Mir 204 5p

Taqman RT and stem-loop real-time assay were from Applied Biosystems: miR-31 (assayID: 002279), miR-130a (assayID: 000454), miR-204-5p (assayID: 000508) and miR-1236 (assayID: 002761). Briefly, 100 ng RNAs were reverse transcribed by specific stem-loop primer and further analyzed by Taqman real-time PCR assay using default setting. U6 snRNA (assayID: 001973) was used as an internal loading control. Data were analyzed by Applied Biosystems 7500 software V2.0.1. MiR-204 hairpin precursor can generate both miR-204-5p (miR-204) and its complementary miR-204-3p (miR-204*) from the other arm. We focused on miR-204-5p here because the LNA experiment (Fig. 2D–F) confirmed the anti-HBV potential from miR-204-5p. In this report, we used the old name miR-204 and the new name miR-204-5p interchangeably.

Antibodies against GAPDH, Bcl-2 and VEGF were obtained from Santa Cruz Biotechnology. Vemurafenib and trametinib were obtained from Selleck Chemicals. TaqMan probes for GAPDH, VEGFA, TIMP2, PTPN14, HIF-1α, Bcl-2, CCL3, IL1b, CCL5, tgfB, CXCL8, CSF3, PDGFb, CCL20, miR-4443, miR-4488, miR-204-5p, miR-199b-5p, miR-630, miR-1234, ts-3676, miR-145-5p and RNU48 were purchased from Applied Biosystems. Primers relative to H3, RAD51, Notch1 and FOXM1 have been used in the work by Canu et al [18 (link)]. Western Blot analysis were performed as previously described [11 (link), 16 (link)].

Nine miRNA were chosen for RT-qPCR validation; miR-29a-3p (TaqMan® ID 007600_mat, Applied Biosystems), miR-99a-5p (TaqMan® ID 006254_mat), miR-126-3p (TaqMan® ID 008451_mat), miR-140-3p (TaqMan® ID 471823_mat), miR-222-3p (TaqMan® ID 000525), miR-223-3p (TaqMan® ID 002295), miR-204-5p (TaqMan® ID 000508), miR-409-3p (TaqMan® ID 002332), miR-6119-5p (Custom TaqMan® small RNA Assay). Reverse transcription was achieved on 10 ng of total RNA using the TaqMan® MicroRNA Reverse Transcription (Applied Biosystems, Foster City, CA, USA) kit following the manufacturer’s instructions. In the thermal cycler (StepOne+, Applied Biosystems, Foster City, CA, USA), each 15 μL RT reaction followed 30 min at 16°C, 30 min at 42°C, 5 min at 85°C. Then, 1.3 μL of miRNA-specific cDNA from the reaction were amplified using the TaqMan® Small RNA Assays (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Amplification was performed at 95°C for 10 min, pursued by 40 cycles of 95°C for 15 s and 60°C for 1 min. All miRNA levels were normalized to the values of U6 snoRNA [47 (link),48 (link)].