Site directed mutagenesis kit

A computer-based program Jefferson (https://cm.jefferson.edu/rna22/Precomputed/) was applied to measure the target binding sites of lncRNAGAS5, which suggested that miR-449b could specifically bind to GAS5. Besides, prediction on TargetScan (http://www.targetscan.org/vert_72/) suggested that miR-449b could directly bind to the 3′-untranslated region of HMGB1. The wildtype GAS5 and HMGB1 3′UTR were amplified and cloned to the pmiR-GLO reporter vector (Promega, U.S.A.), respectively. The mutants of GAS5 (5′-ACUGCUU-3′→5′-UGACGUA-3′) and HMGB1 3′UTR (5′-ACUGCCA-3′→5′-UGACGGA-3′) were constructed using the site-directed mutagenesis kit (YEASEN, Shanghai, China) according to the instructions. Well-constructed vectors were co-transfected with either mimic-NC or mimic-miR-449b into HEK293T cells (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academic of Science, Shanghai, China). The relative luciferase activity was detected 48 h later. The experiment was performed in triplicate. The mimic-miR-449b and mimic-NC were synthesized by GenePharma Co, Ltd.

The targeted miRNAs of NEAT1 were predicted using starBase v.3 software at http://starbase.sysu.edu.cn/. The partial sequences of NEAT1 harboring the complementary sequence for miR-206 or miR-599 were cloned into the dual luciferase psiCHECK2 vector (Promega, Southampton, UK) to construct NEAT1 wild-type reporter (WT-NEAT1 or WT-NEAT1′). Site-directed mutants (MUT-NEAT1 or MUT-NEAT1′) of the target region were generated using the Site-Directed Mutagenesis kit (Yeasen, Shanghai, China). Each reporter construct was cotransfected into SW1736 and KAT-18 cells and miR-206 mimic, miR-599 mimic, or miR-NC mimic. Luciferase activity was detected after 48 h transfection using the Dual-luciferase Reporter Assay System (Promega).

The binding sites of circRBM33 and miR-542-3p and that of miR-542-3p and HIF-1α were predicted online using http://starbase.sysu.edu.cn/. Wild-types circRBM33 (WT-CircRBM33) and HIF-1α (WT-HIF-1α) reporters were constructed by cloning the sequence of circRBM33 or HIF-1α containing miR-542-3p complementary sequence into the pmirGLO vector (Promega, WI, USA). The site-directed mutant reporters MUT-CircRBM33 and MUT-HIF-1α were produced by site-directed mutagenesis kit (Yeasen, Shanghai, China). The reporter, along with miR-542-3p mimic or miR-NC, were co-transfected into cells to detect luciferase activity on the dual luciferase reporter gene detection system (Promega).

The PCT (NM_001741) promoter fragments nt −700 to +299, −202 to +299, and −700 to −202 were amplified by PCR using human whole-blood genomic DNA as a template and inserted into the KpnI and HindIII sites of the luciferase reporter plasmid pGL3-basic vector (Promega), yielding the reporter constructs PCT (−700/+299), PCT (−202/+299), and PCT (−700/−202), respectively. The primer sequences are listed in Table 1. In the NF-κB point mutants, the potential NF-κB binding site (nt-53 to −44) in the PCT (−202/+299) reporter was replaced with a non-functional sequence using a Site-Directed Mutagenesis Kit (Yeasen, Shanghai, China), yielding the reporter construct PCT (−202/+299) Mut. All constructs were confirmed by DNA sequencing.
The 3′-UTR of the PCT gene containing the miR-513b binding site predicted by TargetScan (http://www.targetscan.org/) was inserted into the XhoI and NotI sites of the psiCHECK-2 dual-luciferase reporter vector and designated PCT-3′-UTR (454 bp). The plasmid PCT-3′-UTR Mut containing the mutated binding site of miRNA-513b in the 3′-UTR generated by PCR-based site-directed mutagenesis was also constructed. All constructs generated were verified by DNA sequencing.

To make vectors expressing full-length or different fragments of HIF1α with Flag, myc, GFP, or glutathione S-transferase (GST) tags, PCR amplification was performed using pfu polymerase (Yeasen) and the amplicons were inserted into p3xflag-CMV-13, pCDNA3-myc, pEGFP-C1, or pEBG. The constructs harboring EGFP-tagged HOIP and mCherry-tagged HIF1α were generated by cloning the DNA fragments encoding HOIP-EGFP and HIF1α-mCherry into p3xflag-CMV-13. Lamin B1 cDNA was subcloned into pTagBFP-C1 to construct plasmid expressing Lamin B1-BFP. The lentivirus expressing HOIP was engineered by cloning HOIP cDNA into pLvx-t2a-mCherry. The constructs harboring HA-tagged Otulin, CYLD or A20 were obtained by subcloning the PCR products into pCDNA3.1. The plasmid expressing GST-specific tandem Ub-binding entity (TUBE) was constructed by subcloning the cDNA encoding UBAN domain of IκB kinase (IKK) γ into pGEX-4T-1. Site-directed mutagenesis of HIF1α mutants was conducted to generate different point mutants using site-directed mutagenesis kit (Yeasen) as described previously [31 (link), 32 (link)]. Other plasmids were described in the previous publications [6 (link), 31 (link)–39 (link)]. All constructs were verified by DNA sequencing.