Glomax 20 20 machine

Dual luciferase reporter assays were performed to verify the direct interactions between KCNQ1OT1 and miR-34a and miR-34a and the 3′-UTR of Atg4B mRNA. PCR was conducted using the PrimeSTAR DNA polymerase (Takara, Kusatsu, Japan) to amplify the KCNQ1OT1 cDNA containing the predicted miR-34a binding site and the 3′-UTR of Atg4B cDNA containing the predicted miR-34a binding site. The primers used are presented in Table S2. Then, the PCR products were purified and cloned to the pmirGLO vector. Afterward, we co-transfected the pmirGLO-WT-KCN1 or -Mut-KCN1 with miR-34a mimics or miRNA-NC into colon cancer cells with Lipofectamine 3000. Also, the pmirGLO-WT-Atg4B or -Mut-Atg4B was transfected in a similar way. The luciferase activity was conducted after 48 h of transfection using the luciferase assay kit (Promega Corporation, Fitchburg, WI, USA) and the Promega GloMax 20/20 machine.

RNA22 (http://www.mybiosoftware.com/rna22-v2-microrna-target-detection.html) was used to predict the targeting miRNA of ZNF667-AS1. TargetScan (http://www.targetscan.org/vert_72/) was applied to predict the targeting mRNA of miR-206. Wild-type (WT) or mutated-type (MT) sequences of ZNF667-AS1 or AKAP13 containing predicted miR-206 binding position were separately cloned into pMIRREPORT luciferase reporter vectors (Ambion, Austin, TX, USA). The transfection reagent lipofectamine™ 2000 was used to co-transfect those vectors with miR-206 mimic or NC, respectively, into HEK-293T. Luciferase activity was detected using luciferase assay kits (Promega, Madison, WI, USA) and GloMax 20/20 machine (Promega) at 48 h post-transfection. Renilla luciferase activity was used for normalization.

Dual luciferase reporter assays were performed to verify the binding between lncRNA DLX6-AS1, miR-26a and the 3ʹ-untranslated region (UTR) of TRPC3. In these experiments, pmirGLO-WT-DLX or pmirGLO-Mut-DLX and miR-26a mimics or miR-NC were co-transfected into laryngeal cancer cells. The pmirGLO-WT-TRPC3 or pmirGLO-Mut-TRPC3 were co-transfected with miR-26a mimics or miR-NC in a similar way. The pRL-TK plasmid was also co-transfected as the internal control. After 48 hrs, cells were lysed, and luciferase measurements were performed using a luciferase assay kit (Promega, WI, USA) and a Promega GloMax 20/20 machine.