Glomax20 20 luminometer fluorescence detector

Bioinformatic website RNA22 was used to analyze the target relationship between lncRNA PVT1 and miR‐145, and dual‐luciferase reporter gene assay was carried out to verify whether miR‐145 was a target of lncRNA PVT1, and whether FSCN1 was a target gene of miR‐145. Initially, the full length of lncRNA PVT1 was ligated into pmirGLO Dual‐Luciferase miRNA Target Expression Vector (Promega Corporation, Madison, WI, USA) to construct wild‐type (WT) pmirGLO‐lncRNA. pmirGLO‐lncRNA mutant (MUT) was also developed in which the binding sites of miR‐145 were mutated. Target sequence and mutation sequence were created according to potential binding sites of miR‐145 on the 3′‐untranslated region (3′UTR) of FSCN1. The cells were seeded in a 6‐well plate at a density of 2 × 10⁵ cells/well and then transfected. After 48 h of transfection, the cells were collected and dual‐luciferase reporter gene assay was performed based off of the instructions of dual‐luciferase detection kit (D0010; Beijing Solarbio Life Sciences Co., Ltd, Beijing, China). Fluorescence intensity was measured by means of a GloMax 20/20 luminometer fluorescence detector in Promega Corporation, Madison, WI, USA (Shanxi Zhongmei Biotechnology Co., Ltd., Shanxi, China).

Firstly, the sequence fragment of LDOC1 3′-untranslated region (3′UTR) was artificially synthesized and introduced into the psiCHECK-2 vector (Promega, Madison, WI, USA). The mutation sites in the complementary sequence of seed sequences were designed on the wild type (WT) of LDOC1. Subsequently, all luciferase reporter plasmids, such as LDOC1 3′UTR-WT and LDOC1 3′UTR-mutant (MUT), were obtained. All aforementioned plasmids were co-transfected with miR-4532 mimic (2 nM, Dharmacon, Lafayette, CO, USA) or mimic-NC (2 nM, Dharmacon, Lafayette, CO, USA) into 4 × 10⁵ HEK-293 T cells (CRL-1415, Shanghai Xin Yu Biotech Co., Ltd., Shanghai, China) and CD34⁺ HSCs, respectively. After 48 h, the cells were lysed and the luciferase activity was determined using Dual-Luciferase Reporter Assay kits (RG005, Beyotime Biotechnology, Shanghai, China) in the Glomax20/20 luminometer fluorescence detector (Promega, Madison, WI, USA). The experiment was repeated three times to obtain the mean value [24 (link)].

Target gene analysis of miR-93-5p was performed using the biological prediction website (http://www.targetscan.org/vert_71). A dual luciferase reporter gene assay was used to verify whether AHNAK was a direct target gene of miR-93-5p. Primers were designed and 3′untranslated region (UTR) sequence of AHNAK gene was amplified and ligated with psiCHECK2 vector to obtain dual luciferase report vectors, named wild type (WT). The miR-93-5p seed sequence and the mutant primers of AHNAK 3′UTR binding region were designed. With the 3′UTR fragment of the AHNAK gene as a template, the forward and reverse fragments of the 3′UTR of the AHNAK gene were obtained after amplification by PCR, from which fragment containing mutant binding site was obtained by PCR. The mutation report vector of AHNAK target site was obtained by ligating of cleavage sites with the psiCHECK2 vector, which was named mutant type (MUT). The correctly sequenced WT and MUT were separately co-transfected with miR-93-5p into HEK-293T cells (Shanghai Beinuo Biotechnology Co., Ltd., Shanghai, China). After 48 h of transfection, cells were harvested and lysed. Luciferase activity change caused by miR-93-5p on AHNAK 3′UTR was measured in the cells according to the method provided by Genecopoeia’s dual luciferase assay kit. The luciferase intensity was measured using a Promega Glomax 20/20 luminometer fluorescence detector.

A dual-luciferase reporter gene assay was adopted to verify whether EGR1 could be targeted by miR-181a–2–3p, and human EGR1 3′UTR WT fragments containing miR-181a–2–3p binding sites were synthesized. Endonuclease sites, SpeI and Hind III, which introduced pMIR reporter, were obtained from Huayueyang Biotechnology, Beijing, China. MUT sites were designed based on the EGR1 WT 3′UTR and miR-125a-5p binding site, and the target fragment was subsequently inserted into the pMIR-reporter plasmid. Lipofectamine 3000 (Invitrogen) was employed to cotransfect the plasmids with miR-181a–2–3p mimic or NC-mimic into HEK293T cells. The luciferase activity at a wavelength of 570 nm was detected using a dual-luciferase reporter system (Promega, Madison, WI) and Glomax20/20 luminometer fluorescence detector (Promega). Relative luciferase activity = Firefly luciferase activity/Renilla luciferase activity

TargetScan (http://www.targetscan.org/vert_71/) was used to predict the potential target genes of miR-29a, and GAB1 was predicted as one of the candidate genes. To further validate the relationship between miR-29a and GAB1, synthetic 3’untranslated region (3’UTR) fragments of GAB1 (GAB1-wild type [Wt]) were introduced into pMIR-reporter plasmids (Huayueyang Biotechnology Co., Ltd., Beijing, China) via endonuclease sites SpeI and Hind III. Afterwards, the target fragments were inserted into pMIR-reporter plasmids using T4 DNA ligase. Therefore, the pMIR-reporter plasmids with Wt GAB1 (pMIR-GAB1-Wt) and GAB1 mutant (Mut) at the putative miR-29a binding sites (pMIR-GAB1-Mut) were designed. The pMIR-GAB1-Wt plasmids and pMIR-GAB1-Mut plasmids were respectively co-transfected with miR-29a mimic or mimic-NC using Lipofectamine 2000 into the cells (Beinuo Biotechnology Co., Ltd., Beijing, China). The cells were transfected with pRL-TK plasmids, which was used as reference. After transfection for 48 h, the cells were collected and lysed. Dual-Luciferase Reporter assay kit (K801–200, Biovision, Milpitas, CA, USA) and Glomax 20/20 luminometer fluorescence detector (Promega, Madison, WI, USA) were applied for luciferase activity detection. The experiment was repeated 3 times independently.

The RNA22 online database (https://cm.jefferson.edu/rna22/Interactive/) was adopted in order to predict the binding relation between GAS5 3′UTR and miR-17-3p, as well as miR-17-3p and Ang-2 3′UTR, and the results of which were further validated with a dual-luciferase reporter assay. The sequences harboring the binding sites or the mutant form (MUT) were cloned into the pUC57 vector and then subcloned into the psiCHECK-2 vector. Following amplification, all plasmids were extracted using plasmid mini kits from Omega (D1100-50T, Solarbio Science & Technology Co., Ltd., Beijing, China). The HEK-293T cells seeded in 6-well plates (density of 2 × 10⁵ cells/well) were submitted to transfection with the luciferase reporter plasmids of GAS5-WT, GAS5-MUT, Ang-2-WT, and Ang-2-MUT, respectively, with miR-17-3p mimics. After a period of 48 h incubation, Genecopoeia's Dual-Luciferase Assay kits (D0010, Solarbio) were adopted for measuring the luciferase activity, while a Glomax 20/20 luminometer fluorescence detector (Promega Corp., Madison, WI, USA) was utilized for measuring the luminance values. The experiment was repeated in triplicate to obtain the mean value.