Renilla luminescence

The concentration of RNAs containing 5′ untranslated regions was determined using A₂₆₀, which was normalized by the intensity of the full-length product on an agarose gel, and then by the molar ratio of the construct to empty pA60. Molar-adjusted amounts of each RNA relative to 100–200 ng of pA60 were transfected into three technical triplicate wells of ~50% confluent cells in a 96-well plate using the TransIT-mRNA reagent (Mirus, Madison, WI). The concentration of RNAs containing 3′ UTRs was determined using a Qubit RNA HS assay (Life Technologies), normalized for the molar ratio of the construct to empty pA60, and molar-adjusted amounts of RNA relative to 7 ng of pA60 were transfected using TransIT-mRNA. A reduced amount of the 3′ UTR RNAs was used due to decreased yield of the longest 3′ UTRs. HEK 293T cells were grown in DMEM + 10% FBS, Hep G2 cells were grown in EMEM + 10% FBS, MCF7 cells were grown in DMEM:F12 + 10% FBS, A549 cells were grown in F12-K media + 10% FBS, and K-562 cells were grown in RPMI media + 10% FBS. Cells were harvested after ~18 hr (Figure 5) or 2 hr (Figure 6) and Renilla luminescence was measured (Promega).

The WT and MUT Socs1 reporter were obtained from Synthgene (Nanjing, China). These plasmids were cotransfected with miR-374b-5p mimic, inhibitor, or control into HEK293T cells (10⁶ cells per well in six-well plate) using Lipofectamine 2000 (Synthgene, China) according to the manufacturer’s protocol. After 48 h, the luciferase activity was detected and normalized by Renilla luminescence using the kit (Promega, United States) followed with the manufacturer’s protocol.