2.1. Software and primer designing
The sequence of the miR34a gene was taken from the NCBI database (GenBank: EF609116.1) and was checked using CLC main version 5 software. The secondary structures of the wild-type and mutant miR34a gene were also analyzed by RNA software. Gene Runner and OLIGO7 software were applied to design six primers to amplify the miR34a gene, site-directed mutagenesis, and cloning. The primers have restriction site sequences compatible with pCDH vector and spacer sequences.
2.2. miR34a gene amplification and site directed mutation
Total DNA was extracted from whole blood cells by QIAamp DNA extraction kit (Qiagen, Germany). Two primer pairs were separately used to amplify the 250bp DNA encoding miR34a by PCR. The F-34a (5-TGGGGAGAGGCAGGACAGG-3) and R-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA. PCR reactions were performed using Takara puf kit for 30 cycles (reaction conditions were as follows: 30s at 95 ºC, 30s at 60 ºC, and 30s at 72 ºC). The first pair, 34a FWD (5'- TGGGGAGAGGCAGGACAGG – 3' and 5'- TCCGAAGTCCTGGCGTCTCC-3') and S2 (5' TTA CTA TTG CTC ACA A CA TCC TC C TAA GAC ACT GC 3′) were used for amplification of the first DNA fragment with the first set of 6 nucleotides deserted point mutation.
In comparison, the second pair S1 (5′CTCGAGTACAACTATGCGGCCGCAGCAGAATG-GGAGATGAATTTCA3′) and 34a REV (5′ACGACGCGTCG TTAGTGTTACAGAGTCT-GATATCC3′) were used for amplification of the second fragment of DNA with a second set of 6 nucleated deserted point mutation encoding miR34a pre-miRNA. PCR reactions were performed using Takara puf kit for 30 cycles (reaction conditions were as follow: 30s at 95 ºC, 30s at 60 ºC, 30s at 72 ºC for the first reaction and 30s at 95 ºC, 30s at 60 ºC, 30s at 72 ºC for the second reaction). Both S1 and S2 primers were designed to include an overlapping sequence. In the next step, these overlapping fragments were used to assemble two amplified sequences into a full-length DNA through an overlap extension of PCR (30s at 95 ºC, 30s at 60 ºC, and 120s at 72 ºC). Finally, a primer pair composed of 34a FWD and 34a REV was used to amplify the assembled fragment following reaction conditions as the 30s at 95ºC, 30s at 60ºC, and 90s at 72º C).
2.3. miR34a gene cloning
pCDH lentiviral vector and the amplified miR34aW and miR34aM gene fragments were digested separately with BamH1 and not1 restriction enzymes (Roche, Germany) and ligated by T4 DNA ligase (Fermentas, Germany). The ligation mixture was transformed to Stbl4 competent bacteria by CaCl2 method. The transformed bacteria were cultured on LB-agar medium containing ampicillin (100µg/ml). Colony PCR was performed to screen the positive colon by universal primers of pCDH vector (pCDH-FWD 5′-CACCAAAATCAACGGGACTT-3′ and pCDH-REV 5′-ATATAGACAAACGCACACCGGCCT-3′). Positive bacterial colonies were harvested and cultured overnight in LB medium for plasmid extraction. DNA sequencing with universal primers was done to confirm the accuracy of the cloned product.
2.4. Lentiviral vector packaging, titration, and cell transduction
Lentiviral particles were produced in HEK293T cells as described by Kutner et al. with some modifications [18]. Briefly, 24 hours before transfection, a 5 million concentration of HEK 293T cells were plated in a 10cm dish in DMEM for confluency of 80% to 90% in the next day. Cell culture medium was replaced with fresh complete DMEM two hours before transfection. Then, cells were transfected with Lenti ORF pcdh-mir34a (wild and mutant separately), pMD2G, and pSPAX by calcium phosphate method. Culture medium was replaced with fresh medium 14 hours post-transfection. For virus isolation, the Culture medium was collected three times at 12 hours intervals.
To increase virus particle concentration, cell debris was removed following centrifugation of culture medium at 300g for 5 minutes, and the supernatant was subsequently filtrated through Millex-HV 0.45μm PVDF filter (Millipore). Lentiviral particles were precipitated via ultracentrifugation at 45000g for 1.5 hours, and the virus particles in the pellet were re-suspended in 1ml of PBS. Concentrated virus particles were used to transduce HEK 293T cells.
Three days post-transduction, flow cytometric analysis was performed for lentivirus titration [15]. Briefly, serial dilution (0.5, 1, 5, 25, and 50) of concentrated virus particles were added to 100000 HEK 293T cells which were cultured in 500 µl of DMED medium. Three days later, cells were harvested and washed twice with PBS, and GFP positive cells population were counted by flow cytometry.
Viral transduction performed by the concentrated virus, briefly HEK293T cells (6.0×104) were cultured in 6 well plates. After 24 hours, 10ulit of concentrated virus dissolved to 2 ml DMEM free FBS medium, and the final solution was added to each well. After 16 hours, the medium was replaced with a fresh DMED complete medium. The GFP fluorescent microscopy was applied for transduction rate detection after 72 hours.
2.5. Puromycin kill curve and stable cell line selection
First, HEK293T cells (6.0×104, not infected with the virus) were cultured in 5 wells of a 24-well plate containing 1 ml of complete DMEM medium. On the next day, when the cells stick to the bottom of the plate, we replace the culture medium of the cells with 1 ml of the culture medium containing zero, 0.5, 1, 1.5 and 2 micrograms of antibiotic puromycin, respectively. On the first day after the treatment, the culture medium of the cells was replaced with the selected medium containing the antibiotic puromycin, and daily the number of living cells in each well was counted by vital staining.
The best result is obtained 1 to 4 days after antibiotic treatment. During this period, the number of living cells for each concentration was shown as a curve. The least amount of antibiotic that destroys the cells at this time was selected for the selection of cells after transduction.
2.6. Isolation of exosomes
To produce miR34a enriched exosomes, HEK293T stable cells were cultured in DMEM supplied with 10% exosome-free FBS. The culture media were periodically collected at 48 hours intervals, and then exosomes were isolated via sequential centrifugation and re-suspended in PBS. Aliquots of exosomes were stored at -80 ºC.
2.7. Characterization of exosome
Transmission electron microscopy (TEM), zeta sizer analysis, and western blot analysis were carried out to characterize the obtained exosome. For electron microscopy imaging, the ultra-concentrated exosomes were fixed with 4% paraformaldehyde in PBS. The fixed samples were air-dried on Formvar-carbon coated TEM grids and stained with 0.5% uranyl acetate in 30% ethanol for 10 min, followed by staining with lead citrate 10 min. The sample was sequentially rinsed in 0.05 M NaOH and distilled water for removing lead precipitates on the grid sections. Then, the grids were dried at room temperature, and the stained sections were scanned under the transmission electron microscope (Philips) operating at 150 kV. According to the manufacturer's instructions, the purified exosomes' dimensions were measured using a zeta sizer (Malvern Corp.).
The transduced cells and exosomes were lysed with RIPA buffer containing protease inhibitor cocktail (Roche, Germany), and the obtained total protein was subjected to Western Blot analysis. Briefly, the cells were washed twice with ice-cold PBS and centrifuged at 4000 rpm for 5min. The cells (and exosomes) were treated with lysis buffer and incubated on ice for 20min with shaking. Cell debris was precipitated by centrifugation at 13000 rpm for 20 min, and the upper phase was collected as a source of the total protein. The number of total proteins was calculated using the Bradford assay. The extracted proteins (10 μg) were boiled for 5 min in 10X SDS buffer (1 μg) and resolved on 10 % polyacrylamide gel for 2 hours. Protein fragments were transferred to nitrocellulose membrane under the constant current of 300 mA for 2 hours, and the membrane was blocked with 5% nonfat dry skim milk in PBS for 2 hours. The membrane was washed with PBS and treated with HRP-conjugated anti-His tag antibody for 16 hours to detect LAMP-DARPin G3 chimeric protein. After three times of washing with PBST, the related band was detected using ECL solution. In addition, the expression of CD63 as an exosome marker was investigated by flow cytometry.
2.8. Revers Transcription and quantitative PCR
Total RNA was extracted using Qiagen RNeasy mini kit from purified exosomes three days post-transduction of HEK293T cells with miR-34a virus. Revert-Aid™ First Strand cDNA Synthesis Kit (Fermentas, Germany) was applied for cDNA synthesis according to the manufacturer's instructions.
The relative qPCR experiments were performed on the ABI thermal cycler in the total volume of 20 µl containing 10 µl of Qiagen qPCR Master Mix (Qiagen), 10 pmol of each primer, and 2 µl of each cDNA. 16 s RNA was used as a reference gene.