Pgl3 luciferase reporter vector

The 3’UTR of LncRNA-RP11-197K6.1 and its mutant containing the miR-135a-5p binding site were cloned into the pGL3 luciferase reporter vector (Invitrogen, Carlsbad, CA, USA). HCT116 cells (5 × 10^3) were seeded in 96-well plates and co-transfected with miR-135a-5p mimic and either wild-type p-GL3-LncRNA-RP11-197K6.1 (LncRNA-RP11-197K6.1-WT) or mutant p-GL3-LncRNA-RP11-197K6.1 (LncRNA-RP11-197K6.1-Mut) vectors. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized to Renilla luciferase activity driven by a constitutive promoter (phRL vector).

pRC/CMV₂ over-expression vector and pGL3 luciferase reporter vector were purchased from Invitrogen (Carlsbad, CA) and Promega (Madison, WI), respectively. Coding sequence (CDS) fragments of ATG9A were PCR-amplified from rat cDNA with two linkers (i.e., Hind III and Xba I) and then inserted into pRC/CMV₂ using T4 DNA ligase. Having been confirmed by DNA-sequencing, this plasmid was named as pRC/CMV₂-ATG9A and used in vitro to produce expression of ATG9A protein. Furthermore, rat genomic DNA containing the 3′-untranslated region (3′-UTR) of ATG9A was extracted from rat blood using an E.Z.N.A™ Blood DNA Kit (Omega, Norcross, USA) according to the manufacturer's instructions. After which the ATG9A 3′-UTR and a mutation sequence were amplified from rat genomic DNA using a fusion PCR and inserted into the ECOR I and Xba I sites of a pGL3 luciferase reporter vector. Following confirmation by DNA-sequence, these plasmids were named as pGL3-ATG9A 3′-UTR-Wild Type and pGL3-ATG9A 3′-UTR-Mutant. The pRL-TK (Promega) was used as an endogenous control.

LOXL1‐AS1 wild type (WT), LOXL1‐AS1 mutants (LOXL1‐AS1 MUT‐1, LOXL1‐AS1 MUT‐2 and LOXL1‐AS1 MUT1/2), MYBL2 WT and MYBL2 mutation (MUT) were subcloned into the pmirGLO dual‐luciferase vector (Promega). A549 or SPC‐A1 cells were co‐transfected with LOXL1‐AS1 WT/MUT vectors or MYBL2 WT/MUT and miR‐423‐5p mimics or miR‐423‐5p inhibitor, as well as respective controls (NC mimics or NC inhibitor). Besides, WT or mutation of LOXL1‐AS1 promoter was subcloned into the pGL3 luciferase reporter vector (Invitrogen) to establish promoter WT vector or promoter MUT vector. The two vectors were respectively co‐transfected into A549 or SPC‐A1 cells with sh‐MYBL2#1, sh‐MYBL2#2, or sh‐NC.