Journal: Frontiers in Genetics

Article Title: Downregulated microRNA-129-5p by Long Non-coding RNA NEAT1 Upregulates PEG3 Expression to Aggravate Non-alcoholic Steatohepatitis

doi: 10.3389/fgene.2020.563265

Figure Lengend Snippet: Successful establishment of the mouse NASH model. (A) Representative images of liver resected from mice with normal diet and 5 weeks of MCD diet. (B) Serum levels of ALT and AST in mice on normal and 5-week MCD diets. (C) Pathological changes of liver tissue in mice on normal and 5-week MCD diets detected by Oil Red O staining (×200) (the upper diagram) and HE staining (×400) (the lower diagram). * p < 0.05 vs. the blank group (mice on normal diet). All data were measurement data and expressed as mean ± standard deviation. Comparison between two groups was analyzed using unpaired t -test. N = 6. NASH, non-alcoholic steatohepatitis; MCD, methionine–choline deficient; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HE, hematoxylin–eosin.

Article Snippet: Six randomly selected mice were fed with normal diet (Harlan Teklad, TD94149) and the remaining 36 mice were fed with MCD diet (Harlan Teklad, TD90262) to establish the NASH model.

Techniques: Staining, Standard Deviation, Comparison

Journal: Frontiers in Genetics

Article Title: Downregulated microRNA-129-5p by Long Non-coding RNA NEAT1 Upregulates PEG3 Expression to Aggravate Non-alcoholic Steatohepatitis

doi: 10.3389/fgene.2020.563265

Figure Lengend Snippet: PEG3 expression is upregulated in liver fibrosis of NASH. (A) Relative expression of PEG3 in liver tissues of mice on normal and 5-week MCD diets. (B) Relative expression of PEG3 in HSCs at different time points of mice on MCD diet from weeks 0 to 6. (C) Relative expression of PEG3 in HSCs at different time points of mice on normal diet from days 0 to 8. * p < 0.05 vs. the blank group (mice on normal diet) or the MCD group (mice on MCD diet) at 0 week or the blank group (mice on normal diet) on day 0. All data were measurement data and expressed as mean ± standard deviation. Comparison between two groups was analyzed using independent sample t -test. One-way analysis of variance was used for data comparison among multiple groups. The experiment was independently repeated three times. N = 6. HSCs, hepatic stellate cells.

Article Snippet: Six randomly selected mice were fed with normal diet (Harlan Teklad, TD94149) and the remaining 36 mice were fed with MCD diet (Harlan Teklad, TD90262) to establish the NASH model.

Techniques: Expressing, Standard Deviation, Comparison

Journal: Frontiers in Genetics

Article Title: Downregulated microRNA-129-5p by Long Non-coding RNA NEAT1 Upregulates PEG3 Expression to Aggravate Non-alcoholic Steatohepatitis

doi: 10.3389/fgene.2020.563265

Figure Lengend Snippet: miR-129-5p negatively regulates the expression of PEG3 in HSCs. (A) Binding sequences between PEG3 and miR-129-5p. (B) Relative luciferase activity of pGL3-PEG3-WT and pGL3-PEG3-Mut. (C) Relative expression of miR-129-5p in liver tissues from mice on 5-week MCD diet ( n = 6). (D) Relative expression of miR-129-5p in HSCs of mice on MCD diet from weeks 0 to 6. (E) Relative expression of miR-129-5p in HSCs of mice on normal diet from days 0 to 8. (F) Relative mRNA expression of PEG3, α-SMA, and Col1A1 after transfection determined by RT-qPCR. (G) Protein bands of α-SMA, Col1A1, and GAPDH after transfection. (H) Relative protein expression of α-SMA and Col1A1 normalized to GAPDH after transfection determined by western blot analysis. (I) HSC proliferation of mice on normal diet detected by EdU assay. * p < 0.05 vs. the blank group (mice on normal diet). # p < 0.05 vs. the pcDNA3-PEG3 + miR-NC group (HSCs transfected with pcDNA3-PEG3 + miR-NC). All data were measurement data and expressed as mean ± standard deviation. Comparison between two groups was analyzed using unpaired t -test. One-way analysis of variance was used for data comparison among multiple groups. The experiment was independently repeated three times. N = 6. Wt, wild-type; Mut, mutant; EdU, 5-ethynyl-2′-deoxyuridine; NC, negative control.

Article Snippet: Six randomly selected mice were fed with normal diet (Harlan Teklad, TD94149) and the remaining 36 mice were fed with MCD diet (Harlan Teklad, TD90262) to establish the NASH model.

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, EdU Assay, Standard Deviation, Comparison, Mutagenesis, Negative Control

Journal: Frontiers in Genetics

Article Title: Downregulated microRNA-129-5p by Long Non-coding RNA NEAT1 Upregulates PEG3 Expression to Aggravate Non-alcoholic Steatohepatitis

doi: 10.3389/fgene.2020.563265

Figure Lengend Snippet: NEAT1 leads to activation of HSCs via the miR-129-5p/PEG3 axis. (A) Relative expression of NEAT1 in liver tissue of mice at 5 weeks post-modeling. (B) Relative expression of NEAT1 in HSCs of mice on MCD diet from weeks 0 to 6; ∗ p < 0.05 vs. mice at 0 week. (C) Relative expression of NEAT1 in primary HSCs of mice on normal diet from days 0 to 8; ∗ p < 0.05 vs. mice on day 0. (D) Relative expression of NEAT1, miR-129-5p, PEG3, α-SMA, and Col1A1 determined by RT-qPCR. (E) Protein bands of PEG3, α-SMA, Col1A1, and GAPDH. (F) Relative protein levels of PEG3, α-SMA, and Col1A1 normalized to GAPDH determined by western blot analysis. In panels (D–F) , * p < 0.05 vs. the blank, shRNA-NC, or vector group (HSCs without any treatment or in the presence of shRNA-NC or vector). (G) Relative expression of α-SMA and Col1A1 in the presence of pcDNA3-NEAT1 determined by RT-qPCR. (H) Protein bands of α-SMA, Col1A1, and GAPDH in the presence of pcDNA3-NEAT1. (I) Relative expression of α-SMA and Col1A1 in the presence of pcDNA3-NEAT1 normalized to GAPDH determined by western blot analysis. In panels (G–I) , * p < 0.05 vs. the pcDNA3-NEAT1 + shRNA-NC group (HSCs in the presence of pcDNA3-NEAT1 + shRNA-NC); # p < 0.05 vs. the pcDNA3-NEAT1 + miR-NC group (HSCs in the presence of pcDNA3-NEAT1 + miR-NC). (J) HSC proliferation of mice on normal diet detected by EdU assay; * p < 0.05 vs. the shRNA-NC or vector group (HSCs in the presence of shRNA-NC or vector); # p < 0.05 vs. the pcDNA3-NEAT1 + shRNA-NC or pcDNA3-NEAT1 + miR-NC group (HSCs in the presence of pcDNA3-NEAT1 + shRNA-NC or pcDNA3-NEAT1 + miR-NC). (K) Quantitative analysis on α-SMA and Col1A1 immunofluorescence intensity in primary HSCs after transfection; * p < 0.05 vs. the blank (HSCs without treatment) or pcDNA3-NEAT1 + shRNA-NC group (HSCs in the presence of pcDNA3-NEAT1 + shRNA-NC); # p < 0.05 vs. the pcDNA3-NEAT1 + miR-NC group (HSCs in the presence of pcDNA3-NEAT1 + miR-NC). All data were measurement data and expressed as mean ± standard deviation. Comparison between two groups was analyzed using unpaired t -test. One-way analysis of variance was used for data comparison among multiple groups. The experiment was independently repeated three times. N = 6 mice per group.

Article Snippet: Six randomly selected mice were fed with normal diet (Harlan Teklad, TD94149) and the remaining 36 mice were fed with MCD diet (Harlan Teklad, TD90262) to establish the NASH model.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, shRNA, Plasmid Preparation, EdU Assay, Immunofluorescence, Transfection, Standard Deviation, Comparison

Journal: Frontiers in Genetics

Article Title: Downregulated microRNA-129-5p by Long Non-coding RNA NEAT1 Upregulates PEG3 Expression to Aggravate Non-alcoholic Steatohepatitis

doi: 10.3389/fgene.2020.563265

Figure Lengend Snippet: NEAT1 expression has a positive correlation with the effects of PEG3 in NASH mice. (A) Relative expression of PEG3, NEAT1, and miR-129-5p determined by RT-qPCR. (B) Pathological changes of liver tissue in mice on normal diet and the 5-week MCD diet detected by Masson staining (×200) (the upper diagram) and HE staining (×200) (the lower diagram). (C) Relative expression of α-SMA and Col1A1 determined by RT-qPCR. (D,E) Relative expression of α-SMA and Col1A1 normalized to GAPDH determined by western blot analysis. * p < 0.05 vs. the blank group (mice on normal diet). # p < 0.05 vs. the MCD group (mice on MCD diet alone). & p < 0.05 vs. the MCD + ad-shNEAT1 group (mice on MCD diet and injected with ad-shNEAT1). All data were measurement data and expressed as mean ± standard deviation. One-way analysis of variance was used for data comparison among multiple groups. The experiment was independently repeated three times. N = 6 mice per group.

Article Snippet: Six randomly selected mice were fed with normal diet (Harlan Teklad, TD94149) and the remaining 36 mice were fed with MCD diet (Harlan Teklad, TD90262) to establish the NASH model.

Techniques: Expressing, Quantitative RT-PCR, Staining, Western Blot, Injection, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Schematic showing intravenous infusion of [trimethyl- H 9 ]choline and [methyl- C 1 ]methionine in mice fed a methionine and choline deficient diet or fed a paired control diet. (b) Rate of appearance of choline calculated from m+9 serum enrichments from 8 h intravenous infusion of [trimethyl- H 9 ]choline. (c) Rate of appearance of methionine calculated from m+1 serum enrichments from 8 h intravenous infusion of [methyl- C 1 ]methionine. (d) Normalized labeling of tissue SAM from 8 hour intravenous infusion of [methyl- C 1 ]methionine. (e) Normalized labeling of tissue SAM from 8 hour intravenous infusion of [trimethyl- H 9 ]choline. The red line represents the fraction of serum methionine that is labeled in control conditions and the green line represents the fraction of serum methionine that is labeled in MCD diet. Labeling below these lines indicates that labeling is potentially sourced from serum methionine pools rather than tissue autonomous production. (f) Normalized labeling of pooled PCs in the liver from 8 hour intravenous infusion of [trimethyl- H 9 ]choline. (g) Ratio of ion counts of pooled PC species to pooled PE species. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two-way ANOVA with Šídák correction for multiple comparisons. All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. PC, phosphatidylcholine; PE, phosphatidylethanoline; PEMT, phosphatidylethanolamine methyltransferase

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Control, Labeling

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Normalized labeling of serum and tissue serine from 8 hour intravenous infusion of [trimethyl- H 9 ]choline.The red line represents the fraction of serum serine that is labeled in control conditions and the green line represents the fraction of serum serine that is labeled in MCD diet. Labeling below these lines indicates that labeling is potentially sourced from serum serine pools rather than tissue autonomous production. (b) Relative ion counts for glycine across tissues in MCD diet and control diet fed mice. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two-way ANOVA with Šídák correction for multiple comparisons (a) or Mixed-effects analysis with Šídák correction for multiple comparisons (b). All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. MCD, methionine and choline deficient

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Labeling, Control

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Schematic of the folate cycle highlighting reactions that support production of 5,10-methylene-THF and SAM. (b) Schematic showing intravenous infusion of [2,3,3- H 3 ]serine and [U- C ]glycine in mice fed a methionine and choline deficient diet or fed a paired control diet. (c) Rate of appearance of serine calculated from m+3 serum enrichments from 8 h intravenous infusion of [2,3,3- H 3 ]serine. (d) Rate of appearance of glycine calculated from m+2 serum enrichments from 8 h intravenous infusion of [U- C 2 ]glycine. (e) Schematic of use of [2,3,3- H 3 ]serine tracing to measure the contribution of the folate cycle to SAM synthesis. (f) Normalized labeling of tissue SAM from 8 hour intravenous infusion of [2,3,3- H 3 ]serine. The red line represents the fraction of serum methionine that is labeled in control conditions and the green line represents the fraction of serum methionine that is labeled in MCD diet. Labeling below these lines indicates that labeling is potentially sourced from serum methionine pools rather than tissue autonomous production. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two tailed T-test (c,d) or two-way ANOVA with Šídák correction for multiple comparisons (f). All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. MCD, methionine and choline deficient; SHMT, serine hydroxymethyltransferase, MTR, 5-methyltetrahyrofolate homocysteine methyltransferase; SAM, s-adenosylmethionine

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Control, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Schematic showing potential sources of circulating serine. (b) Normalized labeled fraction of serum serine from infusion of [U- C 2 ]glycine. (c) Normalized labeled fraction of serum serine from infusion of [U- C 6 ]glucose. (d) Rate of appearance of essential amino acid phenylalanine from intravenous infusion of [ N]phenylalanine. (e) Serine production fluxes from different sources measured using the serine rate of appearance and infusions of U- C 2 ]glycine, U- C 6 ]glucose, and [ N]phenylalanine. (f) Relative ion counts for serine across tissues in MCD diet and control diet fed mice. (g) Schematic of proposed mechanism. When methionine and choline are deficient in the diet, there is increased release of serine from the kidney that supports maintained methionine, PC, and choline synthesis in the liver. Thus, methionine and choline fluxes are maintained by one-carbon metabolism buffering. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two tailed T-test (b), two-way ANOVA with Šídák correction for multiple comparisons (e), or mixed-effects analysis with Šídák correction for multiple comparisons (f). All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. SAM, s-adenosylmethionine; MCD, methionine and choline deficient

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Labeling, Control, Two Tailed Test