Dual luciferase activity detection kit

An online website (https://cm.jefferson.edu/rna22/Precomputed/) was in application to prediction of the possible binding sites of miR‐497 and LRG1. The 3’ untranslated region (UTR) fragment of LRG1 was cloned into a pmirGLO plasmid (Promega, Wisconsin, USA) to construct a pmirGLO‐LRG1‐wild‐type (WT) plasmid. A site‐directed mutation kit (Takara, Tokyo, Japan) was indicated to mutate the miR‐497 binding site on the LRG1 3’UTR to construct a pmirGLO‐LRG1‐mutant (MUT) plasmid. The plasmids and miRNA were transfected into rat osteoblasts with reference to the instructions of Lipofectamine 2000. The pmirGLO‐LRG1‐WT plasmid or pmirGLO‐LRG1‐MUT and miR‐497 mimic or mimic NC were cotransfected into cells. Cells were collected 48 h later, and the dual‐luciferase activity detection kit (Promega) was implied to detect luciferase activity.

The bioinformatics online database TargetScan was used to forecast the targeted gene of miR-141-3p. The results showed that there was a targeted binding site between the 3′-UTR of BRD4 and miR-141-3p, indicating that BRD4 is probably the targeting gene of miR-141-3p. The luciferase recombinant vectors containing wild-type BRD4 3′-UTR (BRD4-Wt) and mutant BRD4 3′-UTR (BRD4-Mut) were amplified and constructed, respectively. The BRD4-Wt or BRD4-Mut recombinant vectors were cotransfected with miR-141-3p mimic and miR-con into TPC-1 cells, respectively. After 48 hours, the luciferase activity was measured using the dual-luciferase activity detection kit (from Promega) to calculate the relative fluorescence activity of cells.

Myeloma cell line RPMI8226, Guangzhou Jennio Biotech Co., Ltd.; fetal bovine serum (FBS), American HyClone Company; RPMI 1640 medium, Annexin V-FITC/PI Apoptosis Kit, and Bicaquinoline Acid (BCA) Protein Detection Kit, Beijing Solarbio Science and Technology Co., Ltd.; Lipofectamine™ 2000 kit, American Invitrogen Company; RNA drawer kit, reverse transcription kit, and PCR kit, Dalian Takara Company; PCR primers, circ_0058063 small interfering RNA (si-circ_0058063), out-of-order nonsense negative sequence (si-NC), miR-635 mimics (mimics), mock control sequence (miR-NC), circ_0058063 wild-type plasmid (WT-circ_0058063), mutant plasmid (MUT-circ_0058063), miR-635 inhibitor (anti-miR-635), and inhibitor negative sequence (anti-miR-NC), Sangon Biotech Shanghai Co., Ltd.; rabbit anti-human proliferating nuclear antigen Ki-67, B lymphocyte tumor-2 (Bcl-2), B lymphocyte tumor-2-related protein (Bax), MMP-9, and glyceraldehyde-phosphate dehydrogenase (GAPDH) polyclonal antibody, Santa Cruz Biotechnology, Inc.; dual luciferase activity detection kit, Promega Corporation.

The bioinformatics web software miRDB was employed to predict target genes and verify if miR-199a-3p and mTOR have binding sites. The wild-type mTOR dual-luciferase reporter vector (WT mTOR) and mutant mTOR dual-luciferase reporter vector (MUT mTOR) was constructed respectively, and then co-transfected into C28/I2 cells with miR-199a-3p mimic and the negative control. A miRNA control was employed as a negative control. To determine whether miR-199a-3p contributes to the effect of Exos^SC on OA model, C28/I2 cells were pretreated with Exos^SC for 24 h, then were transfected with the antagomir-199a-3p and the reporter plasmids. The activity of luciferase in C28/I2 cells was measured 48 h after transfection, and reporter tests were carried out according to the manufacturer’s instructions of a dual luciferase activity detection kit (Promega, E1910, USA). The activity of renilla luciferase was normalised to that of firefly luciferase and represrnted as % of the control.

The binding sites of ROR and ROCK1 with miR-145-5p were predicted using STARBASE, an online bioinformatics software. The wild-type (WT) and mutated (MUT) ROR or Rho-associated kinases 1 (ROCK1)-dual luciferase reporter vectors were constructed, and co-transfected into MC3T3-E1 cells with miR-145-5p mimic or inhibitor, respectively. After incubation for 48 hours, the luciferase activity was tested by dual luciferase activity detection kit (Promega, USA).