Pgl3 promoter vector

The 3′-UTR sequence or the mutant sequence of LIMD1 was cloned into pGL3 promoter vector (Genscript, Nanjing, China). A549 cells were cultured in 24-well plates transfected miR-550a-5p mimics or negative control (NC). Then, cells were transfected with pGL3-LIMD1 3′-UTR or pGL3-LIMD1-MUT by Lipofectamine 3000 reagent (Invitrogen). Twenty-four hours later, the cells were collected and Renilla luciferase activity was considered as a normalization using Dual-Luciferase Assay Kit (Promega).

The 3′‐UTR sequences of ARP2 and ARP3, containing the putative target sites for miR‐24‐1* and let‐7a*, respectively, were inserted into the KpnI and SacI sites of pGL3 promoter vector (Genscript, Nanjing, China). The resulting constructs were named pGL3‐ARP2, pGL3‐ARP2‐mut, pGL3‐ARP3 and pGL3‐ARP3‐mut. Cells were plated in 24‐well plates and cotransfected with 100 ng of pGL3‐ARP2, pGL3‐ARP2‐mut and 50 nM miR‐24‐1* mimics and negative control using Lipofectamine 2000 (Invitrogen). For pGL3‐ARP3, pGL3‐ARP3‐mut and let‐7a* mimics, we performed the same procedure towards pGL3‐ARP2, pGL3‐ARP2‐mut and miR‐24‐1*. A renilla luciferase vector pRL‐SV40 (5 ng) was also cotransfected to normalize the difference in transfection efficiency. Luciferase activity levels after 48 hrs incubation were measured using a Dual‐Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to manufacturer's instructions. Transfection was repeated in triplicate.

In the Dual-luciferase reporter assay, the wild-type and mutated 3’-UTR sequence of SRGAP1 mRNA were inserted into the KpnI and SacI sites of pGL3 promoter vector (Genscript, Nanjing, China). We transfected with pRL-SV40, negative control, miR-770-5p mimics, pGL3-SRGAP1 and pGL3-SRGAP1-mut by using Lipofectamine 2000 (Invitrogen Corp, CA, USA). After collection, cells were assessed by using the Dual Luciferase Assay (Promega, Madison, WI) after 48 h transfection following the manufacturer’s instructions. All experiments were repeated three times independently.

Then, AMER-1-wild and -mut were inserted into the pGL3 promoter vector (GenScript Co., Ltd., Nanjing, China), which was transfected into 7901 and MKN-45 cells using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.), along with IRF-2 overexpression vectors or NC vectors. The cells were seeded in 24-well plates. After 48 h, firefly luciferase signals and Renilla luciferase (internal reference) were detected using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA.) according to the manufacturer’s protocol. All experiments were performed in triplicate.