Effects of miR-15a modulation in gene expression in human aortic SMCs

Among in silico predicted or experimentally confirmed miR-15a targets are many genes of relevance to AAA pathology and known to be down-regulated in AAA conditions. To better understand which of these targets could be linked to the observed detrimental effects of miR-15a on SMC dynamics in vitro, we performed RNA-sequencing of hAoSMCs transfected with miR-15a-modulators (mimic, inhibitor, and scrambled control). Upon transfection with miR-15a-mimic, 1030 genes were significantly down-regulated and 741 were significantly up-regulated, whereas miR-15a-inhibition led to 23 down-regulated and 82 up-regulated genes. Overall design: Primary human aortic smooth muscle cells (hAoSMCs, lot nr 3003) were purchased from Cell Applications (San Diego, USA) and cultured in complete Human SMC Growth Medium (Cell Applications; 311-500), supplemented with 1% PenStrep. Cells were seeded in 6-well plates at 125.000 cells/well and kept in growth medium overnight. Next morning, cells were starved for 24h in OptiMEM (Gibco, ThermoFisher Scientific) + 2% FBS + 1% PenStrep. Shortly before transfection, medium was changed back to full growth medium as per above. Transfection of miRNA-modulators (miRVana miRNA mimic/inhibitor/scrambled control, ThermoFisher Scientific) was performed using Lipofectamine RNAiMAX (ThermoFisher Scientific) in accordance with manufacturer's manual and using 7.2 µl RNAiMAX per well and a final modulator concentration of 50nM. Cells were then cultured for 48h. RNA was harvested by pooling two wells of each condition to assure adequate RNA concentration. Extraction was performed using miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen), following the manufacturer's protocol, including an on-column DNAse digestion step. Extracted RNA from cell lysates stemming from hAoSMCs 48h post-transfection with miR-15a-mimic (n=3), miR-15a-inhibitor (n=3) and scrambled control oligo (n=3) was analysed by a commercial vendor (Novogene UK Company Limited, Cambridge, United Kingdom) using following procedures. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dUTP. The directional library was complete after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and Bioanalyzer for size distribution detection. The clustering of the index-coded samples was performed according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq PE150 platform and >40M paired-end reads were generated for each sample. Raw data (raw reads) of fastq format were cleaned by removing reads containing adapter sequence, reads containing poly-N and low-quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data was calculated. All downstream analyses were based on the clean, high-quality data. Paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. FeatureCounts v1.5.0-p3 was used to quantify reads mapped to each gene. FPKM of each gene was calculated based on the length of the gene and its mapped reads. Differential expression analysis was performed using the DESeq2 R package (1.20.0). The resulting p-values were adjusted for false discovery rate using the Benjamini and Hochberg's approach. At this point, data was released by the commercial vendor and all subsequent analyses were performed independently by our group. Genes with an adjusted p-value <=0.05 and absolute fold-change >=1.5 were defined as differentially expressed.