Normal HDFs (NHDFs) were purchased from PromoCell GmbH (NHDF-cryovial adult cell line, cat. no. C-12302) and were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 1% Gibco^® Antibiotic-Antimycotic Solution (10,000 U/ml penicillin, 10,000 µg/ml streptomycin and 25 µg/ml Gibco Amphotericin B; Gibco; Thermo Fisher Scientific, Inc.) in an incubator (5% CO₂, 37°C).

Hsa-miR-302b-3p (GenBank LM379226.1, chromosomal location 4q25, approved symbol MIR302B) was used to transfect NHDFs. The miRCURY LNA miRNA Mimic-5′ FAM (miR sequence 5′-UAAGUGCUUCCAUGUUUUAGUAG-3′) and its inhibitor (miRCURY LNA Inhibitor 5′ TACTAAAACATGGAAGCACT-3′) were provided by Qiagen GmbH. As a negative control, Negative Control 5 miRCURY LNA miRNA Mimic (Qiagen GmbH; cat. no. YM00479904-ADB; product no. 339173) was used. The guide strands of this control have no homology to any known miRNA or mRNA sequences in mice, rats or humans, and have been tested for adverse effects in multiple cell lines.

Three non-viral transient transfection methods were employed for the transfection of NHDFs: Transfection with i) Nucleofector Amaxa™ 4D-Nucleofector™ System with X Unit apparatus (hereafter referred to as nucleofection) (Lonza Group Ltd.); ii) Viromer^® Blue (Lipocalyx GmbH) and iii) INTERFERin^® (Polyplus-transfection SA). As a physical/mechanical method of transfection, nucleofection is an electroporation-based transfection method that allows nucleic acids, such as DNA or RNA, to be transferred into cells by applying a specific voltage and using specific reagents (36). Techniques using Viromer Blue and INTERFERin are chemical transfection methods. According to the manufacturer's protocol, Viromer Blue is a polymer-based transfection reagent featuring a viral mechanism of membrane fusion. The reagent is capable of forming a complex with miRNA and transporting it into cells by endocytosis. By contrast, INTERFERin, a non-liposomal cationic amphiphilic lipid-based transfection reagent, is one of the most efficient reagents for small interfering RNA delivery. The transfection protocols are described hereafter. The reagent to nucleic acid ratio was consistent with the manufacturers' protocols. Different cell densities were used for the three transfection methods because all reagents were used in the cell density range recommended by the manufacturer.

Transfection efficiency was assessed using a fluorescence microscope (Leica DM IL LED; Leica Microsystems, Inc.) by counting the total number of observed cells and the number of cells that express fluorescence of a FAM™ dye-labeled synthetic miRNA.

Briefly, for nucleofection, cells were passaged 2 days before transfection. Cells (1×10⁵) were seeded in 96-well plates. The cells were transfected upon reaching a confluence of 90%. The cells were washed with phosphate-buffered saline and dissociated with 0.25% Trypsin-EDTA solution at 37°C for 5 min. The enzyme was neutralized with DMEM containing 10% FBS. Aliquots of the cells were taken and cell density was determined, and then the required number of cells was resuspended in 20 µl 4D-Nucleofector™ Solution; 5 and 50 nM miRNA mimic and negative control (10% of final sample volume) was added to each sample. For transfection, the P2 Primary Cell 4D-Nucleofactor™ X Kit for Human Dermal Fibroblasts Basic Nucleofector Kit (Lonza Group Ltd.) and 4D-Nucleofector System apparatus (Lonza Group Ltd.) were used according to the manufacturer's protocol. After transfection, the Nucleocuvette™ was incubated at room temperature for 10 min and resuspended in pre-warmed medium to a total volume 100 µl. Next, 25 µl of each sample was transferred to a 96-well culture plate with 175 µl medium in each well (four wells for each sample and negative control). The cells were then harvested after 4, 24, 48 and 72 h and gene expression was measured.

The standard complexation protocol was employed for Viromer^® Blue. Briefly, 8×10⁴ cells were seeded 1 day before transfection in one well of a 24-well plate in 0.5 ml DMEM. The medium was changed to fresh medium of the same type immediately before transfection. Cells were transfected upon reaching a confluence of 60–80%. Next, the transfection complex was prepared: 45 µl Viromer solution mixed with 5 µl of 5 and 50 nM miRNA mimic. Then, 50 µl of the transfection complex was added to the cells in each well. Effects were monitored 24, 48 and 72 h post-transfection.

Briefly, ~2.5×10⁴ cells were seeded 1 day before transfection in a 24-well plate in 1 ml DMEM. The cells were transfected upon reaching a confluence of 30–50%. Three different miRNA mimic concentrations were tested: 1, 3 and 7 nM. Each concentration of miRNA was diluted in 100 µl medium without FBS and mixed with 2 µl INTERFERin. Afterwards, the DMEM was removed, and 0.5 ml fresh medium was added to each well, followed by 100 µl of the transfection mix. CROT gene silencing was assessed between 24 and 72 h.

Metabolic activity and the viability of transfected cells were measured via mitochondrial dehydrogenases, by means of the colorimetric MTT assay (Invitrogen™ CyQUANT™ MTT Cell Viability Assay; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The absorbance was measured at a wavelength of 570 nm using an Infinite M200 Pro Microplate Reader (Tecan Group Ltd.).

To investigate the cytotoxic effects of transfection, the CyQUANT lactate dehydrogenase (LDH) Cytotoxicity Assay (Thermo Fisher Scientific, Inc.) was used. The assay measures the LDH released from cells with a damaged membrane, whereby the amount of LDH released into the medium is quantified via the level of formazan formation. For this purpose, transfection with INTERFERin^® and Viromer^® Blue was compared with non-transfected NHDFs. A total of 24 h after transfection, the CyQUANT LDH cytotoxicity assay was performed according to the manufacturer's instructions. A sample of Triton-X-100 was used as the positive control for 100% cytotoxicity.

RNA was isolated from NHDFs using the Total RNA Mini Plus (A&A Biotechnology) according to the manufacturer's protocol. The quality of isolated RNA was assessed via electrophoresis using a 1.5% agarose gel (MilliporeSigma) and it was quantified on a NanoDrop 2000c (Thermo Fisher Scientific, Inc.). RT was performed using 0.5 µg total RNA for each sample using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's recommendations. CROT (GenBank accession no. NM_021151) was analyzed as a target gene of hsa-miR-302b-3p, according to miRBase (https://mirbase.org). The expression of CROT was normalized to the housekeeping genes GAPDH (GenBank accession no. NM_002046.3) and 18S ribosomal RNA (18S; GenBank accession no. X03205.1). Target and housekeeping gene probes were provided by Applied Biosystems (Thermo Fisher Scientific, Inc.) as ready to use assays: CROT TaqMan™ Gene Expression Assay (FAM), Assay ID: Hs00221733_m1; GAPDH Human GAPD (GAPDH) Endogenous Control (FAM™/MGB probe, non-primer limited), Assay ID: Hs99999905_m1; 18S Eukaryotic 18S rRNA Endogenous Control (FAM™/MGB probe, non-primer limited), Assay ID: Hs99999901_s1. qPCR reaction was performed using TaqMan^® Gene Expression Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) as recommended by the manufacturer: 50°C for 5 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Gene expression was assessed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.).

The prediction of downstream target gene sequences of miR-302b-3p was performed using the miRDB website (https://mirdb.org). pmirGLO reporter vectors (Promega Corporation) comprising CROT−3′UTR wild type (WT) and CROT−3′UTR mutant (MUT) were transfected by nucleofection into NHDF cells along with a miR-302b-3p inhibitor, miR-302b-3p mimics or a miRNA negative control. The Dual-Luciferase^® Reporter Assay System (Promega Corporation) was used for the luminescence intensity determination 48 h after transfection. Comparison with Renilla luciferase activity was used as a method of normalization.

The individual gene expression level was calculated by relative quantitative analysis and the Pfaffl model, including the reaction efficiency for individual genes (37). The normality of the gene expression distribution of all analyzed variables was assessed using the Shapiro-Wilk test. To compare experimental groups, one-way analysis of variance (ANOVA) was used. The significant differences between the analyzed groups were calculated using the Tukey-Kramer HSD post-hoc comparison in Statistica version 10 (StatSoft, Inc.). The results are presented as the mean ± standard deviation of three experimental repeats. P<0.05 was considered to indicate a statistically significant difference.

The MTT assay revealed that NHDFs transfected with hsa-miR-302b-3p mimics delivered by INTERFERin, Viromer Blue or nucleofection exhibited lower but not significantly reduced viability when compared with non-transfected control cells (Fig. 1A).

The cytotoxicity assay results demonstrated that, after a 24-h incubation, both of the investigated miRNA delivery agents (Viromer Blue and INTERFERin) had relatively low toxic effects on NHDFs, i.e., 7.5 and 10.7% for Viromer Blue and INTERFERin, respectively (Fig. 1B).

In NHDFs, ~77.8 and 68.3% efficiencies were achieved with nucleofection of 50 nM hsa-miR-302b-3p, after 4 and 24 h, respectively (Fig. S1A). The transfection efficiency with Viromer Blue after 48 h was ~67.8% (50 nM) and 57.9% (5 nM) (Fig. S1B). Efficiencies of ~61.5% (7 nM), 58.2% (3 nM) and 53.3% (1 nM) were achieved with INTERFERin 24 h after transfection. The percentage of transfection was maintained up to 48 h after transfection but decreased after 72 h (Fig. S1C).

To confirm that CROT is a target of hsa-miR-302b-3p, CROT 3′UTR luciferase reporter plasmids containing WT or MUT potential binding sites for miR-302b-3p were designed (Fig. 2A) and a DLR assay was performed (Fig. 2B and C). Co-transfection of NHDFs with the WT CROT 3′-UTR construct and hsa-miR-302b-3p mimic resulted in a significant decrease in cellular luciferase activity compared with cells transfected with the control mimic (P<0.05; Fig. 2B). Overexpression of miR-302b-3p did not affect the luciferase activity of MUT CROT 3′-UTR (Fig. 2B). By contrast, transfection of NHDFs with the miR-302b-3p inhibitor increased the luciferase activity of the WT CROT 3′-UTR construct (P<0.05, Fig. 2C), whereas it had no impact on MUT CROT 3′-UTR. Overall, CROT was revealed to be a target gene of miR-302b-3p.

Using RT-qPCR, the mRNA expression levels of CROT were detected following transfection with hsa-miR-302b-3p. As expected, 50 nM hsa-miR-302b-3p nucleofection caused a decrease in the expression levels of the CROT gene in NHDFs; the greatest decline (21.4-fold; P<0.01) was observed after only 4 h. Furthermore, 24 and 48 h after nucleofection, CROT transcript expression was 7.5- and 4.7-fold lower compared with the negative control (P<0.05). After 72 h, the expression of the CROT gene reached the same level as the negative control. However, transfection with 5 nM miRNA mimic had no effect on CROT (Fig. 3).

Viromer Blue-mediated transfection with 50 nM hsa-miR-302b-3p also resulted in a significant decrease in CROT gene expression, i.e., 3.8-, 3.7- and 2.2-fold at 24, 48 and 72 h, respectively. In the 5 nM group, a decrease in target gene expression was only demonstrated after 48 h (P<0.05, Fig. 4).

Transfection using INTERFERin was tested at three different concentrations of hsa-miR-302b-3p (1, 3 and 7 nM). Notably, a significant 2.7-fold decrease in CROT expression was observed only after 48 h in the 1 nM group, whereas higher concentrations of hsa-miR-302b-3p (3 and 7 nM) decreased target gene expression at all measured time points compared with in the control group (P<0.05; Fig. 5). Moreover, 4.7- and 6-fold lower CROT transcript levels were detected in the 3 nM group at 24 and 72 h post-transfection, respectively (P<0.05). Unexpectedly, a weaker effect of 3 nM hsa-miR-302b-3p on CROT expression (3-fold decrease) was observed after 48 h (P<0.05). The impact of 3 nM hsa-miR-302b-3p on CROT expression was strongest (6-fold decrease) after 72 h (P<0.05).