High performance liquid chromatography (hplc)

HSF were grown in 6-well plates and the synthesized RNAs (100 mM of diluted siRNA, miR-101 mimics or miR-101 inhibitor per well) were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 or 48 h transfection, the cells were harvested for analysis. Carboxy Fluorescein (FAM) modified 2′-OMe-oligonucleotides were chemically synthesized and purified using high-performance liquid chromatography (GenePharma, Shanghai, China). All transfections were performed in triplicate. The primer sequences were as follows: siRNA-EZH2: 5′-UAGCUUAUCAGACUGAUGUUGA-3′; siRNA-control: 5′-UAGCUUAUCAGACUGAUGUUGA-3′; miR-101 mimics: 5′-CUACAGUACUGUGAUAACUGAA-3′, mimics control: 5′-UAGCUUAUCAGACUGAUGUUGA-3′, miR-101 inhibitor: 5′-GAUGUCAUGACACUAUUGACUU-3′, miR-101 inhibitor control: 5′-CAGUACUUUUGUGUAGUACAA-3′.

MALAT1 siRNAs were specifically designed and synthesized by Shanghai GenePharma Co, Ltd. (Shanghai, China) by targeting MALAT1 sequences (MALAT1-1, 5’-CACAGGGAAAGCGAGTGGTTGGTAA-3’ and 5’-TTACCAACCACTCGCTTTCCCTGTG-3’; and MALAT1-2, 5’-GAGGUGUAAAGGGAUUUAUTT-3’ and 5’-AUAAAUCCCUUUACACCUCTT-3’). A negative control siRNA was also synthesized with targeting sequences of 5’-GGCCUAAAGUAGUAGCUAUTT-3’ and 5’-AUAGCUACUACUUUAGGCCTT-3’.
To assess the effects of miR-320a on regulation of HUVEC proliferation, we had a miR-320a mimic, a miR-320a inhibitor, and control RNA chemically synthesized and purified using high-performance liquid chromatography (GenePharma). The concentration of these synthesized siRNAs (mimic, inhibitor, and negative control) were all 20 μM and the working concentration was 20 nM diluted at 1:1000. Plasmids containing MALAT1 siRNAs were transfected into cells for 48 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and then the cells were used for our experiments.