Dual luciferase reporter vector

The wild‐type and mutant‐type circPTPRA and LMNB1 fragments were constructed and cloned into the dual‐luciferase reporter vector by GenePharma. Dual‐luciferase reporter plasmids (WT or Mut‐circPTPRA/LMNB1) were cotransfected into PDAC cells with miR‐140‐5p mimics or miR‐140‐5p mimics NC, respectively. After 48 h, Firefly and Renilla luciferase activities were assayed according to the Dual‐Glo^® Luciferase Assay System (Promega).

The theoretical binding sequence of miR-346(or miR-425-5p)in MIR17HG gene and its mutant sequence were amplified by PCR, synthesized and cloned into a pmirGlo Dual-luciferase vectors (Promega, Madison, WI, USA) to construct dual luciferase reporter vector (GenePharma). HEK293T cells were seeded in 96-well plates and cotransfected with wildtype pmirGLO-MIR17HG (or MIR17HG mutant) reporter plasmid and pre-miR-346 (or pre-miR-425-5p) or pre-NC. Through the Dual-Luciferase Reporter System (Promega), luciferase activity was measured 48 h after transfection. The 3’-UTR sequence of TAL1 containing the putative miR-346(or miR-425-5p)binding sites and their mutant sequences were cloned into Dual-luciferase vectors. The transfection procedure and Luciferase activities measurement were performed similarly as described above.
The MIR17HG and DEC1 promoter regions were amplified from human genomic DNA by PCR. In addition, putative TAL1 binding sites in the PCR conducts were deleted one by one. The PCR products were subcloned into the pGL3-Basic vector (Promega). Human full-length TAL1 gene was constructed in pEX3 vector (GenePharma). Firefly luciferase activity was normalized to renilla luciferase activity for each individual analysis.