Pgl3 basic plasmid

The OIP5-AS1 wild-type (WT) and mutant (mut) untranslated region (3’-UTR) containing the miR-92a-3p binding site were constructed and subcloned into the pGL3 basic plasmid (Addgene, Inc.). Then, 293 T cells were grown in 24-well plates and incubated at 37°C. Subsequently, the miR-92a-3p mimic and miR-NC were co-transfected into 293 T cells with pGL3-WT or mut-OIP5-AS1 for 48 h. The cells were lysed, and the firefly luciferase activity was measured and normalized to Renilla luciferase activity with the adoption of a Dual-Luciferase Reporter assay system (Promega Corporation) as per the operating protocols.

MiR-129-5p or miR-129-5p-mut mimics were purchased from RiboBio Co., Ltd. (Guangzhou, China). DNA fragments from the 3′-untranslated region (UTR) of OTX1 containing the predicted complementary sites of miR-129-5p were constructed into a pGL3-basic plasmid (Addgene, Cambridge, USA). LSCC cells (1× 10⁴) were seeded in 48-well plates in triplicate and settled for 24 h. Next, the pGL3-OTX1–3′-UTR reporter plasmids (100 ng) plus 5 ng of pRL-TK Renilla plasmid (Promega, Madison, USA), and increasing amounts (10 and 50 nM) of negative control (NC) or miR-129-5p or miR-129-5p-mut mimic were co-transfected into LSCC cells using Lipofectamine LTX reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s recommendation. Luciferase and Renilla signals were measured 24 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, USA) according to a protocol provided by the manufacturer. The sequences of miR-129-5p and miR-129-5p-mut were 5′-cuuuuugcggucugggcuugc-3′ and 5′-cuuuuugcggucugguugugc-3′, respectively.