Journal: Cells

Article Title: MEG3 Promotes Differentiation of Porcine Satellite Cells by Sponging miR-423-5p to Relieve Inhibiting Effect on SRF

doi: 10.3390/cells9020449

Figure Lengend Snippet: MEG3 inhibits myoblast proliferation. ( A , B ) EdU staining assays after MEG3 knockdown ( A ) or overexpression of the two MEG3 variants ( B ) showing the EdU incorporation in proliferous porcine satellite cells. The S phase of mitosis cells were stained with EdU. The nuclei were stained with DAPI. Scale bar: 50 μm. ( C , D ) CCK-8 cell proliferation assays of porcine satellite cells suggested that MEG3 knockdown significantly promoted myoblast proliferation after transfection with antisense oligonucleotide (ASO)– MEG3 for 24, 36, and 48 h, compared with the negative control groups ( C ), while overexpression of MEG3 v1 or MEG3 v2 inhibited myoblast proliferation ( D ). ( E , G ) Flow cytometry analysis after MEG3 knockdown ( E ) or overexpression of the two MEG3 variants’ overexpression plasmids for 36 h ( G ). ( F , H ) Histogram bar graph summarizing the flow cytometry analysis showing the number of cells in each cell cycle phase. Statistical results showing that MEG3 knockdown significantly increased the percentage of S-phase cells ( F ), while MEG3 overexpression significantly decreased the percentage of S-phase cells ( H ). Error bars represent mean ± SEM of three biological replicates. Statistical significance of differences was assessed by Student’s t -test. * p < 0.05, ** p < 0.01. NC, negative control.

Article Snippet: The miR-423-5p mimic, mimic negative control (NC) and the antisense oligonucleotide (ASO) oligo against MEG3 and scrambled oligo were purchased from RiboBio (Guangzhou, China).

Techniques: Staining, Knockdown, Over Expression, CCK-8 Assay, Transfection, Negative Control, Flow Cytometry

Journal: Cells

Article Title: MEG3 Promotes Differentiation of Porcine Satellite Cells by Sponging miR-423-5p to Relieve Inhibiting Effect on SRF

doi: 10.3390/cells9020449

Figure Lengend Snippet: MEG3 promotes myogenic differentiation of porcine satellite cells. ( A , B ) The real-time PCR ( A ) and Western blot analysis ( B ) showing that MEG3 knockdown significantly decreases expression levels of myogenic marker genes ( MyoD , MyoG , MyHC ) and SRF . ( C , D ) Immunofluorescence staining of MyoG ( C ) and MyHC ( D ) in porcine satellite cells after transfection with ASO– MEG3 or ASO–NC showing that MEG3 knockdown significantly reduces the positively stained cells of MyoG and MyHC . ( E ) The detection of overexpression efficiency of MEG3 v1 and MEG3 v2. ( F – I ) qPCR ( F ), Western blot ( G ), and immunofluorescence staining of MyoG ( H ) and MyHC ( I ) results showing that MEG3 overexpression promotes satellite cell differentiation. Scale bar of ( C , H ): 50 μm. Scale bar of ( D , I ): 100 μm. Porcine satellite cells were harvested after transfection with MEG3 ASO or overexpression plasmid and differentiation for 48 h. The relative mRNA levels were normalized to those of the control 18S rRNA . The relative protein levels were normalized to those of the control β-tubulin . Error bars represent mean ± SEM of three biological replicates. Statistical significance of differences was assessed by Student’s t -test. * p < 0.05, ** p < 0.01. N.S. means that there was no significant difference. NC, negative control. qPCR, quantitative polymerase chain reaction.

Article Snippet: The miR-423-5p mimic, mimic negative control (NC) and the antisense oligonucleotide (ASO) oligo against MEG3 and scrambled oligo were purchased from RiboBio (Guangzhou, China).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Expressing, Marker, Immunofluorescence, Staining, Transfection, Over Expression, Cell Differentiation, Plasmid Preparation, Control, Negative Control

Journal: Cells

Article Title: MEG3 Promotes Differentiation of Porcine Satellite Cells by Sponging miR-423-5p to Relieve Inhibiting Effect on SRF

doi: 10.3390/cells9020449

Figure Lengend Snippet: Effect of MEG3 downregulation on myogenesis transcriptome. ( A ) PCA analysis of four datasets (30 hNC, 30 hASO and 40 hNC, 40 hASO groups) for MEG3 knockdown. ( B ) Hierarchical clustering of differentially expressed genes after transfection of porcine satellite cells with ASO– MEG3 or ASO–NC and differentiation for 40 h. The color-ratio bar at the right indicates intensity of gene upregulation (blue) and downregulation (white). ( C – F ) GO enrichment and KEGG pathway scatterplot analysis for differentially expressed genes for cells differentiated for 30 ( C , E ) and 40 h ( D , F ). ( G , H ) Validation of differentially expressed genes involved in muscle development by qPCR. The y -axis indicates the fold change of RNA-Seq and qPCR. The relative mRNA levels were normalized to those of the control 18S rRNA . Error bars represent mean ± SEM of three biological replicates. Statistical significance of differences was assessed by Student’s t -test. ** p < 0.01. NC, negative control. ASO, antisense oligonucleotide. PCA, Principal component analysis. GO, Gene ontology. KEGG, Kyoto Encyclopedia of Genes and Genomes. qPCR, quantitative polymerase chain reaction.

Article Snippet: The miR-423-5p mimic, mimic negative control (NC) and the antisense oligonucleotide (ASO) oligo against MEG3 and scrambled oligo were purchased from RiboBio (Guangzhou, China).

Techniques: Knockdown, Transfection, Biomarker Discovery, RNA Sequencing, Control, Negative Control, Real-time Polymerase Chain Reaction

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: The list of differentially expressed genes(node degree > 5).

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques:

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A)Representative micro-CT images showing the midshaft architectures of femurs from young and aged mice. N = 3. Scale bar, 1 mm. (B) Bone histomorphometric analysis of BMD and BV/TV in femurs from young and aged mice. N = 3. The mean ± s.e.m. is shown. (C) BMSCs showed a typical cobblestone-like morphology (a). Differentiation potential of BMSCs assessed by Alizarin Red staining (b) and Oil Red O (c). (D) The expression blot of lncRNA Malat1 and targets miRNA. At least three animals in each group were analysed, each experiment was repeated twice, and representative images are shown.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Micro-CT, Staining, Expressing

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A) FISH analysis of Malat1 in BMSCs (The nuclei were stained with DAPI, and 18S rRNA was used as a cytoplasmic marker). (B) Cell nuclear/cytoplasmic fractionation and qRT-PCR showed the cellular districution of lncRNA Malat1 in BMSCs ( U6 and Gapdh were used as separation quality standards and endogenous controls). Three independent experiments were performed for qRT-PCR assays.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Staining, Marker, Fractionation, Quantitative RT-PCR

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A) qRT-PCR analysis was used to detect the expression of lncRNA Malat1 during osteoblast differentiation of BMSCs at days 0,3,7,14 and 21. RNA expression at the indicated time points was normalized to day 0. Gapdh was used as an internal control. (B) qRT-PCR detection shows the expression of osteoblastic marker Runx2 on selected days. Gapdh was used as internal control. (C) qRT-PCR detection shows the expression of osteoblastic marker Ocn on selected days. Gapdh was used as internal control. (D) qRT-PCR detection shows the expression of osteoblastic marker Alp on selected days. Gapdh was used as internal control. Data are presented as means ± SD. Asterisk indicates P < 0.05, double asterisks indicate P < 0.01, triple asterisks indicate P < 0.001, quadruple asterisks indicate P < 0.0001 vs. control. Runx2 , runt-related transcription factor 2; Ocn , osteocalcin; Alp , alkaline phosphatase. Three independent experiments were performed for qRT-PCR assays.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Quantitative RT-PCR, Expressing, RNA Expression, Control, Marker

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A) qRT-PCR detection shows expression of lncRNA Malat1 in BMSCs after transfection with siRNA against lncRNA Malat1 (ASO) or the negative control (NC) vector for 24 h. Gapdh was used as an internal control. Data are presented as means ± SD. (B) qRT-PCR detection shows expression of miR-129-5p in BMSCs after transfection with siRNA against lncRNA Malat1 (ASO) or the negative control (NC) vector for 24 h. U6 was used as an internal control. Data are presented as means ± SD. Relative mRNA expression of Stat1 (C), Alp (D), Runx2 (E), Ocn (F) and Opn (G) was measured by qRT-PCR at day 7 of OM induction. Gapdh was used for normalization. Data are presented as means ± SD. (H) The expression of STAT1 and RUNX2 was detected by Western blot analysis when lncRNA Malat1 was knocked down in BMSCs. (I) Protein expression of STAT1 and RUNX2 in BMSCs transfected with ASO or NC vector. Asterisk indicates P < 0.05, double asterisks indicate P < 0.01 vs. Blank. (J) Images of ALP staining in BMSCs after culture in GM or OM for 7 days. Three independent experiments were performed for all of the blots and qPCR assays, and representative images are shown. (K) Histograms show ALP activity by spectrophotometry. Data are presented as means ± SD.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Plasmid Preparation, Control, Western Blot, Staining, Activity Assay, Spectrophotometry

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A) Immunofluorescence staining was applied to test the effects of siRNA against lncRNA Malat1 (ASO) on the protein levels of RUNX2. (B) Western blot assay was conducted to test protein levels of total, nuclear, cytoplasmic RUNX2 in BMSCs transfected with ASO or NC vector. Three independent experiments were performed, and representative images are shown. (C) Quantitative analysis of protein expression of RUNX2 in BMSCs transfected with ASO or NC vector. Double asterisks indicate P < 0.01, triple asterisks indicate P < 0.001, quadruple asterisks indicate P < 0.0001 vs. blank group. (D) Quantitative analysis of protein expression of RUNX2 in BMSCs transfected with ASO or NC vector. An asterisk (*) indicates P < 0.05 vs. blank group.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Immunofluorescence, Staining, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A) qRT-PCR detection shows expression of miR-129-5p in BMSCs after transfection with miR-129-5p inhibitors or NC for 24 h. Gapdh was used as an internal control. Data are presented as means ± SD. (B) qRT-PCR detection shows expression of Malat1 in BMSCs after transfection with miR-129-5p inhibitors or NC for 24 h. U6 was used as an internal control. Data are presented as means ± SD. Relative mRNA expression of Stat1 (C), Alp (D), Runx2 (E), Ocn (F) and Opn (G) was measured by qRT-PCR at day 7 of OM induction. Gapdh was used for normalization. Data are presented as means ± SD. (H) The expression of STAT1 and RUNX2 was detected by Western blot analysis when miR-129-5p was knocked down in BMSCs. (I) Protein expression of STAT1 and RUNX2 in BMSCs transfected with miR-129-5p inhibitor or NC vector. An asterisk (*) indicates P < 0.05, double asterisks indicate P < 0.01 vs. control. (J) Images of ALP staining in BMSCs after culture in GM or OM for 7 days. Three independent experiments were performed for all of the blots and qPCR assays, and representative images are shown. (K) Histograms show ALP activity by spectrophotometry. Data are presented as means ± SD.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot, Plasmid Preparation, Staining, Activity Assay, Spectrophotometry

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A) Complementary bases between the sequences are labeled with red font. The sequence of the mutant lncRNA Malat1 construct is also shown as underlined. (B) Dual-luciferase reporter assay of 293T cells co-transfected with lncRNA Malat1 -wt, or lncRNA Malat1-mut and with miR-129-5p mimic or NC. (C) MiR-129-5p expression in BMSCs was measured by qRT-PCR following transfection with ASO for 24 h. Data are presented as means ± SD. Two independent experiments were performed.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Labeling, Sequencing, Mutagenesis, Construct, Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR

Journal: PeerJ

Article Title: lncRNA MALAT1 mediates osteogenic differentiation of bone mesenchymal stem cells by sponging miR-129-5p

doi: 10.7717/peerj.13355

Figure Lengend Snippet: (A) RIP assay shows that the miRNAs were successfully pulled-down using an Ago2 antibody. IgG was used as the negative control. (B) RNA levels of lncRNA Malat1 and miR-129-5p after immunoprecipitation were determined by qRT-PCR. Gapdh was the reference gene of lncRNA Malat1 . U6 was used as the reference gene of miR-129-5p. Numbers are mean ± SD. Three independent experiments were performed.

Article Snippet: The miR-129-5p inhibitor/mimic vector and Antisense oligonucleotide (ASO) -MALAT1 were designed and synthesized by Ribobio Co., Ltd.

Techniques: Negative Control, Immunoprecipitation, Quantitative RT-PCR

Journal: Current opinion in neurology

Article Title: Therapeutic strategies for C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1097/WCO.0000000000000984

Figure Lengend Snippet: Expression of sense and antisense expanded C9 transcript RNA and dipeptide repeat (DPR) proteins affect a wide array of downstream cellular pathways including ER stress, nucleocytoplasmic defects, altered stress granule dynamics, autophagy dysfunction, translational defects, proteasome inhibition, RAN translation, nucleolar stress, RNA foci formation. Different therapeutic approaches (green boxes) target the C9 RNAs and RAN DPRs directly as well as the downstream pathways. Additionally, external factors, such as patient lifestyle, stress, comorbid diseases and environmental factors (bottom red bar) can influence cellular events with exercise and lifestyle changes offering mixed therapeutic potential. Some content modified from Servier Medical Art ( smart.servier.com ) under creative common license.

Article Snippet: While there are multiple therapeutic approaches for C9-ALS/FTD in the pre-clinical pipeline, there are only two C9orf72-ALS/FTD specific clinical trials registered in ClinicalTrials.gov : (1) an antisense oligonucleotide (ASO)-based clinical trial of BIIB078 (Biogen), which targets C9orf72 expansion containing transcripts for ASO-induced RNase H-mediated degradation ( NCT03626012 ) and; (2) a clinical trial ( NCT04220021 ), to test the safety and tolerability of metformin in C9orf72 ALS patients and the drug’s ability to decrease RAN protein levels.

Techniques: Expressing, Inhibition, Modification

Journal: bioRxiv

Article Title: Repeat associated non-AUG translation as a common mechanism for the polyGln ataxias

doi: 10.1101/2025.10.14.682372

Figure Lengend Snippet: (A) Schematic diagram depicting polyGln encoding genes and their corresponding repeat expansion mutations in the polyGln-SCAs (left) and antibodies designed to test if sense-encoded polySer and antisense-encoded polyLeu RAN proteins are expressed across these polyGln encoding CAG expansion mutations. (B-C) Constructs used to express Flag-polySer and Flag-polyLeu expansion proteins. (D-E) Representative IF images showing co-localization of signal from newly developed α -polySer or α -polyLeu repeat antibodies (green) and α-Flag (red) in transfected HEK293T cells (upper panels). No polySer or polyLeu signal was detected in cells transfected with empty vector (center) or pre-immune serum (lower panels). (F) Our newly developed α-polySer antibody detects nuclear staining (b) in representative images from white matter striatal bundles similar to staining using a previously validated HD-Ser-Ct antibody (d) in HD but not unaffected controls (a,c) or preimmune serum (e) (G) Similarly, IHC using newly developed α-polyLeu and previously validated HD-Leu-Ct antibodies show similar representative staining in HD (g,i) but not control caudate (f,h) or HD tissue stained with pre-immune sera (j). Red, positive staining; purple, counterstain; WMB, white matter bundles.

Article Snippet: While antisense oligonucleotide (ASO) strategies for SMA – and SOD1 – ALS have been tremendously successful, unfortunately, ASO clinical trials for several repeat expansion diseases including HD and C9orf72 ALS/FTD ( https://investors.biogen.com/news-releases/news-release-details/biogen-and-ionis-announce-topline-phase-1-study-results ) have failed.

Techniques: Construct, Transfection, Plasmid Preparation, Staining, Control

Journal: Nucleic Acid Therapeutics

Article Title: Integrated Assessment of Phase 2 Data on GalNAc 3 -Conjugated 2′- O -Methoxyethyl-Modified Antisense Oligonucleotides

doi: 10.1089/nat.2022.0044

Figure Lengend Snippet: Ligand-conjugated antisense technology. All ASOs are conjugated to the same GalNAc 3 cluster and linker at the 5′ terminal 2′MOE-modified nucleotide by a phosphodiester group that hydrolyzes after receptor-mediated cellular uptake to release the unconjugated ASO [ , ]. 2′MOE, 2′- O -methoxyethyl; ASOs, antisense oligonucleotides; GalNAc 3 , triantennary N -acetylgalactosamine.

Article Snippet: Receptor-mediated delivery of an antisense oligonucleotide (ASO) using the ligand-conjugated antisense technology is establishing a new benchmark for antisense therapeutics.

Techniques: Modification