Quikchange site directed mutagenesis kit

miRanda software was used to predict target genes of cli-miR-205b by searching the 3’ UTR sequences of genes identified from RNA-seq. Combining the results of target genes of differentially expressed microRNAs and DEGs from RNA-seq, HSD11B1 was selected as a putative target gene of cli-miR-205b. Expression levels of cli-miR-205b and HSD11B1 in ovaries were then examined with RT-qPCR.
The 3’ UTR regions of HSD11B1 fragments were amplified from genomic DNA and the pmirGLO vector (Promega, Madison, WI, USA) with NheI and XhoI restriction enzyme sites at the 3’ UTR region of the luciferase gene to construct the luciferase reporter plasmid pmirGLO-Wt/pmirGLO-Mut. Primer sequences used to amplify 3’ UTR regions are listed in Additional file 1. Mutagenesis of the miR-205b target site in the HSD11B1 3’ UTR was carried out using a QuikChange site-directed mutagenesis kit (Promega) with pmirGLO-Wt as the template, resulting in the mutant reporter plasmid pmirGLO-Mut. For the luciferase reporter assay, 293 T cells were seeded in 24 well plates and transfected with 50 nM mimics of miR-205b or scrambled microRNA (mimics-NC) and 1 μg luciferase reporter plasmid pmirGLO-Wt/pmirGLO-Mut using an X-tremegene HP (Roche, Basel, Switzerland). At 48 h after transfection, cells were harvested, and luciferase activity was measured using the dual-luciferase reporter assay (Promega) [16 (link)].

To construct a luciferase reporter vector, 3’-untranslated region (3’UTR) of Sirt4 was synthesized by PCR with the involvement of restriction enzyme cutting site. For sequence point mutation, site-directed mutagenesis of potential target site in the Sirt4 3’UTR was performed using a QuikChange Site-Directed Mutagenesis kit (Promega, Madison, WI, USA). The wild-type and mutant 3’UTR of Sirt4 were cloned into downstream of the luciferase open reading frame in the pMIR-report vector (Ambion, Carlsbad, CA, USA).

MiR-195 mimics and negative control (NC) were purchased from GenePharma (Shanghai, China). The specific sequences are as follows: miR-195 sense: 5′-UAGCAGCACAGAAAUAUUGGC-3′; antisense: 5′-CAAUAUUUCUGUGCUGCUAUU-3′; NC sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′. pcDNA 3.1-CCND1 and pcDNA 3.1-FGF2 were purchased from GeneRay (Shanghai, China). The 3′-untranslated regions (3′-UTRs) of CCND1 and FGF2 were cloned into the pGL3-basic vector (Promega, USA) with XbaI and PciI at the downstream of the luciferase gene. To mutate the binding sequence of miR-195 in the 3′-UTRs, a QuikChange, Site-Directed, Mutagenesis kit (Promega, USA) was used following the instruction. The mature sequence of miR-195 was amplified and cloned into the lentiviral vector LV3-GFP-puro (GenePharma, Shanghai, China) to generate LV3-miR-195 cell, and negative control LV3-NC was conducted as the same way.

The full length mRNA sequence of TUSC3 (3888 bp, NCBI Reference Sequence: NM_006765.3) was synthesised and then inserted into the HindΙΙΙ and MluΙ restriction sites downstream of the luciferase open reading frame of the pMIR-REPORT luciferase vector (Ambion, Carlsbad, CA, USA). For sequence point mutation, site-directed mutagenesis of the potential target site in the TUSC3 mRNA was performed using the QuikChange Site-Directed Mutagenesis kit (Promega, Madison, WI, USA). The correct clones were confirmed by sequencing. HEK 293 and GBM cells were seeded into a 24-well plate (5 × 10⁴ per well) and co-transfected with miR-UL112-3p mimics/inhibitor (50 nM) and the wild-type/mutant pMIR-REPORT luciferase vector (200 ng) using Lipofectamine^TM 2000 reagent according to the manufacturer’s instructions. Luciferase activity was detected 48 h after co-transfection using the dual luciferase system kit (Promega, Madison, WI, USA). The relative luciferase activity was calculated by normalising firefly luminescence to that of renilla.

The 3′UTR region of Cthrc1 was amplified from human genomic DNA and inserted into the pmirGLO vector (Promega, Madison, WI, USA) with XhoI and SalI at the 3′ end of the luciferase gene to construct the luciferase reporter plasmids pmirGLO-Cthrc1-Wt and pmirGLO-Cthrc1-Wt. Site-directed mutagenesis of the miR-30b target site in the Cthrc1 3′UTR was performed using a QuikChange Site-Directed Mutagenesis kit (Promega) and pmirGLO- Cthrc1-Wt as the template. For the luciferase reporter assay, A549 and Calu-3 cells were seeded into 96-well plates and transfected with 50 nM of miR-30b mimic or Scrambled and 200 ng of luciferase reporter plasmid (pmirGLO-Cthrc1-Wt/pmirGLO-Cthrc1-Mut) using Lipofectamine™2000 (Invitrogen). 48 h after transfection, cells were harvested and luciferase activity was measured with a Dual-Luciferase assay kit (Promega) according to the manufacturer’s instructions.