Journal: Theranostics

Article Title: Circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/EZH2 axis

doi: 10.7150/thno.42417

Figure Lengend Snippet: CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against AGO2 on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.

Article Snippet: After blocking for 1 h, the membranes were incubated with the primary anti-EZH2, C/EBP-α, PPAR-γ or AGO2 antibodies (1:000; Cell Signaling Technology, Danvers, MA, USA) at 4ºC overnight, followed by a 2 h incubation with HRP-conjugated secondary antibody at room temperature.

Techniques: Quantitative RT-PCR, RNA In Situ Hybridization, Staining, Luciferase, Transfection, Plasmid Preparation, Negative Control, Expressing

Journal: Oncology Research

Article Title: Long Noncoding RNA LINC0086 Functions as a Tumor Suppressor in Nasopharyngeal Carcinoma by Targeting miR-214

doi: 10.3727/096504017X14865126670075

Figure Lengend Snippet: LINC0086 interacts with miR-214 and regulates its expression. (A) qPCR was performed to detect the expression of miR-214 in C666-1 and HK-1 cells after LINC0086 transfection. (B) The wild type (WT) and mutant type (MT) of LINC0086 3′-UTR, and the binding sites were shown. (C) The relative luciferase activities were inhibited in the C666-1 and HK-1 cells cotransfected with wild-type LINC0086 3′-UTR and miR-214, and not with the mutant type. Firefly luciferase activity was normalized to Renilla luciferase. (D) Association of LINC0086 and miR-214 with Ago2 in C666-1 and HK-1 cells. Cellular lysates from C666-1 and HK-1 cells were used for RNA immunoprecipitation (RIP) with antibody against Ago2. LINC0086 and miR-214 expression levels were detected using qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group.

Article Snippet: RIP experiments were performed using the Magna RIP RNA-Binding Protein IP Kit (Millipore, Bedford, MA, USA) and the Ago2 antibody (Cell Signaling, Danvers, MA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Immunoprecipitation, Quantitative RT-PCR

Journal: Theranostics

Article Title: Arterial cyclic stretch regulates Lamtor1 and promotes neointimal hyperplasia via circSlc8a1/miR-20a-5p axis in vein grafts

doi: 10.7150/thno.69551

Figure Lengend Snippet: CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the Ago2 or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.

Article Snippet: Then the cell lysis was incubated with magnetic beads conjugated with ani-Ago2 antibody (CST) or negative control IgG, at 4 °C overnight.

Techniques: Expressing, Western Blot, Negative Control, Immunoprecipitation, Quantitative RT-PCR, Luciferase, Reporter Assay, Mutagenesis, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Endocrinology

Article Title: CREB3 Transactivates lncRNA ZFAS1 to Promote Papillary Thyroid Carcinoma Metastasis by Modulating miR-373-3 p /MMP3 Regulatory Axis

doi: 10.1155/2021/9981683

Figure Lengend Snippet: ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with AGO2 antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).

Article Snippet: AGO2 (anti-AGO2, 1 : 50, Cell Signaling, USA) antibody or IgG was used and immunoprecipitated by 25 μ l protein A/G ligated beads.

Techniques: Luciferase, Expressing, Immunoprecipitation