Optimized workflow to modify microRNA expression in primary human intravascular cells

Abstract

Background A comprehensive dissection of the role of microRNAs (miRNAs) in gene regulation and subsequent cell functions requires a specific and efficient knockdown or overexpression of the miRNA of interest; these are achieved by transfecting the cell of interest with a miRNA inhibitor or a miRNA mimic, respectively. Inhibitors and mimics of miRNAs with a unique chemistry and/or structural modifications are available commercially and require different transfection conditions. Here, we aimed to investigate how various conditions affect the transfection efficacy of two miRNAs with high and low endogenous expression, miR-15a-5p and miR-20b-5p respectively, in human primary cells. Results MiRNA inhibitors and mimics from two commonly used commercial vendors were employed, i.e., mirVana (Thermo Fisher Scientific) and locked nucleic acid (LNA) miRNA (Qiagen). We systematically examined and optimized the transfection conditions of such miRNA inhibitors and mimics to primary endothelial cells and monocytes using either a lipid-based carrier (lipofectamine) for delivery or an unassisted uptake. Transfection of LNA inhibitors with either phosphodiester (PE)- or phosphorothioate (PS)-modified nucleotide bonds, delivered using a lipid-based carrier, efficiently downregulated the expression levels of miR-15a-5p already 24 h following transfection. MirVana miR-15a-5p inhibitor displayed a less efficient inhibitory effect, which was not improved 48 h following a single transfection or two consecutive transfections. Interestingly, LNA-PS miR-15a-5p inhibitor efficiently reduced the levels of miR-15a-5p when delivered without a lipid-based carrier in both ECs and monocytes. When using a carrier, mirVana and LNA miR-15a-5p and miR-20b-5p mimics showed similar efficiency 48 h following transfection to ECs and monocytes. None of the miRNA mimics effectively induced overexpression of the respective miRNA when given to primary cells without a carrier. Conclusion LNA miRNA inhibitors efficiently downregulated the cellular expression of miRNA, such as miR-15a-5p. Furthermore, our findings suggest that LNA-PS miRNA inhibitors can be delivered in the absence of a lipid-based carrier, whereas miRNA mimics need the aid of a lipid-based carrier to achieve sufficient cellular uptake.

Cell Proliferation, Viability, Differentiation, and Apoptosis of Iron Oxide Labeled Stem Cells Transfected with Lipofectamine Assessed by MRI

“…The important role of lipofectamine as a nanocarrier to enable encapsulation of nucleic acids into cells [ 19 ] and to enhance the delivery of nanoparticles into cells was described before [ 20 ]. Caglayan et al found that elevated concentrations of mirVana or locked nucleic acid miR-15a-5p mimic that were delivered to endothelial cells using lipofectamine RNAiMAX as a carrier specifically increased the expression of miR-15a-5p [ 41 ]. Magro et al revealed that nanomaterials can be successfully internalized into mesenchymal stem cells transfected with lipofectamine by an increase in GFP expression [ 42 ].…”

PTEN, PTENP1, microRNAs, and ceRNA Networks: Precision Targeting in Cancer Therapeutics

“…Thus, in a cancer-dependent context, lncRNAs may be overexpressed to either inhibit or decrease the level of PTEN targeting microRNAs, thereby increasing PTEN expression and activity as a tumour suppressor. Furthermore, miRNAs can be depleted using miRNA inhibitors, or 'sponges' [177], in order to increase PTEN or PTENP1 levels in cells.…”

“…Estrogen plays an essential role in the occurrence of endometrioid carcinoma and affects the negative feedback loop of PTEN-miR-200c-PTENP1 [114]. Thus, the upregulation of certain miRNAs using miRNA mimics [177] could be a useful potential cancer therapy depending on the cancer type.…”

“…The methods utilised have involved the use of lipids, polymers, inorganic and extra-cellular vesicle carriers [178]. There are still many challenges with the use of miRNAs in human trials [167] despite the advancements in miRNA delivery systems, and miRNA drugs have yet to reach phase III human trials [177]. For example, in solid tumours such as hepatocellular carcinomas, the trial of the MRX34 drug, which delivers miR-34 via liposomes intravenously to patients, was terminated due to immune-related severe adverse events [179].…”