Rat aortic smooth muscle cell line A7r5 was maintained at 37 °C in DMEM supplemented with 10 % FBS, 2 mmol/L l-glutamine, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin, in a 5 % CO2 incubator.
Fast Lane cell cDNA kit (Qiagen, 21651, Germany) was used for first strand cDNA synthesis directly from cultured cells. Housekeeping gene GAPDH was amplified by PCR using the cDNA synthesized as a template to verify the quality of the cDNA. Verified cDNA was used in Exo70 RT-PCR detection. The primer sequences used in this experiment were the following: for Exo70 gene, Exo70-f: 5’-CCCCAACAAGAGGAAAGA-3’ and Exo70-r: 5’-CCTGACAAAGGCACTAACG-3’; for GAPDH gene, GAPDH-f : 5’-AGAGACAGCCGCATCTTCTTG-3’ and GAPDH-r : 5’-GGTAACCAGGCGTCCGATAC-3’. Gel electrophoresis was performed to detect the PCR products. Exo70 PCR product size was 115 bp.
A7r5 cells during their logarithmic phase, were dissolved to form a cell suspension and seeded in 24-well plates containing the coverslips in each well. When the cells reached 80 % confluence in the coverslips, the original culture was discarded, cells were washed with PBS, fixed with room-temperature 4 % paraformaldehyde (PFA)/PBS for 30 min, washed, permeabilized with 0.1 % Triton X-100/TBS for 30 min, and blocked with 0.5 % BSA. Next they were washed and sequentially incubated with primary antibody at 4 °C overnight and secondary antibody at room-temperature for 60 min. Cells were observed with a confocal microscope and recorded.
Rat Exoc7 gene (NM_022691) was used as the target gene. According to the complete rat Exoc7 mRNA sequence in Gene Bank, four shRNA interference sequences were designed: pLV-ratExoc7-sh1 (GGAACCAAGATTTCATGAATG CTCGAG CATTCATGAAATCTTGGTTCC TTTTT), pLV-ratExoc7-sh2 (GGATAACATCAAGAATGATCC CTCGAG GGATCATTCTTGATGTTATCC TTTTT), pLV-ratExoc7-sh3 (GCCTAAAGATGGCACCGTTCA CTCGAG TGAACGGTGCCATCTTTAGGC TTTTT), and pLV-ratExoc7-sh4 (GCGCCATCTTCCTACACAACA CTCGAG TGTTGTGTAGGAAGATGGCGC TTTTT). These sequences were inserted into a lentivirus vector to construct four shRNA expression vectors and they were used to transfect A7r5 cells to verify the efficiencies of interference. The highest efficiency vector pLV-ratExoc7-sh2 was selected and the corresponding lentivirus was used for subsequent experiments. Cells were cultured for 12 h and then the culture medium was removed. Three ml DMEM high glucose complete medium with 10 % FBS without P/S was added, and the corresponding virus solution was added directly to the culture plate with MOI = 50. Twelve hours post-infection, the culture medium was changed with 5 ml DMEM high glucose complete medium with 10 % FBS. The cells were continuously cultured for 4 days with medium replacement every 2 days. Ninety-six hours post-infection, the infected cells were observed under the microscope. Fresh DMEM high glucose complete medium with 10 % FBS was added and 1 μg/ml of puromycin was added for selection. The cells were cultured for additional 4 days with daily medium change. After 4 days’ selection, the cells were basically stabilized. When the resistant cells were 90 % confluent, they were digested and passed with at a 1:4 ratio and cultured with 1 μg/ml puromycin for 4 days. After continuous subcultyre and selection, cells were finally carrying the stable resistance gene and could be passed on. Cells were then digested and harvested and the interference efficiencies were analyzed using Western blot and qRT-PCR technology.
A7r5 and A7r5-ratExoc7-KD cells were lysed and scraped off the culture plates in protein sample buffer containing 4 % SDS, 20 % glycerol, and 25 mM Tris, pH 7.6. Proteins were extracted and quantified. The resulting cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes for Western blot analysis, using a goat monoclonal anti-rat antibody (1:1000, sc-135082, Santa Cruz). A secondary antibody was applied and proteins were visualized by ECL chemiluminescence reagent kit, stabilization of the membrane, and photographed using a gel imaging system.
qRT-PCR analysis was performed using BioRad qReal-time PCR detection system and THUNDERBIRD SYBR qReal-timePCR kit. The GAPDH gene was used as an internal reference to ensure relatively accurate mRNA expression and to avoid system and random errors during sample processing. Based on qPCR primer design principles and the complete ratExoc7 mRNA sequence from Gene Bank, qPCR primers were designed. The sequences were the following: for ratExoc7 gene: ratExoc7-f: 5’-AGCGACCAGCTCACTAAGAA-3’, RatExoc7-r: 5’-CACAGGGATGATGGAGTTCTCCA-3’, PCR product was 87 bp in length; for GAPDH gene: GAPDH-f: 5’-TGCACCACCAACTGCTTAGC-3’, GAPDH-r: 5’-GGCATGGACTGTGGTCATGAG-3’, PCR product was 87 bp in length. The melting curve was used for the analysis of the immune specificity of amplified products. The standard curve was used to compare the relative content of the target genes.
Wound healing assay
A7r5 cells were seeded in 24-well plates (~1 × 104 cells per well) for 24 h. When cells reached the confluence, the culture medium was removed, 2 ml fresh culture medium containing 10 μg ml−1 mitomycin were added, and the cells were cultured for additional 2 h. Next, scratches were introduced using a sterile 100 μl pipette tip. Cells were washed with DMEM to remove debris and the remaining cells were incubated with regular growth medium. Cells were observed after wounding at 0, 6, 12, 24 and 48 h by inverted microscope until the time point of complete wound closure.
A7r5 cells were cultured until 80 % confluence. The cells were digested with trypsin, washed with serum-free culture medium 3×, counted, and added to cell culture wells as cell suspensions with 1 × 104 cells per well. Matrigel stored at −80 °C was transferred to a 4 °C refrigerator and was liquefied overnight. Sixty microliter Matrigel were then added into 300 μl serum-free culture medium and mixed. One hundred μl of this mixture was added to the above culture wells, and incubated at 37 °C for 4–5 h. The Matrigel was washed once with serum-free culture medium. One hundred μl cell suspensions were added to the wells. At the lower culture wells, 600 μl of 20 % FBS culture medium were added and the Transwell plate was incubated at 37 °C for 20–24 h. The Transwell was removed and washed with PBS twice, fixed with 5 % pentanediol at 4 °C. Crystal violet (0.5 %) was added for 5–10 min for staining. The wells were washed with PBS 2×, observed under the microscope, and cells were counted.
Results are expressed as means ± SD. The comparisons between the two groups were made by one-way ANOVA using GraphPad PRISM 5.0. Values of *p < 0.05 were considered statistically significant.