Primerstar

LncRNA UCA1 siRNA, CXCR4 siRNA, and miR-204 mimics were purchased from GenePharma (Shanghai, China). UCA1 was amplified from the cDNA of PC3 cells using PrimerSTAR (TaKaRa) and cloned into the pcDNA3.1(+) vector. All cells were transfected with 100 nM miR-204 mimics, UCA1 siRNA, CXCR4 siRNA, or 2 µg pcDNA3.1(+)-UCA1 expression vector using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The WT and MT 3′UTR of CXCR4 or the UCA1 fragment containing the miR-204 binding sites were synthesized and then cloned into the luciferase reporter vector p-Luc.

The genomic DNA of C2C12 cells was isolated for the PCR amplification template. The promoter of the Camk2b gene was amplified by PCR using PrimerSTAR^® (TaKaRa, Dalian, Liaoning, China). Then, we cloned the promoter of the Camk2b gene into the eukaryotic expression vector pGL3-Basic (named as pGL3-Basic-Camk2b). Similarly, we amplified the full length of the Myod gene and cloned it into the pcDNA3.1 plasmid (pcDNA3.1-Myod). According to Promega’s dual luciferase reporter assay kit (Promega, Madison, WI, United States), we transfected the pGL3-Basic, pGL3-Basic-Camk2b and pcDNA3.1, pGL3-Basic-Camk2b and pcDNA3.1-Myod into C2C12 cell by Lipofectamine^TM 3000 Transfection Reagent (Thermo Fisher Scientific, MA, United States). At last, we identified the double-luciferase activity by BioTek Synergy 2 multifunctional microplate reader (BioTek, Winooski, VT, United States). The ratio of the expression of firefly luciferase to renilla luciferase was the promoter activity. The PCR primer pairs were listed in Supplementary Table 1.

The mouse SHP1 mRNA sequence was amplified from cDNA by PCR using PrimerSTAR (TAKARA, Dalian, China), and the PCR product was inserted into the PLVX-IRES-PURO plasmid after double digestion. The constructed plasmid combined with PMD2G and PSPAX2 plasmids was transfected to 293T cells by LIPO2000 (Invitrogen, Carlsbad, CA, USA), and the supernatant was collected after 48 h. The supernatant was filtered through a 0.22 μm filter to eliminate cell debris, and then was concentrated by centrifugation. The lentivirus with polybrene (5 μg/ml) was applied to transfect WT MSCs for 12 h, and the transfected MSCs were purified using puromycin (2 μg/ml). The expression of SHP1 protein in purified MSCs was detected by western blotting.

The putative promoter region of miR-421 (−1 kb) harboring predicted hypoxia response element (HRE) sequences were synthesized by PCR (PrimerSTAR, TaKaRa, Japan) and cloned into pGL3 vector between SacI and NheI. CoCl₂ or DMOG treated HEK293T cells were co-transfected with cloned pGL3 vector and renilla luciferase vector with or withour Si-HIF-1α. After 48h transfection, the relative luciferase activity was calculated by Dual-Luciferase Reporter Assay System (Promega, USA).
The 3′-UTR (untranslated region) of E-cadherin and caspase-3, containing putative target region of miR-421, was synthesized (Sangon, Shanghai, China) and cloned into sites between SacI and SalI downstream of luciferase reporter gene of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, USA), respectively. Additionally, the mutant miR-421 putative target region was inserted into pmirGLO Dual-Luciferase Vector in the same way. All insertions were verified by sequencing (Sangon, Shanghai, China). HEK293T cells were seeded in a 24-well plate for 24h before co-transfected with 50nM of either miR-421 or NC and 200ng reporter plasmid containing wild type (WT) or mutant type (Mut) of E-cadherin or caspase-3 3′-UTR. After 48h transfection, the relative luciferase activity was calculated by Dual-Luciferase Reporter Assay System (Promega, USA).

TRIzol (Invitrogen, Carlsbad, CA, United States) was used to extract total RNA from the peripheral blood of healthy family members. First strand cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Jiangsu, China). Primers containing EcoRI and SmaI (forward primer: 5′-gga​att​ctATG​GCT​ACG​CCC​CTG​GTG​GC-3′, reverse primer: 5′-tccccc​gggTCA​CTC​CTT​TTG​CTC​TGT​GG-3′) sites were designed to amplify the coding sequence of the TINF2 gene. The PCR products were cloned into the pEGFP-C1 vector (GENEWIZ, New Jersey, United States) through double digestion with EcoRI and SmaI (New England Biolabs, Beijing, China). The mutant pEGFP-C1-TINF2-MUT vector was obtained by PCR-based site-directed mutagenesis using PrimerStar (Takara, Dalian, China). The primers were as follows: forward primer: 5′-GGT​GTG​GAC​ATG​GGT​GGC​TGC​TTC​CAG​AG-3′ and reverse primer: 5′-CTC​TGG​AAG​CAG​CCA​CCC​ATG​TCC​ACA​CC-3’. The constructs were sequenced to confirm that no secondary mutation was introduced. Plasmids were extracted using Endofree Plasmid Midi Kit (Tiangen, Beijing, China).

All constructs used in the study are listed in Additional file 2: Table S1. Sequences of the primers, crRNAs, and oligonucleotides used in the study are listed in Additional file 3: Table S2. Plasmids and chromosomal DNA were extracted using AxyPrep kits (Corning, China). DNA polymerases used were PrimerSTAR (Takara, Japan) or Taq DNA polymerases (Tsingke, China). Restriction endonucleases and T4 DNA ligase were from Thermo Scientific (USA), and the isothermal assembly method was used in this work [35 (link)]. Gene deletions were confirmed by PCR and Sanger sequencing (Tsingke, China).

The putative promoter region of miR-148a-3p (-1 kb) harboring the predicted myc-binding sequences were synthesized by PCR (PrimerSTAR, TaKaRa) and cloned into the pGL3 vector between HindIII and NheI. pENTER-c-myc vector (pC-MYC) or pENTER vector (pNull)-treated T24 or UM-UC-3 bladder cancer cells were co-transfected with the cloned pGL3 vector and renilla luciferase vector in the absence or presence of the c-myc inhibitor (c-myc-Inh, 10058-F4, Selleck, Houston, TX, USA). A total of 48 h after transfection, the relative luciferase activity was calculated using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
The 3′-UTRs of DNMT1, ERBB3 and AKT2 containing putative miR-148a-3p target regions were synthesized (Sangon, Shanghai, China) and cloned between the SacI and SalI sites downstream of the luciferase reporter gene in pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). In addition, the mutant miR-148a-3p putative target region was generated using the same approach. All insertions were verified by sequencing (Sangon). The dual-Luciferase report assay was performed as previously described.^{14 (link)}