Journal: Genes

Article Title: Hsa_circ_0006692 Promotes Lung Cancer Progression via miR-205-5p/CDK19 Axis

doi: 10.3390/genes13050846

Figure Lengend Snippet: hsa_circ_0006692 played its roles via sponging hsa-miR-205-5p and regulation of CDK19. ( A ) After the detection of ago2 or IgG RIP assay, hsa_circ_0006692 and miR-205-5p levels was determined by qRT-PCR; ( B ) After RNA pull-down analysis using Bio-miR-NC and Bio-miR-205-5p in A549 and H1299 cells, expression of hsa_circ_0006692 was measured by qRT-PCR; ( C – E ) CDK19 expression level was increased in A549/circ-0006692-OE cells and decreased in A549/circ-0006692-SH cells analyzed by qRT-PCR and WB; ( C , D ) miR-205-5p expression was decreased in A549/circ-0006692-OE cells and increased in A549/circ-0006692-SH cells analyzed by qRT-PCR; ( F ) miR-205-5p expression in A549 cells transfected with miR-205-5p mimics or inhibitors was analyzed using qRT-PCR; ( G , H ) qRT-PCR analysis of hsa_circ_0006692 and CDK19 expression in A549 cells transferred with miR-205-5p mimics or inhibitor. *** p < 0.001; **** p < 0.0001.

Article Snippet: The cell lysates were added to antibodies against Ago2 or IgG (CST) and were treated overnight at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Transfection

Journal: Endocrinology

Article Title: Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery

doi: 10.1210/endocr/bqab007

Figure Lengend Snippet: Effect of hepatic Argonaute 2 (Ago2) deficiency on glucose metabolism of diabetic obese mice. A, Body weight of liver-specific Ago2-deficiency (L-Ago2) wild-type (WT)- ob/ob (n = 16), and L-Ago2 knockout (KO)- ob/ob (n = 13) mice on normal chow (CD) from age 5 to 12 weeks. B, Food intake normalized by body weight per day of L-Ago2 WT- ob/ob (n = 21), and L-Ago2 KO- ob/ob (n = 24) mice at age 11 weeks. C, Blood glucose levels after 6 hours of fasting of L-Ago2 WT- ob/ob (n = 20) and L-Ago2 KO- ob/ob (n = 16) at age 13 weeks. D, Glucose tolerance test (GTT) and its area under the curve (AUC) analysis in L-Ago2 WT- ob/ob (n = 18) and L-Ago2 KO- ob/ob (n = 18) mice at age 7 weeks. E, Serum insulin levels after 6 hours of fasting of L-Ago2 WT-Lean (n = 3), L-Ago2 WT- ob/ob (n = 4), L-Ago2 KO-Lean (n = 3), and L-Ago2 KO- ob/ob (n = 7) mice at age 13 weeks. F, Insulin tolerance test (ITT) and its AUC analysis in L-Ago2 WT- ob/ob (n = 18) and L-Ago2 KO- ob/ob (n = 18) mice at age 9 weeks. G, Messenger RNA (mRNA) expression levels of de novo lipogenesis and gluconeogenesis in livers of L-Ago2 WT- Lean (n = 5), L-Ago2 WT- ob/ob (n = 10), L-Ago2 KO- Lean (n = 5), and L-Ago2 KO- ob/ob (n = 4) mice after overnight fasting at age 13 weeks. H, Metabolic disease–associated microRNAs (MD-miRNAs) and I, their target mRNA expression levels in livers of L-Ago2 WT- Lean (n = 5), L-Ago2 WT- ob/ob (n = 10), L-Ago2 KO- Lean (n = 5), and L-Ago2 KO- ob/ob (n = 4) mice after overnight fasting at age 13 weeks. Data are shown as mean ± SEM of group size (n). Statistical analyses were performed by unpaired 2-tailed t test for C, D, and F or by 2-way analysis of variance followed by Tukey post hoc test for E, G, H, and I. * P less than or equal to .05, ** P less than or equal to .01, *** P less than or equal to .001.

Article Snippet: Ago2 primary antibody ( ) and α-Tubulin primary antibody ( ) were purchased from Cell Signaling Technology.

Techniques: Knock-Out, Expressing

Journal: Endocrinology

Article Title: Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery

doi: 10.1210/endocr/bqab007

Figure Lengend Snippet: Effect of hepatic Argonaute 2 (Ago2) deficiency on glucose metabolism in a long-term high-fat diet (HFD) condition. A, Body weight of liver-specific Ago2-deficiency (L-Ago2) wild-type (WT) and L-Ago2 knockout (KO) mice on an HFD at age 23 (n = 22 and 25, respectively), 30 (n = 22 and 25, respectively), and 34 (n = 9 and 10, respectively) weeks. B, Blood glucose levels after 6 hours of fasting of L-Ago2 WT (n = 9) and L-Ago2 KO (n = 10) mice at age 34 weeks. C, Serum insulin levels of L-Ago2 WT (n = 9) and L-Ago2 KO (n = 10) mice at age 34 weeks. D, Glucose tolerance test (GTT) and its area under the curve (AUC) analysis in L-Ago2 WT (n = 9) and L-Ago2 KO (n = 10) mice at age 30 weeks. E, Insulin tolerance test (ITT) and its AUC analysis in L-Ago2 WT (n = 9), and L-Ago2 KO (n = 10) mice at age 31 weeks. F, Metabolic disease–associated microRNAs (MD-miRNAs) and their G, target messenger RNA (mRNA) expression levels in livers of L-Ago2 WT (n = 9) and L-Ago2 KO (n = 9) mice at age 34 weeks. Data are shown as mean ± SEM of group size (n). Statistical analyses were performed by ordinary one-way analysis of variance, followed by Tukey post hoc test for A or unpaired 2-tailed test for B to G. * P less than or equal to .05, ** P less than or equal to .01, *** P less than or equal to .001.

Article Snippet: Ago2 primary antibody ( ) and α-Tubulin primary antibody ( ) were purchased from Cell Signaling Technology.

Techniques: Knock-Out, Expressing

Journal: Endocrinology

Article Title: Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery

doi: 10.1210/endocr/bqab007

Figure Lengend Snippet: Effects of vertical sleeve gastrectomy (VSG) surgery on glycemic control and metabolic disease–associated microRNA (MD-miRNA) expression in liver-specific Ago2-deficiency (L-Ago2) knockout (KO) mice. A, Body weight gain of L-Ago2 wild-type (WT)- SHAM (n = 7) and L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 7), and L-Ago2 KO- VSG (n = 6) mice post surgery. B, Body weight loss of L-Ago2 WT- SHAM (n = 7) and L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 7), and L-Ago2 KO- VSG (n = 5) mice pre surgery and post surgery. C, Blood glucose levels after 6 hours of fasting of L-Ago2 WT- SHAM (n = 7), L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 7), and L-Ago2 KO- VSG (n = 6) mice 4 weeks post surgery. D, Serum insulin levels after 6 hours of fasting of L-Ago2 WT- SHAM (n = 7), L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 7), and L-Ago2 KO- VSG (n = 6) mice 4 weeks post surgery. E, hematoxylin-eosin–stained liver section of L-Ago2 WT- SHAM , L-Ago2 WT- VSG , L-Ago2 KO- SHAM , and L-Ago2 KO- VSG mice 4 weeks post surgery. F, Plasma alanine aminotransferase (ALT) levels of L-Ago2 WT- SHAM (n = 4), L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 6), and L-Ago2 KO- VSG (n = 6) mice 4 weeks post surgery. G, MD-miRNAs and their target messenger RNA (mRNA), de novo lipogenesis, and H, gluconeogenesis gene expression levels in livers of L-Ago2 WT- SHAM (n = 6), L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 7), and L-Ago2 KO- VSG (n = 6) mice 4 weeks post surgery. Data are shown as mean ± SEM of group size (n). Statistical analyses were performed by multiple t test for A or by unpaired 2-tailed test for C and F, or by 2-way analysis of variance followed by Tukey post hoc test for B, G, and H. * P less than or equal to .05, * P less than or equal to .01, *** P less than or equal to .001.

Article Snippet: Ago2 primary antibody ( ) and α-Tubulin primary antibody ( ) were purchased from Cell Signaling Technology.

Techniques: Expressing, Knock-Out, Staining

Journal: Endocrinology

Article Title: Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery

doi: 10.1210/endocr/bqab007

Figure Lengend Snippet: Hepatic Argonaute 2 (Ago2) deficiency enhances the expression of the peroxisome proliferator–activated receptor α (PPARα) pathway. A, Messenger RNA (mRNA) expression of Tfam -mitochondrial genes in livers of liver-specific Ago2-deficiency (L-Ago2) wild-type (WT) (n = 9) and L-Ago2 knockout (KO) (n = 9) mice at age 34 weeks. B, Western blot analysis of PPARα protein expression in the liver of L-Ago2 WT (n = 6) and L-Ago2 KO (n = 6) mice at age 34 weeks. C, miR-27a and miR-27b expression in livers of L-Ago2 WT (n = 9) and L-Ago2 KO (n = 9) mice at age 34 weeks. D, PPARα and its target mRNA expression in livers of L-Ago2 WT (n = 9) and L-Ago2 KO (n = 9) mice at age 34 weeks. E, Western blot analysis of PPARα protein expression in the liver of L-Ago2 WT- SHAM (n = 3), L-Ago2 WT- VSG (n = 3), L-Ago2 KO- SHAM (n = 3), and L-Ago2 KO- VSG (n = 3) mice 4 weeks post surgery. F, miR-27a and miR-27b expression in livers of L-Ago2 WT- SHAM (n = 6), L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 7), and L-Ago2 KO- VSG (n = 6) mice 4 weeks post surgery. G, PPARα and its target mRNA expression in livers of L-Ago2 WT- SHAM (n = 6), L-Ago2 WT- VSG (n = 6), L-Ago2 KO- SHAM (n = 7), and L-Ago2 KO- VSG (n = 6) mice 4 weeks post surgery. NS, nonspecific band. Data are shown as mean ± SEM of group size (n). Statistical analyses were performed by unpaired 2-tailed test for A to D, by multiple t test for E, or by 2-way analysis of variance followed by Tukey post hoc test for F and G. * P less than or equal to .05, * P less than or equal to .01, *** P less than or equal to .001.

Article Snippet: Ago2 primary antibody ( ) and α-Tubulin primary antibody ( ) were purchased from Cell Signaling Technology.

Techniques: Expressing, Knock-Out, Western Blot

Journal: Endocrinology

Article Title: Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery

doi: 10.1210/endocr/bqab007

Figure Lengend Snippet: Effects of peroxisome proliferator-activated receptor (PPAR) agonist in Argonaute 2 (Ago2)-deficient primary hepatocytes. A, messenger RNA (mRNA) expression of Tfam -mitochondrial genes in liver-specific Ago2-deficiency (L-Ago2) wild-type (WT) (n = 6), and L-Ago2 knockout (KO) (n = 6) primary hepatocytes treated with or without 10-µM WY14643 (a PPARα agonist) for 24 hours. B, miR-27a and miR-27b expression in L-Ago2 WT (n = 6) and L-Ago2 KO (n = 6) primary hepatocytes treated with or without 10-µM WY14643 (PPARα agonist) for 24 hours. C, Oxygen consumption rate (OCR) of L-Ago2 WT (n = 12) and KO (n = 10–12) primary hepatocytes were measured in the presence or absence of a PPARα agonist (WY14643: 10 μM) pretreatment for 48 hours. D, PPARα target mRNA expression in L-Ago2 WT (n = 6) and L-Ago2 KO (n = 6) primary hepatocytes treated with or without 10-µM WY14643 (PPARα agonist) for 24 hours. Data are shown as mean± SEM. Statistical analyses were performed by 2-way analysis of variance for A, B, and D; and ordinary 1-way analysis of variance followed by Tukey post hoc test for C. * P less than or equal to .05, * P less than or equal to .01, *** P less than or equal to .001.

Article Snippet: Ago2 primary antibody ( ) and α-Tubulin primary antibody ( ) were purchased from Cell Signaling Technology.

Techniques: Expressing, Knock-Out

Journal: Endocrinology

Article Title: Hepatic Ago2 Regulates PPARα for Oxidative Metabolism Linked to Glycemic Control in Obesity and Post Bariatric Surgery

doi: 10.1210/endocr/bqab007

Figure Lengend Snippet: A proposed molecular mechanism of the role of hepatic Argonaute 2 (Ago2) in obesity, post bariatric surgery, and in peroxisome proliferator–activated receptor α (PPARα) response. Hepatic Ago2 is required for the expression of metabolic disease–associated microRNA (MD-miRNA), which is enhanced in obesity but suppressed post bariatric surgery. Hepatic Ago2-dependent RNA silencing plays a critical role in gene regulation relevant to mitochondrial functions, including PPARα, adenosine 5′-monophosphate–activated protein kinase α (AMPKα1), and PPAR γ coactivator (PGC1α), and PPARα-mediated oxidative metabolism in obesity, post–vertical sleeve gastrectomy (VSG), and PPARα response.

Article Snippet: Ago2 primary antibody ( ) and α-Tubulin primary antibody ( ) were purchased from Cell Signaling Technology.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Long Non-coding RNA GAS5 Regulates T Cell Functions via miR21-Mediated Signaling in People Living With HIV

doi: 10.3389/fimmu.2021.601298

Figure Lengend Snippet: Differential expression of GAS5 and miR-21 in CD4 T cells after TCR stimulation. (A,B) Relative expression of GAS5 ( n = 8) and miR-21 ( n = 8) in CD4 T cells isolated from HS and PLHIV was determined by RT-qPCR. (C) Correlation between GAS5 and miR-21 expressions in CD4 T cells isolated from HS or PLHIV was determined by Pearson correlation analysis. (D,E) Correlation between CD4 T cell counts and relative GAS5 ( n = 8) or miR-21 ( n = 8) expressions in CD4 T cells from PLHIV was determined by Pearson Correlation analysis. (F,G) Relative expression of GAS5 in CD4 T cells from HS and PLHIV in the absence or presence of TCR stimulation for 1, 3, and 5 days ( n = 6). (H,I) Relative expression of miR-21 in HS and PLHIV CD4 T cells with or without TCR stimulation for 1, 3, and 5 days ( n = 6). (J) Relative expression of miR-21 in TCR-stimulated CD4 T cells from HS and PLHIV in 1, 3, and 5 days ( n = 6). (K) Kinetic changes in miR-21 expression in CD4 T cells from both PLHIV and HS following TCR stimulation for 1, 3, and 5 days. (L) Relative expression of miR-146a in CD4 T cells from HS and PLHIV with or without TCR stimulation for 24 h ( n = 6). (M) Representative dot blot and relative expression of AGO2 (a major component of RISK complex) in CD4 T cells from HS and PLHIV with or without TCR stimulation for 24 h, determined by Western blotting ( n = 6).

Article Snippet: The primary antibodies used were rabbit PARP-1 Ab (clone 46D11; Cat #9532), rabbit phospho-histone H2A.X Ab (Ser139) (clone 20E3; Cat #9718), rabbit phospho-S6 ribosomal protein (Ser235/236) Ab (clone D57.2.2E; Cat #4858), rabbit phospho-Akt (Ser473) XP Ab (clone D9E; Cat #4060), mouse AGO2 Ab (clone 2E12-1C9; Cat #57113), and rabbit GAPDH XP mAb (clone D16H11; Cat #5174) (all from Cell Signaling Technology; Danvers, MA).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Dot Blot, Western Blot

Journal: Frontiers in Immunology

Article Title: Long Non-coding RNA GAS5 Regulates T Cell Functions via miR21-Mediated Signaling in People Living With HIV

doi: 10.3389/fimmu.2021.601298

Figure Lengend Snippet: Cell cycle progression, AGO2 expression, and effect of anti-PD1/IL-2 in TCR-stimulated CD4 T cells on GAS5-miR-21 axis. (A) Cell cycle progression in CD4 T cells from HS and PLHIV following TCR stimulation at day 1, 3, and 5 measured by PI flow cytometric analysis ( n = 4). Representative histogram of cell cycle phases of CD4 T cells from HS and PLHIV with and without TCR stimulation at day 3 are shown above and summary data at day 1, 3, 5 are shown below. (B) Relative expression of GAS5 and miR-21 in CD4 T cells from HS and PLHIV following anti-PD1 and IL-2 treatments for 24 h, determined by RT-qPCR ( n = 3). n.s, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: The primary antibodies used were rabbit PARP-1 Ab (clone 46D11; Cat #9532), rabbit phospho-histone H2A.X Ab (Ser139) (clone 20E3; Cat #9718), rabbit phospho-S6 ribosomal protein (Ser235/236) Ab (clone D57.2.2E; Cat #4858), rabbit phospho-Akt (Ser473) XP Ab (clone D9E; Cat #4060), mouse AGO2 Ab (clone 2E12-1C9; Cat #57113), and rabbit GAPDH XP mAb (clone D16H11; Cat #5174) (all from Cell Signaling Technology; Danvers, MA).

Techniques: Expressing, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2

doi: 10.15252/embj.2020104719

Figure Lengend Snippet: A Changes in WT and variant FOXL2 mRNA levels in KGN cells were assessed by real‐time RT–PCR after transfecting siRNAs against the indicated factors for 48 h. The data are presented as the mean ± SEM from three independent experiments, performed in triplicate. B FOXL2 protein‐expression levels were determined by Western blotting after transfecting KGN cells with control, DHX9, or GW182 siRNAs for 48 h. Quantification of FOXL2 protein expression is presented in the bottom panel. The data are presented as the mean ± SEM from three independent experiments. The P values were analyzed by unpaired, two‐tailed Student’s t ‐test (** P < 0.01, *** P < 0.001). C Relative binding affinities of DHX9 and GW182 to AGOs. 293T cells were transfected with expression vectors encoding the indicated FLAG/HA‐tagged AGOs, and cell extracts were prepared and immunoprecipitated with an anti‐FLAG antibody, followed by immunoblot analyses (top). The empty p3XFLAG‐CMV‐10 vector was used as a control. The band intensities of immunoprecipitated DHX9 and GW182 were quantified and normalized following pulldown with the indicated AGOs (bottom). The data are presented as the mean ± SEM from three independent experiments. * and # indicate statistically significant differences in the respective amounts of DHX9 or GW182 bound to AGO1. The P values were analyzed by unpaired, two‐tailed Student’s t ‐test ( P < 0.05). D Association of endogenous miR‐1236 with RISC components in KGN cells was determined via pulldown assays using immobilized 2′‐ O ‐methylated oligonucleotides (2′‐ O ‐Me oligos) complementary to miR‐1236 followed with a pulldown using streptavidin‐coupled Dynabeads and Western blot analyses (top). Relative quantification of bound proteins compared with proteins from the input is presented as fold enrichment (bottom). Efficient pulldown of endogenous miR‐1236 using the 2′‐ O ‐Me oligos was confirmed with depleted miR‐1236 in the discarded supernatant following the pulldown (Appendix Fig ). As a control, 2′‐ O ‐Me oligos not complementary to miR‐1236 were used. The data are the means ± SEM from three independent experiments. The P values were analyzed by unpaired, two‐tailed Student’s t ‐test (* P < 0.05, ** P < 0.01). E Following transfection of control siRNA or siDHX9 into KGN cells, AGO3‐mediated RISC‐associated RNAs were immunoprecipitated using an anti‐AGO3 antibody. IgG was used as a control. Co‐immunoprecipitated mRNAs were reverse transcribed using a FOXL2 ‐430‐R primer binding downstream of the 402C>G site. The cDNA products were used for FOXL2 allele‐specific PCR analysis with a FOXL2 ‐279F primer (Appendix Fig ), and a representative result obtained by RT–PCR (top) is shown. Quantitative real‐time RT–PCR results (middle) are also presented as fold enrichment of FOXL2 mRNAs normalized using the level of GAPDH mRNA. Western blots of immunoprecipitated AGO3 and the inputs are shown in the bottom panel. The data are presented as the mean ± SEM from three independent experiments. Different letters denote statistically significant differences ( P < 0.05; Student–Newman–Keuls test). F We examined whether DHX9 affected the association between miR‐1236 and AGOs. After transfecting KGN cells with control siRNA or siDHX9, the total RNA and AGOs‐mediated RISC‐associated RNAs were isolated following immunoprecipitations using anti‐AGO3 or anti‐AGO2 antibodies. The AGOs‐immunoprecipitated RNAs were extracted using an acidic phenol:chloroform mixture (5:1, pH 4.3) and precipitated with isopropanol using 10% of 3 M NaOAc (pH 5.2). The enrichment of miR‐1236 within miRISCs was detected using the TaqMan® microRNA assay in the immunoprecipitated RNAs and normalized using the level of total miR‐1236. The data (means ± SEM) are presented as the fold enrichment calculated from three independent experiments. Different letters denote statistically significant differences ( P < 0.05; Student–Newman–Keuls test). G, H Luciferase activities of the reporter constructs presented in Fig were measured in KGN cells after transfecting the miR‐1236 mimic, indicated siRNAs, and either pGL3c‐CDS‐ FOXL2 MT or pGL3c‐UTR‐ FOXL2 MT for 48 h. The data are expressed as the means ± SEM from three independent experiments and were performed in triplicate. The P values were analyzed by unpaired, two‐tailed Student’s t ‐test (*** P < 0.001). Source data are available online for this figure.

Article Snippet: The following antibodies were used in this study: rabbit anti‐FOXL2 (Park et al , ), rabbit anti‐AGO1 (5053S; Cell Signaling Technology, Danvers, MA, USA), rat anti‐AGO2 (11A9; Helmholtz Zentrum München, Germany), rabbit anti‐AGO3 (5054S; Cell Signaling Technology), rabbit anti‐AGO4 (6913S; Cell Signaling Technology), mouse anti‐FLAG (F1804; Sigma‐Aldrich), rabbit anti‐DHX9 (ab26271; Abcam, Cambridge, MA, USA), rabbit anti‐GW182 (NBP1‐28751; Novus Biologicals, Littleton, CO, USA), mouse anti‐Myc (2276S; Cell Signaling Technology), rabbit anti‐GAPDH (sc‐25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit control‐IgG (sc‐2027; Santa Cruz Biotechnology).

Techniques: Variant Assay, Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test, Binding Assay, Transfection, Immunoprecipitation, Plasmid Preparation, Methylation, Reverse Transcription Polymerase Chain Reaction, Isolation, TaqMan microRNA Assay, Luciferase, Construct