection of pMD2.G, psPAX2 and pGFP-C-shLenti plasmid in 293T packaging cells. Calu-6 cells were transduced by recombinant lentiviruses using polybrene. GFP-fluorescence microscopy and MTT assay were used to assess the Calu-6 target cell transduction and rate of lentiviral transduced cells proliferation, respectively. Real time PCR was performed to compare the expression of Cdc42 gene before and after shRNA delivery. Results GFP-fluorescence microscopy analysis showed that Calu-6 cells were successfully transduced with recombinant lentiviral expressing shRNA-Cdc42. The viability of transduced cells was reduced within 72, 96 and 120 hours of transduction process. Real time PCR analyze showed the significant reduction of Cdc42 gene expression. Conclusions: Lentiviral vectors may be reasonable tools for this gene delivery due to stable expression of silencing RNA. Inhibition of Cdc42 expression by lentiviral mediated shRNA delivery could be an effective method to inhibit proliferation of lung cancer cells. Significance: Over expression of Cdc42 gene have been seen in lung cancer. This make Cdc42 gene a key target in treatment of cancer. On the other hand, gene therapy is a proper method to modulate gene expression. It seems modulation of cdc42 gene expression by gene therapy accompany with proper vehicles creating hopes in treatment of lung cancer.


Introduction
Lung cancer is the most leading cause of cancer death in the worldwide (Dellaire et al. 2014).
Surgery, chemotherapy and radiotherapy are three common lung cancer treatments and sometimes have been used in combination; these methods usually work in early stages of cancer and is not very efficient in late stages or metastatic form of lung cancer and have many adverse effects (Sakiragaoglu et al. 2013).
Gene therapy is a method for modifying the defective genome function. The earliest strategy of Gene therapy was providing a functional version of defective gene (Bolhassani & Saleh 2013). More recently, new strategies have been developed in gene therapy which manage cell pathways by target gene correction or disruption. (Humbert et al. 2012).
Using a short hairpin RNA (shRNA) as a part of RNAi technology can be used in cancer targeted therapy. In shRNA-based RNAi technology, sequence-specific gene shRNA as known Pri-shRNA is synthesized and processed by Drosha/DGCR8 complex in nucleus and transported into cytoplasm by Exportin 5 protein. In cytoplasm, pri-shRNA processed by dicer and incorporated into the RNA-interfering silencing complex (RISC) and activated specifically based on gene sequence, leading to targeted mRNA cleavage and degradation (Donald D. Rao et al. 2009).
Although gene therapy has been developed remarkably in last two decades, choosing a suitable vector for transfer of interest gene is still a challenge in gene therapy and efforts keep going on design a safer and long-term gene expression vectors. Among viral and non-viral vectors, Lentivirus mediated vectors have their own advantages for gene delivery purposes.
Lentiviral vectors are a type of retrovirus that can infect both dividing and non-dividing cells.
Lacking of genes required for self-replication in lentiviral vectors lead to their replicate defection and make them as safe vehicle for gene therapy trials. On the other hand, lentiviruses capability of stable transduction make them highly effective and suitable in gene therapy techniques (Song & Yang 2010).
Cdc42 is a member of Rho GTPases family whit a wide range function in the cell including filo podia formation, cell movement, and rearrangement of cytoskeleton, cell migration and proliferation. Upregulation of Cdc42 have been shown in some cancers such as non-small cell lung cancer, colorectal adenocarcinoma, melanoma, breast cancer and testis cancer. The function of Cdc42 gene as a key target in cancer therapy Elevated via collapse degradation of EGFR by c-Cbl and cause EGFR more function and increase cell proliferation (Stengel & Zheng 2011;Gómez Del Pulgar et al. 2008;Chou et al. 2003;Qadir et al. 2015).
In this investigation, we transferred Cdc42 sequence specified shRNA into lung cancer cell line Calu6 by lentiviral vector for stably production of inhibitory shRNA and the expression of Cdc42 gene as a suitable method and target in lung cancer therapy and Calu-6-transduced cells proliferation rate were assayed.

Cell lines and Culture
Calu-6 lung cancer cell line and 293T embryonic cell line were purchased from National Cell Bank of Iran (NCBI, Pasture Institute of Iran, Tehran). Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The medium was changed every 3 days.

Assessment of cytotoxicity
Cytotoxic effect of lentivirus infection on calu-6 cells was determined by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, UK) reduction assay in both shRNA containing and non-containing groups Which relays on the mitochondrial dehydrogenase activities. Briefly, lentiviruses in 1, 10, 50, 100 and 200 MOI was added to 6000 Calu-6 cells in 96-well plate. After 48 h, 10 ul of MTT (5mg/ml) solution was added to culture medium and incubation was done for 4 h. The dark blue formazan crystals were dissolved in 100 μl DMSO and absorbance was recorded at 490 nm. Percentage of viable cells was calculated as: OD test / OD control×100 formulas.

Antibiotic selection of infected cells
The log growth phase Calu-6 cells were seeded in 6-well plates in 1 × 10 5 density and cultured overnight. For infection, the shRNA containing and non-containing lentiviruses were diluted in complete medium containing Polybrene (8 μg/mL) in 50 MOI and treated for 24 h at 37 °C. By the time of incubation, the virus containing medium was replaced with fresh medium and puromycin selection of infected cells was done; Minimal lethal dose of puromycin for Calu-6 cell line was determined during 2 days; For this aim, Calu-6 cell line treated by 0 (as control), 0.5-10 μg/ml of puromycin. Cells observed each day by invert microscope (Micros, Austria). GFP expression in survived cells was verified by fluorescent microscopy. Non-infected cells treated with puromycin used as control group for antibiotic selection. Images captured by USB DCam and iViewCap application.
None transduce cells were subjected as control and LV-shRNA without Cdc42 silencing sequence infected cells were selected as negative control groups.

Proliferation assay
The effect of Cdc42 silencing on cell proliferation process was measured via MTT colorimetric assay. Lentivirus infected cells were seeded at a density of 2000 cells/well in 24well plates. Experiments were designed as CON, NC and Lv-shRNACdc42F. After 24, 48, 72, 96 and 120 h incubation at 37 °C, 50 μL of sterile MTT dye (5 mg/mL; Sigma-Aldrich Corp) was added to each well and incubated for another 4 h at 37 °C. Medium was removed from the wells and dimethyl sulfoxide (DMSO) was added to each well for dissolving the formazan product. Spectrometric absorbance was measured at the wavelength of 490 nm.

Statistical analysis
All experiments were performed in triplicate. The values obtained from this study were expressed as mean ± standard deviation (SD). Statistically significant differences between groups were determined by One-way ANOVA using SPSS 16 software. Differences were considered statistically significant at P <0.05.

Virus production in 293T cells
Recombinant lentivirus packaging and production was done in 293T cells and fluorescence microscopy observation showed that 24 h after infection, 80% of 293T Cells received virus production constructs and were expressing GFP marker ( Figure 2).  (Figure 3). Based on these results, different MOIs calculated. Toxicity of lentiviruses (not shRNA) was assessed with MTT assay which showed no toxic effects on Calu-6 cells (Figure 4)

Antibiotic selection of transduced cells
Calu-6 cells transduced by Cdc42-shRNA encoding lentivirus and control lentivirus purified by puromycin antibiotic. Results showed that after two days of antibiotic treatment, all cells died at 0.5 μg/ml of puromycin, then this dose was selected as minimal lethal dose of puromicyn against Calu6 cells ( Figure 5). To remove all non-infected cells, antibiotic treatment continued for 4 weeks at 0.5 μg/ml µg/ml of puromycin which led to acquiring pure infected Calu6 cells ( Figure 6).

Cdc42 gene expression reduced in Calu-6 cells infected by tGFP-C-shLenti
To investigate the effect of shRNACdc42 on Cdc42 gene expression in calu-6 cells, real-time PCR assay performed and the expression of Cdc42 mRNA was compared between shRNACdc42 containing and non-containing cells. A Delta CT analysis indicated that the expression of Cdc42 gene in transduced cells was significantly decreased in compare to both control and negative control groups. Statistical analysis showed that the amount of Cdc42 mRNA was 0.25-fold in shRNACdc42 expressing cells compared to non-expressing cells ( Figure 7).  Cancer (NSCLC) and progress cell cycle G1/S phases that make it a main target in cancer therapy (Li et al. 2013;Liu et al. 2011;Arias-Romero & Chernoff 2013;Qadir et al. 2015).

Knockdown of Cdc42 inhibits proliferation of calu-6 cells
In last 20 years, change in gene expression to target diseases, have been attracted many attention. Based on human genome sequencing and researches understands about molecular agents generating diseases, silencing pathogen genes is an attractive approach in treatment of wide range of diseases (Cejka et al. 2006). Gene therapy defined as the treatment of a disorder by the introduction of therapeutic genes into the appropriate cellular targets. These therapeutic genes can correct deleterious consequences of defected gene, or re-programme cell functions (Escors & Breckpot 2010). RNAi have many advantage compare to some treatment such as molecular drugs. RNAi technology have more specificity and wide range target capacity (Sakiragaoglu et al. 2013).
The determination of new cancer-specific targets and pathways amenable to RNAi therapy would provide a greater impetus to the promotion of targeted therapies. Several in vitro and in vivo proof-of-concept studies have mentioned that, RNAi has achieved from bench to bedside with several RNAi-based drugs in clinical trials (Bora et al. 2012). Inhibition of multiple genes is an effective approach to prevent or treat cancer as well as reduction of the possibility of multiple drugs' resistance caused by overdose of chemical drugs (Mansoori et al. 2014).
RNAi-based drugs with a gain of function genetic lesion or overexpression of disease causing gene(s), can be effective in the treatment of various diseases, such as cancer (Bora et al. 2012).
conducted on animal models emphasis that targeting essential proteins in the cell cycle, such as kinesin spindle protein (KSP) and polo-like kinase 1 (PLK1) by using specific siRNA have   h, significant differences observed in grows rate of Lv-shRNACdc42 cells compare to NC cells indicated by ** and P<0.01 and compare to CON cells by *** and P<0.001. In 96h and 120h, grows rate of Lv-shRNACdc42 cells compare to NC cells and CON cells was significant indicated by *** and P<0.001. Significant differences don't observed between CON cells and NC cells.