Cell lines, growth conditions and cell's transfection
The following cell lines were used in the study: Acute T cell leukemia (Jurkat, kindly provided by Dr. Galit Granot, FMRC), primary human foreskin fibroblast (pHFF, kindly provided by Dr. Selig from the Israel Institute of Technology); human foreskin fibroblast (hFF). The hFF cell line was immortalized by the ectopic expression of hTERT as previously described [8] and kindly provided by Dr. Selig from the Israel Institute of Technology.
Jurkat cell line was cultured inRPMI-1640 with 20% fetal bovine serum (FBS), containing 100 units/ml L-Glutamine and 1% streptomycin (BiologicalIndustries Beit Haemek, Israel).hFF andpHFF cells were cultured in Dulbecco's minimal essential medium (DMEM) with 20% FBS containing 100 units/ml L-Glutamine and 1% streptomycin (BiologicalIndustries). The cells were incubated at 37°C in 5% CO2. For exosome isolation an exosome free media was prepared by ultracentrifugation of FBS at 100,000g for 16 hours at 4°C (Optima™ XPN, Beckman coulter, USA).
For cell transfection,2.5pHFF cells/well were seeded in 6-well plate for 24hr and were subsequently transfected by using HiPerFect transfection reagent (Qiagen, Germany) according to the manufacturer’s instructions. Briefly, miRNA mimics was diluted (150 ng) in 100 µl of culture medium without serum for a final concentration of 5 nM. Then it was mixed with 12 µl of HiPerFect transfection reagent to allow the formation of transfection complexes in room temperature for 10 min. The resulting transfection solutions were then added to each well containing 2.3 ml of culture medium. Cells were grown at 37oC for 72h before another transfection was performed as above.
Cells were transfected with the following miScript miRNAs:
- hsa miR-342-3p miRNA (miR342 mimic)
- miRNA Negative Control with a scrambled sequence.
- miRNA Inhibitor
- miRNA Inhibitors Negative Control
Isolation of exosomes
Exosomes were isolated by serial ultracentrifugation as previously described [9]. Briefly, a total of 5x Jurkat cells were grown in exosome depleted media for 3 days. Then, the cells debris was removed by 2 consecutive centrifugations at 1,000 RCF 2,000 RCF each. Subsequently, the media was centrifuged at 10,000 RCF for 30 min at 4°C and the supernatant collected and filtered through 0.22 µm filters (Merck Millipore, USA). The clarified medium was than centrifuged at 100,000 RCF at 4°C for 70 minutes (Beckman Coulter, CA, USA). The crude exosome-containing pellets were resuspended in 1 mL and a second round of ultracentrifugation at 100,000 RCF at 4°C for 120 minutes was carried out. The resulting exosome pellet was resuspended in 200 µL of PBS and was referred to as the first pellet. A second pellet was obtained after centrifuging the supernatant of the first pellet at 100,000 RCF at 4°C for 120 minutes.
Nanosized tracking analysis of Jurkat derived exosomes
Exosomes were quantified by using the Nanosight tracking analysis, according to the manufacturer's instructions (Malvern Panalytical, Cambridge UK) and following published method [10]. The analysis settings were optimized and kept constant between samples.
BCA Protein quantification
Exosomes were suspended in RIPA buffer and centrifuged for protein extraction. Total protein was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific, MA, USA) in accordance with the manufacturer's instructions.
Exposure of fibroblasts to Jurkat derived exosomes
200μls of the resuspended exosomes were added to 3X105 pHFF cells per well in a 6-well culture plate. 6 hours later the fibroblast cells were harvested and RNA was extracted for the measurement of hTERT expression by q-PCR. Previous studies conducted in our laboratory have suggested that exosomes affected the recipient cells in a dose and a time dependent manner. Therefore, we used these conditions in all further experiments.
RNA isolation
Total RNA was extracted using the EZ-RNA II Isolation Kit reagent (Biological Industries Beit Haemek, Israel) according to manufacturer's instructions. Briefly, cells were lysed with guanidine thiocyanate detergent solution, followed by organic extraction and alcohol precipitation of the RNA. Quantification and the purity of total RNA were assessed by using the NanoDrop1000®spectrophotometer (Thermo Fisher Scientific, MA, USA).
RNA isolation from exosomes was performed by using Total Exosome RNA and Protein Isolation Kit (Invitrogen, CA, USA). Exosomes were lysed with denaturing solution and then precipitated by acid-phenol: chloroform solution followed by several washes and centrifugation steps. The supernatant was then placed on filter cartridge tubes for further purification using numerous washing solutions provided by the kit. RNA was eluted with PCR grade water.
cDNA formation
Total RNA extracts from cells were reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem, CA, USA) according to the manufacturer's instructions. Briefly, 1,000ng of template RNA was added to 10μL reaction mixture containing dNTP's, random primers, RNase inhibitor, reaction buffer and reverse transcriptase. The incubations steps included 10 min at 250c followed by 120 min at 370C and the reaction was terminated by a heating step at 850c for 5 min.
Q-real time PCR reaction
Real-time-PCR was employed to estimate the levels of the hTERT mRNA in exosomes secreted from the growth media of Jurkat cells based on the Taqman methodology (Applied Biosystems, CA, USA) and for the validation of the results of the microRNAs analysis. The expression of the HPRT-1 (Hypoxanthine-guanine-phosphoribosyltransferase-1) gene was used as the endogenous control. PCR reactions were carried out using the Step One Plus detection system (Applied Biosystems, CA, USA). Taqman real time PCR reactions were performed with 50ng of cDNA, specific primers and Taqman gene expression master mix (Applied Biosystems, CA, USA). The incubations steps included 2 min at 500C followed by 10 min at 950C and 40 cycles of 15 seconds at 950C and 1 min at 600C.
PCR reactions were prepared with the following custom made Taqman fluorochrome labelled primers (Applied Biosystems, CA, USA).
hTERT primers: Forward: 5′-CGTCCAGACTCCGC TTCATC-3′
Reverse: 5′-GAGACGCTCGGCCCTCTT-3′;
HPRT-1 primers: Forward: 5-′TTATGGACA GGACTGAACGTCTTG-3′
Reverse: 5′-TGTAATCCAGCAGGTCAGCAAA-3′
Western blot
The expression of CAFs markers in exosomal exposed fibroblast cells was assessed by Western immunoblot analysis. Proteins were extracted and quantified using the Bradford assay (Bio-Rad Laboratories, CA, USA). 25μg of proteins extracted from treated and untreated cells were subjected to Poly-Acrylamide Gel Electrophoresis and transferred to a nitrocellulose membrane. Membrane was hybridized with antibodies against the different proteins: αSMA (1:500, Bio-Rad, California, USA); Vimentin (1:200, Santa Cruz Biotechnology, Texas, U.S.A.); and subsequently to fluorescent labeled secondary antibodies. Visualization was done by the Odyssey analysis software (Odyssey IR imaging system; LI-COR).
ELISA assay
The expression of IL-6 by exosome exposed fibroblast cells was evaluated by ELISA assay. Culture supernatants were collected centrifuged to pellet any detached cells and analyzed using a Human IL-6 Quantikine ELISA Kit (R&D Systems Inc., MN, USA) according to the manufacturer's instructions. Culture supernatants were diluted 150- 1000-fold for IL-6 measurements.
miRNA profiling
microRNA expression levels were determined by using the Rosetta Genomics proprietary microarrays platform containing 2167 microRNA probes and various control probes. RNA was extracted and labeled; samples were processed according to standard protocols. Data was stored in the Microarray Database. Statistical probability of significance (adjusted p values, set at p < 0.05) and a fold change of >2 were used to select the miRNAs that were significantly differentially expressed between our samples. The miRBase website [12] was used to retrieve the mature sequences for each miR.
Validation of the results of the microRNA analysis
q-PCR was performed to validate and quantify the expression of the results of the microRNA analysis (miR-342-3p, miR-125b-1, miR-128-3p and miR-92a-3p). Prior to RNA isolation as described above, pHFF cells were exposed to Jurkat cells derived exosomes for 6 h and the levels of microRNA were determined by Q-PCR. pHFF cells without exosome exposure served as controls. Specific commercial primers sequences were used for the validation of each miRNAs (ABI,Foster City, CA, USA).
miRNA cDNA formation
For miRNA-specific cDNA formation we used 20ng total RNA from the RNA extraction samples. Taqman® small RNA Assay was used for miRNA reverse transcription reaction. 1-10 ng of total RNA sample were used per 15 µl RT reaction. Taqman® MicroRNA probe was used as the Reverse Transcription primers. Each reaction included 7 µl master mix, 3 µl 5 X RT primers (miRNA specific primers) and 5 µl of RNA sample (1-10 ng).
miRNA Q-real time PCR reaction
Taqman® Small RNA Assays kit was used for Real-time quantitative PCR according to the provided manual (ABI,CA, USA), run and analyzed by the Step- One Q-PCR device(ABI).
miRNA specific primers were chosen for the following miRNAs: miR-342-3p, miR-125b-1, miR-128-3p and miR-92a-3p. U3 was selected as a housekeeping gene to normalize the miRNA levels.
The incubation steps included: 10 min at 95°C, followed by 40 cycles of 15 seconds at 950C and 60 seconds at 60°C.
Proliferation assay
Cellular proliferation of pHFF cells was assessed by the Sulphorodamine B (SRB) assay. Post transfection, cells/ml were cultured in 24-well plates for 72h. Cells were then fixated to the plate with 10% Tetrochloric Acid and incubated at 40C for 1h, then washed with dH2O. Subsequently, 1ml of SRB coloring agent (Sigma Aldrich Israel Ltd., Rehovot, Israel) was added to the wells which were incubated at roomtemperature for 30 mins and then washed with 1% acetic acid. After adding 10nM TRIS.HCl buffer absorbance was measuredat 515nm by an ELISA microplate reader (Biotek InstrumentsInc., VT, USA). Experiments were performed in triplicates.
Cell cycle analysis
The cell cycle status was analyzed by flow cytometry. Cells were processed by standard methods using propidium iodide staining of the DNA. Briefly, after cells' transfection upon reaching 60% confluence, the cells were trypsinized, resuspended in 5 ml cold PBS and centrifuged at 2,500 rpm for 5 mins at 4°C. Cells were subsequently pelleted and fixed by adding 4.5 ml 70% cold ethanol dropwise to 0.5 cold PBS, with gentle mixing. The cells were then incubated for >2 h at -20°C. Cells were subsequently washed by PBS and centrifuged at 2,500 rpm for 5 minutes 4°C. Staining with propidium iodide (P4170, Sigma Aldrich) was conducted prior to flow cytometry analysis. Cells were pelleted and resuspended in 0.5 ml PBS containing 0.025 ml PI (50 μg/ml in PBS and 5μl of 100 mg/ml DNase-free RNase (Sigma, St Louis, MO). Cells were then incubated at 37°C for 15 minutes. Samples were analyzed on a Beckman-Coulter Epics XL-MCL apparatus. The parameters were adjusted for the measurement of single cells using the forward and side scatter plots.
Prediction of miRNA targets
To determine potential mRNA targets for specific miRNAs we have used several publicly available target prediction algorithms based on sequence complementarity between the miRNA and its potential mRNA target 3’ untranslated region (3’UTR).
TargetScan[11] was used to determine potential miR-342 targets involved in cell proliferation and cellcycle. In addition to TargetScan, a combination of bioinformatics tools for miRNA target prediction, includingmiRBase [12],miRanda [13], DIANAmt [14], miRDB [15], miRWalk [16] and PITA [17] were used.
Functional annotation and gene set enrichment analysis
Several bioinformatic tools have been used for the biological interpretation and the analysis of pathways associated with defined lists of genes. These included DIANA miRPath and DAVID. mirPath is a web-based computational tool developed to identify molecular pathways potentially altered by the expression of single or multiple microRNAs. The software performs an enrichment analysis of multiple microRNA target genes comparing each set of microRNA targets to all known pathways. Pathways with p-value <0.05 providing statistically significant values are selected [18]. mirPath was used to gain insight into global molecular networks and canonical pathways specific to miR-342.
The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 bioinformatic algorithm [19] was used for the functional annotation analysis of Gene Ontology (GO) terms associated with a given gene list of confirmed mRNA targets within each miRNA cluster specific for miR-342. We analyzed the list of the most significantly regulated genes using the ClueGO Cytoscape plugin [20]. GO functional categories with p-values under 0.05 were considered statistically significant. To study the putative interactions between the four selected miRs and telomerase, we identified proteins that are upstream to these miRs. These proteins were subjected to the Advanced Network Analysis Tool (ANAT) software, a tool for constructing and analyzing functional protein networks. ANAT provides access to up-to-date large-scale physical association of protein data, advanced algorithms for network reconstruction and several tools for exploring and evaluating the obtained network models. We used the network-based analysis inferring an anchored network (telomerase) that connects a given set of proteins (proteins that regulate the four selected miRs expression) to designated anchor set of proteins [21].
Statistical analysis
Quantitative data were expressed as means ± SD. Statistical significance was determined by the Student’s t-test. P-value < 0.05 was considered as statistically significant.