Human hepatocellular carcinoma (HCC) cell lines HLE (RRID:CVCL_1281) and HLF (RRID:CVCL_2947), intrahepatic cholangiocarcinoma (CC) cell line HuH-28 (RRID:CVCL-2955) and extrahepatic CC cell line TFK-1 (RRID:CVCL_2214) were a kind gift provided by Stephanie Rössler (Institute of Pathology, Heidelberg University). HLE and HLF were maintained in DMEM (D6046, Sigma-Aldrich, St. Louis, MO, United States), HuH-28 and TFK-1 were cultured in RPMI 1640 (21875034, Life Technologies of Thermo Fisher Scientific Inc., Paisley, UK), supplemented with 10% fetal bovine serum (P40–37500, Pan-Biotech, Aidenbach, Germany), 100 U penicillin/0.1 mg streptomycin (P0781, Sigma-Aldrich, St. Louis, MO). RPMI 1640 was further supplemented with 2 mM L-Glutamine (G7513, Sigma-Aldrich, St. Louis, MO). Culturing was maintained at 37 °C in a humidified atmosphere containing 5% CO2.

Sodium selenite and MSC were supplied by Sigma-Aldrich (214485 and M6680, Sigma-Aldrich, St. Louis, MO). Both drugs were dissolved in double distilled water (50 mM for sodium selenite and 250 mM for MSC), then, aliquoted and kept at −20 °C.

For cell viability measurement, 5000 cells of HLE, HLF, HuH-28 and 8000 cells of TFK-1 were seeded in 96-well plates one day ahead of treatment. Regarding MSC, the applied concentrations ranged from 7.5 to 480 μM with doubling paces, except for TFK-1, where the final concentration was 1920 μM. The treatment concentrations for sodium selenite ranged from 0.625 μM to 40 μM with doubling paces for each cell line. The final treatment volume was 150 μl for HLE, HLF, HuH-28, and 200 μl for TFK-1.

Concerning the measurement of miRNA expression, 240,000 cells of HLE, HLF, HuH-28 and 400,000 cells of TFK-1 were seeded in 6-well plates in advance. Next day, HLE, HLF and HuH-28 cells were treated with 15, 30, 60 and 120 μM of MSC, whereas 240, 480 and 960 μM concentrations were further applied for TFK-1. Regarding sodium selenite, treatment concentrations of 2.5, 5, 10 and 40 μM were applied, except for HuH-28, which received 1.25, 2.5, 5 and 10 μM of the drug. The final treatment volume was 3 ml for each cell line.

Each treatment lasted for 72 h. The cell viability experiments were repeated 3 times and three biological replicates were applied in measuring miRNA expression.

The inhibitory effect of MSC and sodium selenite on cell proliferation was measured by sulforhodamine B (SRB) assay. At 72 h following treatment, cell culture media was withdrawn and the cells were washed with 1xPBS. For fixation, the cells were treated with 70 μl of 10% trichloroacetic acid (TCA) for 1 h at 4 °C, rinsed five times with very gently running tap water and air-dried. Then, the cells were stained with 0.4% SRB (S1402, Sigma-Aldrich, St. Louis, MO), 1% acetic acid solution for 20 min at RT. Following the withdrawal of the stain, the cells were washed five times with 1% acetic acid and air-dried. Finally, the stain attached to cellular proteins of TCA-fixed cells was dissolved in 200 μl of 10 mM Tris-HCl (pH 8). The plates were stirred for 20–30 min and the color development was measured at 570 nm using an EL-800 microplate reader (BioTek Instruments, Winooski, VT). For each treatment, the data were normalized to the absorbance value of untreated cells.

At 72 h following treatment, the 6-well plates were placed on ice. Following removal of treatment culture media, the cells were washed with 1xPBS and RNA was isolated with TRIzol (Life Technologies of Thermo Fisher Scientific Inc., Carlsbad, CA) according to the instructions of the manufacturer. Briefly, the cells were lysed in 360 μl of TRIzol, collected in an Eppendorf tube and incubated for five min at RT. Following the addition of 72 μl of chloroform, the tubes were gently shaken by inversion for 15 s and incubated for three min at RT. The aqueous phase was separated by centrifugation at 12,000 x g for 15 min at 4 °C and removed into a new Eppendorf tube. When 180 μl of isopropanol had been added, the mixture was incubated for 10 min at 4 °C and centrifuged at 12,000 x g for 10 min at 4 °C. After the withdrawal of the fluid, the pellet was washed with 360 μl of 75% ethanol, vortexed briefly and centrifuged at 7500 x g for 5 min at 4 °C. Then all fluid was removed and the pellet was air-dried for 10 min. Finally, the pellet was dissolved in nuclease-free double distilled water. RNA concentration was quantified using an ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). RNA samples were kept at −80 °C until further use.

We selected miRNAs that are abundantly expressed in normal liver (miR-21, -22, -24, -122, -125b, -143, -194, -199a, let-7a) according to Table 1 in Chen et al. [27]. Additionally, two further miRNAs related to cancer were selected, miR-210, involved in surviving hypoxia [28] and miR-224, promoting proliferation by AKT activation [29] as controls with hypothesized altered miRNA expression upon treatment. The expression of individual miRNAs was determined using the following TaqMan MicroRNA Assays (Life Technologies of Thermo Fisher Scientific Inc., Foster City, CA): miR-21-5p (ID: 000397), miR-22-3p (ID:000398), miR-24-3p (ID:000402), miR-miR-122-5p (ID:002245), miR-125b-5p (ID:000449), miR-143-3p (ID:002249), miR-194-5p (ID:000493), miR-199a-5p (ID:000498), miR-210-3p (ID:000512), miR-224-5p (ID:000599), let-7a-5p (ID:000377) and RNU48 (001006). Reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) were performed according to the instructions of the manufacturer. Briefly, RT reaction was carried out using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies of Thermo Fisher Scientific Inc.) in a final volume of 7.5 μL containing 10 ng total RNA. The qPCR was performed using TaqMan Universal Master Mix II, no UNG (Life Technologies of Thermo Fisher Scientific Inc.) in a final volume of 10 μL containing 0.65 μL RT product. The amplification reaction was run in triplicates on a LightCycler 480 Instrument II (Roche Diagnostics, Indianapolis, IN). Relative expression was calculated by the 2-ΔΔCq formula, applying RNU48 as the reference and normalized to the average ΔCq value of untreated cells. Fold change higher than 1.5 and lower than −1.5 (0.6) was regarded as an altered miRNA expression.

Results are expressed as mean ± SD. The analysis was performed by Student t-test or one-way ANOVA with 95% confidential interval followed by Tukey’s multiple comparison test (significant differences are indicated as *p < 0.05, **p < 0.01 & ***p < 0.001) compared with control and within the treatments. Statistical differences between IC50 values were determined by fitting nonlinear regression slopes on independent experiments (n ≥ 3). Data were analyzed with GraphPad Prism software, version 8.3.3 (538) (GraphPad Software Inc., San Diego, CA).