Malignant melanoma is a neoplasm of melanocytes. It is the third most common malignant skin cancer and is the most serious cancer regarding local invasiveness and mortality rate [1
]. Among the different stages of tumor progression, the metastatic type has the worse prognosis and is one of the most aggressive and drug-resistant cancers, with a median survival time of 6 months and a 5-year survival rate of less than 5% [2
]. In general, the process of tumor cell invasion starts through the detachment of the cancer cells and proteolysis of the basement membranes by a group of extracellular matrix proteases that are known as the matrix metalloproteinases (MMPs) family of enzymes [3
Collagenases enzymes are a subgroup of the MMPs family of proteases capable of degrading native fibrillary collagens (type I, II, III, V and IX) [5
]. This subgroup of enzymes includes MMP1, MMP8, and MMP13 also known as collagenases-1, -2 and -3 respectively [6
]. These enzymes are normally expressed at minimal tissue level to regulate important biological functions such as angiogenesis, bone development and wound healing [6
]. Interests in these enzymes have grown considerably in cancer research because of their direct contribution in the degradation of extracellular matrix (ECM) components when expressed in high levels [8
]. MMP1 is interstitial collagenases, initially, detected in highly invasive malignant melanoma together with MMP13 [3
]. High expression of these MMPs has been implicated in the promotion of the metastatic potential of many types of cancer cells [9
]. While the role of MMP8 has provoked some controversy in which some studies reported its effect on cytokine release in the cancer microenvironment in breast cancer and some ovarian cancer cell lines [13
], other reports have shown that MMP8 possess some antitumor properties [10
The expression of these collagenases is tightly regulated at transcriptional level by the existence of several cis
-elements within the gene promoter sequence [5
]. MMP1 and MMP13 share many cis
-elements within their gene promoter and are therefore usually co-expressed or co-repressed within the context of the special tissue function [6
]. MMPs with different cis
-elements structure in their promoters will have a different expression pattern such as MMP8 gene promoter which is considerably different from MMP1 and MMP13 gene, containing the Sp-1 transcription factor binding site and lacks the AP-1 and AP-2 promoter sequence [6
]. In pathological conditions, the expression level of these MMPs changes mainly due to activation and binding of multiple transcription factors to specific MMPs gene promoter sequence causing significant changes in the expression levels of respective MMPs [6
]. Among these transcription factors, YB-1 (Y-box-binding protein 1) commands a special concern in cancer progression. This multifunctional protein belongs to the cold-shock protein family, which is characterized by having a highly conserved nucleic-acid-binding motif binding to both DNA and RNA [15
]. This enables YB-1 to have versatile roles in regulating cellular DNA repair, RNA splicing, exon skipping [15
]. However, YB-1 is also considered as an oncoprotein because it is found to enhance uncontrolled proliferation, the evasion from immune recognition and growth suppression, cancer cell immortality, sustained angiogenesis, invasion, and metastasis in several malignancies [15
Accordingly, elevated levels of YB-1 protein were highly correlated with cancer progression and poor prognosis in many malignancies such as breast cancer, lung cancer, osteosarcoma, melanoma and multiple myeloma [15
]. The relationship between YB-1 protein and the expression of collagenases MMPs, in general, is not well elucidated; however, some studies had reported some interference among them [17
]. YB-1 was reported to have an insulating effect on the expression of MMP13 through interaction with the AP-1 sequence of the gene promoter [20
]. However, this interaction was contradicted in the previous study [21
]. Therefore, the effect of YB-1 in regulating the expression of collagenases MMPs is yet poorly revealed. This study aimed to establish a stable A375 cell line with constant knockdown of YB-1 protein and to investigate its effect on the expression of collagenases MMPs, in addition to the exploration of other anti-tumor properties.
4. Materials and Methods
4.1. Establishing the Stable YB-1 Silenced Malignant Melanoma Cell Line
The A375 malignant melanoma cell line was purchased from American Type Culture Collection ATCC (Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Gibco). The cells were maintained in a humidified incubator at 37 °C with 5% CO2. To establish the stable YB-1 silenced A375 cell line, two short hairpin RNA (shRNA) plasmid constructs cloned into retroviral pGFP-V-RS vector (OriGene, Rockville, MD, USA) were used with targeting sequences to YB-1 mRNA in the first plasmid (P1) as follows: 5′-TTCATCAACAGGAATGACACCAAGGAAGA-3′ and second plasmid (P2) as follows: 5′-GACAGCCTAGAGAGGACGGCAATGAAGAA-3′. In parallel, a non-targeting scrambled sequence plasmid construct in a retroviral pGFP-V-RS vector (OriGene) was used as a negative control (Pc).
The plasmids were amplified in JM109 competent cells (Promega, Madison, WI, USA) grown in Miller’s LB Broth (Sigma-Aldrich, Steinheim am Albuch, Germany) supplemented with 25 µg/mL of Kanamycin sulfate (Sigma-Aldrich). Then the plasmids were extracted using PureYield™ Plasmid Miniprep kit (Promega) followed by plasmid quantification and qualification by estimating the A260/A280 and the A260/A230 ratios using Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis.
The A375 cells were grown in 24 well plates. After reaching 75% confluence, the transfection was performed with ViafectTM Transfection kit (Promega) following the manufacturer instructions by mixing 1 µg of the plasmid in 100 µL of serum-free media with 4 µL of transfection reagent. The cells were then incubated for two days before validating the transfection by analyzing the GFP reporter gene function in A375 cells in each well using Cytell™ Cell Imaging System (GE Healthcare, Marlborough, MA, USA). After confirmation of GFP fluorescence as shown in Figure 1
, the cells were then cultured by changing the media every three days with complete media containing (1 µg/mL)and (0.5 µg/mL) of puromycin in alternate weeks for one month during which stable clones were selected and passaged for later experiments. The expression of YB-1 was determined in the selected clones using real-time PCR, immunofluorescence and immunoblotting analysis.
4.2. Real-Time PCR
The untreated A375 cells, P1, P2 and Pc clones were maintained in complete non-selective media till reaching 70% confluence in 25 cm3 cell culture flasks. Then the complete media was replaced with serum-free media for 24 h followed by detaching the cells using triple E-express (Gibco, Carlsbad, CA, USA). The cells were washed twice with Phosphate-buffered saline (PBS) (Thermo Fisher, Waltham, MA, USA) (pH 7.4) followed by centrifugation and mRNA extraction using AurumTMTotal RNA Mini kit (BioRad, Hercules, CA, USA) following the manufacturer protocol. Before proceeding, the RNA integrity was tested on a 1% denaturing agarose gel stained with ethidium bromide by visualizing the 18S and 28S bands with no evident smearing in addition to Nanodrop spectrophotometer determination of RNA purity (Thermo Scientific, Waltham, MA, USA) by assessing the A260/A280 and the A260/A230 ratios being higher than 2.00. The RNA samples were then normalized with RNA elution buffer based upon the Nanodrop measures. The iScript™ Advanced cDNA Synthesis Kit (BioRad, Hercules, CA, USA) was used to synthesize cDNA from each of the mRNA samples following the manufacturer protocol. Each of cDNA samples was then subjected to real-time PCR using SsoAdvUniver SYBR Green master mix (BioRad, Hercules, CA, USA) with specific primer sequences for YB-1, MMP1, MMP8 and MMP13 (BioRad, Hercules, CA, USA). After completion of amplification, the Ct values of each well was determined and used for relative gene expression analysis following ΔΔCt method by normalizing the mean Ct values of target proteins with housekeeping genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin and alpha tubulin among the experimental and control samples to determine the fold difference in the relative mRNA expression of each protein. Each of samples experiments involved triplicate repeats with multiple replicates for validation of the results.
4.3. Protein Extraction and Western Blot Analysis
The four cell lines were treated similarly as in the real-time PCR cell whereby the cell samples were harvested and washed twice with PBS buffer followed by centrifugation each time. The cell pellets of different samples were mixed with 400 µL of Pierce RIPA lysis buffer (25 mM Tris-HCl (pH 7.6), 0.1% sodium dodecyl sulfate (SDS), % sodium deoxycholate, 150 mM NaCl, 1% NP-40) (Thermo Fisher, Waltham, MA, USA) on ice by pipetting and vortexing. The mixtures were also subjected to water bath sonication for 30 s every 10 min while keeping the protein samples on ice for 30 min. Each of the protein lysates was then centrifugated at 12,000 RPM for 30 min at 4 °C followed by careful aspiration of the protein lysate solution without aspirating the precipitate in the bottom of the tube. The protein lysates concentration was determined in each sample using the colorimetric Pierce bicinchoninic acid (BCA) assay kit (Thermo Fisher, Waltham, MA, USA) following the standard protocol to plot the standard curve using the standard protein solutions followed by quantification of total protein in each of the experimental and control samples. 15 µg of protein in Laemmli Sample Buffer (BioRad, Hercules, CA, USA) was loaded into each lane of the mini proteon 12% TGX free SDS-polyacrylamide gel (BioRad, Hercules, CA, USA) followed by electrophoresis for 90 min at 100 V. After confirming the presence of protein bands in the gels, the proteins were blotted to 0.45 µm pore size Polyvinylidene fluoride (PVDF) membrane using Trans-Blot® SD Semi-Dry Transfer Cell (BioRad) at 18 Volt for 45 min. After confirming the presence of protein bands in the membranes, the membranes were then blocked with Blocking One solution (Nacalai, Nakagyo-ku, Kyoto, Japan) for 30 min followed by overnight incubation with primary specific monoclonal mouse antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C with aggression. Then the membranes were washed three to five times with PBS buffer including 0.1% Tween 20 (PBST) buffer (pH 7.4) and were then incubated with horseradish peroxidase (HRP)-conjugated secondary Rabbit anti-mouse antibodies solutions (Santa-Cruz, Germany) for 1 h at room temperature. After five times of more aggressive washing, Luminata Forte Western HRP substrate (Millipore, Burlington, MA, USA) was added to the membrane, and the protein bands were detected and analyzed using ChemiDoc™ XRS (BioRad, Hercules, CA, USA).
Densitometric analysis of the western blotting results was performed using ImageJ software (NIH, Bethesda, MD, USA) [30
4.4. MTT Assay and Cell Counting
The proliferative potential of cell samples was determined using 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and conventional cell counting. In MTT colorimetric assay, approximately 7.5 × 103
cells were plated to each well of 96 well plate in 100 µL of complete media. Next day the complete media was replaced with 100 µL of serum-free media and kept for 24 h in the humidified CO2
incubator at 37 °C. The next day, the media was carefully removed from each well followed by washing with PBS buffer and adding 100 µL media containing MTT reagent (0.5 mg/mL). The cells were then incubated for 4 h in the in a humidified CO2
incubator at 37 °C, followed by aspirating the media containing MTT and adding 100 µL of dimethyl sulfoxide and the 96 well plate was covered with aluminum foil and agitated for two hours. Then the plate was read at 590 nm with a reference filter of 620 nm using TECAN Infiniti plate reader (TECAN, Männedorf, Switzerland).
Cell Viability = (experimental sample absorbance − blank sample absorbance)/(control sample absorbance − blank sample absorbance) × 100
In parallel, the proliferation of each cell sample was assessed by conventional cell counting with trypan blue staining.
4.5. Immuno-Fluorescence Staining
In this experiment, approximately 1 × 104 cells from different cell samples were plated in 24 well plate with 500 µL of complete media and kept in the humidified CO2 incubator at 37 °C. The next day, the complete media was replaced with 500 µL of serum-free media and kept for 24 h in the humidified CO2 incubator at 37 °C followed by washing the wells twice with PBS buffer. The cells were then fixed with 4% formaldehyde in PBS buffer for one hour at room temperature followed by aspiration and washing with PBS buffer containing 5% FBS for three times. The fixed cells were then permeabilized with 0.1% Triton X-100 in PBS buffer solution containing 5% FBS for 20 min at room temperature followed by washing for three times with PBS buffer solution containing 5% FBS. The cell samples were then incubated with specific anti-YB-1 monoclonal mouse antibodies (Santa-Cruz, Germany) diluted in PBS buffer solution containing 5% FBS for one hour at room temperature, followed by aspiration and washing with PBS buffer solution containing 5% FBS for three times. Then the cell samples were incubated with secondary goat anti-Mouse IgG H&L (FITC) (Abcam, Cambridge, UK) antibody diluted in PBS buffer solution containing 5% FBS for one hour at room temperature followed by washing with PBS buffer solution containing 5% FBS for 3 times and the samples were then examined by Cytell™ Cell Imaging System (GE Healthcare, Marlborough, MA, USA).
4.6. Hoechst Nuclear Staining
Hoechst is a nuclear counterstain that permeates through the cell membrane and nuclear membrane without killing the cells to binds to DNA emitting a blue fluorescence stain. In the experiment, one drop of EasyProbes™ Hoechst 33342 Live Cell Stain (Genecopoeia, Rockville, MD, USA) was infused into each well of cell samples in the 24 well plate containing 500 µL of media. Then the plate was incubated at room temperature for 30 min followed by analyzing the plate with Cytell™ Cell Imaging System (GE Healthcare, Marlborough, MA, USA).
4.7. Flow Cytometry for Cell Cycle Interference
To determine the effect of YB-1 silencing on cell cycle, the DNA of different samples was stained with propidium iodide using Guava Cell Cycle Reagent (Millipore, Burlington, MA, USA). In this experiment, approximately 1 × 106 cells from each cell sample were grown for 24 h in serum-free media and were then harvested and collected in 1 mL of media in 15 mL Falcon conical polypropylene tube. The cells were washed twice with PBS buffer solution and were centrifuged and resuspended in cold 70% ethanol and kept overnight at 4 °C. The cells were then washed twice with PBS buffer solution and resuspended and carefully mixed in dark environment with 600 µL of cell cycle reagent. 200 µL in triplicates from each sample was then transferred to each well in flat bottom 96 well plate and incubated in aluminum foil at 37 °C in a humidified incubator for 30 min followed by running the plate in Guava easyCyte flow-cytometer (Millipore, Burlington, MA, USA).
4.8. Wound Healing Assay
One of the valid methods to determine the invasion and cell migration in vitro is through creating a wound in the cells monolayer with subsequent evaluation of their migration towards the empty zone [31
]. In this experiment, approximately 1× 105
cells of the different cell strains were seeded to the wells of 24-well plate in triplicates and were grown in complete DMEM supplemented with 10% FBS. Upon reaching 60% confluence, the media was replaced with serum-free media for 24 h. The next day, a sagittal wound was created by gently scratching the monolayer cells across the center of the well in the plate by a 200 µL pipette tip followed by washing the wells twice with PBS buffer solution to remove the detached cells. After confirming the width of the wound, the cells were replenished with complete DMEM media supplemented with 10% FBS and were evaluated for cell migration.
4.9. Statistical Analysis
The statistical analysis was completed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The samples numerical data were recorded as the mean ± standard error of the mean value, and the. Comparison between groups was made by one-way ANOVA test with Bonferroni post hoc test analysis. p-Values of less than 0.05 were considered statistically significant.