Pmirglo

miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) was used to predict the target mRNAs of miRNA. Sequences of SYT1 and PTTG1 were acquired from Ensembl (http://asia.ensembl.org/index.html). Interacting sequences between miRNAs and mRNAs were analyzed by Global Align in Blast (https://blast.ncbi.nlm.nih.gov).
The PTTG1 sequence that incorporated binding sites with miR-423-5p was amplified through performing PCR at first and were then inserted into pmirGLO (Promega, Madison, WI, USA) in order to establish the reporter vector of wild-type pmirGLO-PTTG1. For another, the reporter vectors of mutation pmirGLO-PTTG1 were constructed through mutating the binding sites of PTTG1 to miR-423-5p. With the help of Lipofectamine 3000 transfection kit (Promega, Madison, WI, USA), pmirGLO-XIST Mut, pmirGLO-XIST Wt, pmirGLO-ROR1 Mut, and pmirGLO-ROR1 Wt were, respectively, cotransfected with miR-423-5p mimic or miR-423-5p-NC into GH3 cells. After 48 h transfection of wild-type or mutation of PTTG1 cotransfected with miR-423-5p, the luciferase reporter gene activity of cells was determined following the guidance of dual luciferase detection kit[20 (link)] (Promega, Madison, WI, USA).

The bioinformatics analysis was executed by the online database CircInteractome (https://circinteractome.nia.nih.gov/) or Targetscan (http://www.targetscan.org/vert_71/). Dual-luciferase reporter assay was used for validating the interaction between miR-637 and circLDLR or LMO4. Wild (wt) type and mutant (mut) circLDLR and LMO4 3′ UTR containing the predicted binding sites of miR-637 were cloned into the pmirGLO Vector (Promega, Shanghai, China) to generate pmirGLO-circLDLR-wt, pmirGLO-circLDLR-mut, pmirGLO-LMO4-wt, or pmirGLO-LMO4-mut. Then, these constructed vectors combined with miR-637 mimic or mimic control (miR-NC) were cotransfected into TPC-1 and IHH-4 cells using Lipofectamine 3000 (Invitrogen). 48 h later, the luciferase activity normalized to Renilla luciferase activity was examined by a dual luciferase assay kit (Promega). The same experiment was repeated three times.

The regulatory role of miR-146 on HDMCP was tested with dual luciferase report system. Firstly, the normal gene fragment containing 3’ UTR region of HDMCP wide type and the specific dual-luciferase miRNA target expression vector-pmirGLO (Promega, the USA) were both cut with SacⅠand SalⅠ. These two segments were then connected with T4 DNA ligase (Fermentas, Lithuania) at 22℃ for 2h. Secondly, the combined vector pmirGLO-HDMCP-3’UTR was used to transfect competent cell with CaCl₂. Thereafter, the transfected cells were cultured at 37℃for 16h, followed by pmirGLO-HDMCP-3’UTR extraction (TIANGEN, China) and verification by gene sequencing. Thirdly, the miR-146 mimics were synthesized and cotransfected both BRL-3A and L02 cells with pmirGLO-HDMCP-3’UTR using lipofectamin 2000 (Invitrogen, The USA). After culturing at 37℃/5% CO₂ for 25h, those cotransfected cells were harvested and the luciferase activity was tested using a dual-luciferase reporter gene detection kit (Promega, the USA).In this step, subjects were divided into four groups as followings: blank cell group, pmirGLO-HDMCP-3’UTR group, negative control miRNA+pmirGLO-HDMCP-3’UTR group and miR-146+pmirGLO- HDMCP-3’UTR group.

Wild-type (WT), mutant (MUT) lnc021545 3′-untranslated region (UTR), and EREG 3′-UTR were amplified and cloned into the luciferase reporter vector pmirGLO (Promega, Madison, WI, USA) to create pmirGLO lnc021545, pmirGLO EREG WT, and MUT plasmids. Then, 1 × 10⁵ MCF-7 cells were co-transfected with 5 ng each of the corresponding plasmids and 20 pmol each of miR-330-3p mimics. A dual-luciferase reporter assay system (Promega) was used in this assay. Each well of luminometer plates was loaded with lysates and the firefly luciferase activity was detected through an EnSpire multifunctional microplate reader (PerkinElmer, Waltham, MA, USA). Each well of the luminometer plates was again loaded with 100 µL Stop&Glo reagent, and renilla luciferase activity was detected. The results were represented as normalized firefly luciferase activity/renilla luciferase activity values.

To construct an elk1 expression plasmid, the full-length coding sequence of grass carp transcription factor elk1 was amplified by PCR and inserted into vector pEGFP-N1 (Promega, Madison, WI, USA) to generate pEGFP-elk1. The sequence of the elk1 promoter was inserted into vector pGL3-basic (Promega) to generate pGL3-pelk1. At the same time, the elk1 and ezra fragment containing presumptive miR-731 target sequences were amplified by PCR. The amplicon was cloned into the dual luciferase vector pmirGLO (Promega) to generate pmirGLO-elk1-WT and pmirGLO-ezra-WT. Mutation of target sequences was performed using a MutanBEST Kit (Takara, Dalian, China) following standard procedures using the corresponding primers in Table 1. Pyrobest DNA Polymerase was used for PCR involving 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min. The resulting DNA fragment was blunted using Blunting Kination Enzyme Mix and ligated using ligation solution I at 16°C for 1 h. The ligation product was transformed into DH5a and all the constructed plasmids were confirmed by Sanger sequencing and were extracted using an Endotoxin-Free Plasmid DNA Miniprep Kit (Tiangen Biotech, Beijing, China) for further luciferase reporter assays. All primer sequences are listed in Table 1.

The targeted regulatory relationship between Smad2 and miR-152-3p was further determined by constructing the reporter gene plasmid of the target gene, Smad2. Specifically, the wild-type (WT) fragment of Smad2 3′ UTR was amplified using the primers containing the miR-152-3p and Smad2 binding site (Table 2). Then, the Smad2 3′ UTR mutant-type (MUT) fragment was synthesized by mutating 4 bases of the binding site using the Stratagene mutation kit (Stratagene, Heidelberg, Germany). Subsequently, these two fragments were connected to the luciferase reporter vector pmiRGLO (Promega, Madison, WI, USA) and the recombinant plasmids pmiRGLO–Smad2-3′ UTR-WT and pmiRGLO–Smad2-3′ UTR-MUT were constructed. The extracted recombinant plasmid was then co-transfected with the miR-152-3p mimic into the HEK-293T cells. After 48 h of transfection, the activity of luciferase was detected using a dual-luciferase detection kit (Promega, Madison, WI, USA).