Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: High expression of MEG3 in SCLC tissues and cell lines. (A) The expression of MEG3 in SCLC tissues (n=48) and normal tissues (n=48) was detected by qRT-PCR. *** p <0.001 vs. the normal tissues. (B) The expression of MEG3 in SCLC cells (NCI-H69 and NCI-H446) and normal bronchial epithelial cells (16HBE) was detected by qRT-PCR. *** p <0.001 vs.16HBE cells. SCLC, small-cell lung cancer.

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: Correlations between MEG3 Expression and Clinicopathological Characteristics in SCLC

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Expressing

Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: MEG3 silencing inhibits cell viability and metastasis in SCLC. (A) The expression of MEG3 after transfection of sh-MEG3/NC or pcDNA3.1-MEG3/NC into SCLC cells (NCI-H69 and NCI-H446) was detected by qRT-PCR. *** p <0.001 vs. the pcDNA3.1-NC group, ### p <0.001 vs. the sh-NC group. (B) The viability of SCLC cells transfected with sh-MEG3/NC was measured by MTT assay. (C) The migration of SCLC cells transfected with sh-MEG3/NC was measured by transwell migration assay. (D) The invasion of SCLC cells transfected with sh-MEG3/NC was measured by transwell invasion assay. * p <0.05, ** p <0.01 vs. the sh-NC group. SCLC, small-cell lung cancer.

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, MTT Assay, Migration, Transwell Migration Assay, Transwell Invasion Assay

Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: Overexpression of MEG3 enhances DDP chemoresistance in SCLC. (A) The viability of SCLC cells transfected with pcDNA3.1-MEG3/NC under different concentrations of DDP (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 µM) was measured by MTT assay. (B) Image of a solid tumor after injection of pcDNA3.1-MEG3/NC. (C) Tumor volume after injection of pcDNA3.1-MEG3/NC. (D) The expression of MEG3 in tumor xenograft tissues injected with pcDNA3.1-MEG3/NC was detected by qRT-PCR. * p <0.05, ** p <0.01 vs. the pcDNA3.1-NC group. SCLC, small-cell lung cancer.

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Over Expression, Transfection, MTT Assay, Injection, Expressing, Quantitative RT-PCR

Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: CAF facilitates metastasis and DDP chemoresistance among SCLC cells. (A) The proteins levels of FAP, FSP1, and α-SMA in CAFs or NFs were determined by Western blot assay. * p <0.05 vs. the NF group. (B) The expression of MEG3 in CAFs or NFs. *** p <0.001 vs. the NF group. (C) The migration of SCLC cells grown in CAF-CM or NF-CM was measured by transwell migration assay. (D) The invasion of SCLC cells grown in CAF-CM or NF-CM was measured by transwell invasion assay. (E) The viability of SCLC cells grown in CAF-CM or NF-CM under different concentrations of DDP (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 µM) was measured by MTT assay. * p <0.05, ** p <0.01, *** p <0.001 vs. the NF-CM group. SCLC, small-cell lung cancer; NF, normal fibroblasts; CAF, cancer-associated fibroblasts; CM, conditioned medium.

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Western Blot, Expressing, Migration, Transwell Migration Assay, Transwell Invasion Assay, MTT Assay

Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: Overexpression of MEG3 is found in CAF-derived exosomes. (A) The expression of MEG3 in SCLC cells under different CM was detected by qRT-PCR. *** p <0.001 vs. the NF-CM group, ## p <0.01 vs. the CAF-CM group. (B) The morphological characteristics of exosomes, which were round membranous vesicle, were observed by transmission electron microscopy. Scale bar: 50 nm. (C) The levels of exosomes surface markers (CD63, HSP70, and GM130) were detected by Western blot assay. * p <0.05 vs. the whole cells group. (D) The expression of MEG3 in CAF-Exo or NF-Exo was detected by qRT-PCR. *** p <0.001 vs. the NF-Exo group. (E) Uptake of CAF-derived exosomes was observed under a confocal microscope. (F) The expression of MEG3 in SCLC cells co-cultured with exosomes from CAFs or NFs was detected by qRT-PCR. *** p <0.001 vs. the NF-Exo+SCLC cells (NCI-H69 and NCI-H446) group. SCLC, small-cell lung cancer; NF, normal fibroblasts; CAF, cancer-associated fibroblasts; CM, conditioned medium.

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Over Expression, Derivative Assay, Expressing, Quantitative RT-PCR, Transmission Assay, Electron Microscopy, Western Blot, Microscopy, Cell Culture

Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: Identification of a miR-15a-5p/CCNE1 axis as a downstream target of MEG3 in SCLC. (A) The predicted complementary binding sites between miR-15a-5p and MEG3/CCNE1. (B) The luciferase activity in NCI-H446 cells co-transfected with pGL3-MEG3 wt/pGL3-MEG3 mut and miR-15a-5p mimics/mimics NC or pGL3-CCNE1 wt/pGL3-CCNE1 mut and miR-15a-5p mimics/mimics NC was determined by dual luciferase reporter assay. ** p <0.01 vs. the mimics NC group. (C) The expression of miR-15a-5p after transfection of miR-15a-5p mimics/mimics NC or miR-15a-5p inhibitor/inhibitor NC into NCI-H446 cells was detected by qRT-PCR. ** p <0.01 vs. the mimics NC group, ## p <0.01 vs. the inhibitor NC group. (D) The expression of miR-15a-5p in NCI-H446 cells transfected with sh-MEG3/NC was detected by qRT-PCR. ** p <0.01 vs. the sh-NC group. (E) The protein levels of CCNE1 in NCI-H446 cells transfected with miR-15a-5p mimics/mimics NC was determined by Western blot assay. ** p <0.01 vs. the mimics NC group. (F) The expression of miR-15a-5p in SCLC tissues (n=48) and normal tissues (n=48) was detected by qRT-PCR. *** p <0.001 vs. the normal tissues. (G) The expression of CCNE1 in SCLC tissues (n=48) and normal tissues (n=48) was detected by qRT-PCR. *** p <0.001 vs. the normal tissues. SCLC, small-cell lung cancer.

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Yonsei Medical Journal

Article Title: MEG3 LncRNA from Exosomes Released from Cancer-Associated Fibroblasts Enhances Cisplatin Chemoresistance in SCLC via a MiR-15a-5p/CCNE1 Axis

doi: 10.3349/ymj.2022.63.3.229

Figure Lengend Snippet: Overexpression of MEG3 promotes DDP chemoresistance and metastasis in SCLC via the miR-15a-5p/CCNE1 axis. (A) The expression of CCNE1 in NCI-H446 cells after transfection with sh-CCNE1/NC was detected by qRT-PCR. ** p <0.01 vs. the sh-NC group. (B) The expression of CCNE1 in NCI-H446 cells after transfection with pcDNA3.1-MEG3 or pcDNA3.1-MEG3+miR-15a-5p mimics was detected by qRT-PCR. ** p <0.01 vs. the pcDNA3.1-NC group, ## p <0.01 vs. the pcDNA3.1-MEG3 group. (C) The viability of NCI-H446 cells transfected with pcDNA3.1-NC, pcDNA3.1-MEG3, pcDNA3.1-MEG3+miR-15a-5p mimics, or pcDNA3.1-MEG3+sh-CCNE1 under different concentrations of DDP (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 µM) was measured by MTT assay. (D) The migration of NCI-H446 cells transfected with pcDNA3.1-NC, pcDNA3.1-MEG3, pcDNA3.1-MEG3+miR-15a-5p mimics, or pcDNA3.1-MEG3+sh-CCNE1 was measured by transwell migration assay. (E) The invasion of NCI-H446 cells transfected with pcDNA3.1-NC, pcDNA3.1-MEG3, pcDNA3.1-MEG3+miR-15a-5p mimics, or pcDNA3.1-MEG3+sh-CCNE1 was measured by transwell invasion assay. * p <0.05, ** p <0.01 vs. the pcDNA3.1-NC group, # p <0.05 vs. the pcDNA3.1-MEG3 group. SCLC, small-cell lung cancer.

Article Snippet: Small hairpin RNA targeting MEG3 (sh-MEG3), sh-CCNE1, and the corresponding negative control (sh-NC), vector overexpressing MEG3 (pcDNA3.1-MEG3), empty vector (pcDNA3.1-NC), miR-15a-5p inhibitor/inhibitor negative control, and miR-15a-5p mimics/mimics negative control were all purchased from VectorBuilder (Guangzhou, China).

Techniques: Over Expression, Expressing, Transfection, Quantitative RT-PCR, MTT Assay, Migration, Transwell Migration Assay, Transwell Invasion Assay

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A and B ) MA plots demonstrate that Meg3 , Rian , and Mirg are postmitotic (day 7 MNs vs. day 4 pMNs; A ) MN signature (day 7 MNs vs. day 7 INs; B ) lncRNAs. X -axis: mean abundance; Y -axis: log 2 fold-change. ( C ) Illustration of the imprinted Dlk1-Dio3 locus. The lncRNAs of the maternally-inherited allele (in red) are on mouse chromosome 12. The miRNA and C/D snoRNA genes are shown by hairpin loops and triangles, respectively. ( D ) Comparison of ChIP-seq for MN transcription factors (Lhx3 and Isl1), H3K4me3, and H3K27ac, together with ATAC-seq of the Dlk1-Dio3 locus. ( E ) In situ hybridization shows that Meg3 is gradually enriched and restricted in postmitotic MNs in the developing spinal cord. ( F ) In situ hybridization together with adjacent sections of immunostaining on E12.5 segmental spinal cords demonstrate that Meg3 is enriched at brachial and thoracic MNs (Isl1/2 on ), but no preference for columnar MN subtypes was revealed for Foxp1 or Lhx3. ( G and H ) RAR binding and recruitment of the elongating form of Pol2 to the Meg3 transcription start site (TSS) occur within 8 hr of retinoic acid (RA) exposure.

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Comparison, ChIP-sequencing, In Situ Hybridization, Immunostaining, Binding Assay

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A ) Illustration of the imprinted Dlk1-Dio3 locus. The lncRNAs on the maternally-inherited allele (in pink) and protein-coding genes from the paternally-inherited allele (in blue) are depicted. miRNA genes are shown by hairpin loops. Arrows indicate the transcription directions. The IG-DMR site is hypomethylated (open circles) on the maternally-inherited allele, but hypermethylated (filled circles) on the paternally-inherited allele. Lower panel shows the qPCR analysis of Meg3 expression in ESCs, NEs, pMNs, MNs and INs (n = 3 independent experiments; FC: fold-change). ( B and C ) qPCR reveals that MN transcription factors ( Neurog2-Isl1-Lhx3 ), as long as adding RA, can induce Meg3 expression in the IG-DMR matΔ ESCs (FC: fold-change; error bars represent SD, n = 3, * p-value<0.05, ** p-value<0.01 by Student’s t -test). ( D ) Panels show single molecule RNA FISH of Meg3 in Hb9::GFP ESC~MNs. ( E ) Subcellular fractionation of ESC~MNs revealed that Meg3 is not only enriched in the nuclei, but also highly chromatin-associated. Two independent Meg3 primers were used. Gapdh , Rnu1 , and Kcnq1ot1 were used to reflect the purity of cytoplasmic, nuclear, and chromatin-associated fractions, respectively.

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Expressing, Fractionation

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A ) Upper panel: Time-series expression of the PRC2 subunits ( Eed , Suz12 , Jarid2 , and Ezh2 ) during ESC~MN differentiation. The levels of PRC2 complex progressively decreased during differentiation, whereas Jarid2 is reactivated in day 7 postmitotic MNs. Lower panel: The abundances of the known lncRNAs that interact with PRC2 in MNs, as revealed by RNA-seq. ( B ) RNA-seq analysis of ESC~MNs. Reads from ESCs, RA-induced nascent neural epithelium (NE at day 2), MN progenitors (pMN at day 4), postmitotic interneurons (IN at day 7), and postmitotic MNs (MN at day 7). Reads are normalized to the total number of mappable reads per sample. RNA-seq revealed that Meg3 v1 (blue boxes) and Meg3 v5 (pink boxes) are the most abundant Meg3 isoforms in postmitotic MNs (GENCODE version M9). ( C ) Radar chart reveals that day 7 postmitotic MNs have the highest distribution of Meg3 v5 during ESC~MN differentiation. ( D ) Histogram plot indicates that the Meg3 v1 and Meg3 v5 isoforms account for more than 99% of Meg3 transcripts during ESC~MN differentiation. ( E ) Schematic diagram of RT-PCR primer locations within the Meg3 v1 and Meg3 v5 regions. Expression of Meg3 according to different primer combinations for Meg3 isoforms suggests that Meg3 v1 and Meg3 v5 are independent transcripts. Gapdh as a loading control. ( F ) Western blot shows that the loss of Meg3 imprinted lncRNAs does not affect the protein abundance of Ezh2 and Jarid2 in ESC~MNs.

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Quantitative Proteomics

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A ) Ezh2, Jarid2, and Suz12 immunoprecipitation specifically retrieves Meg3 RNA isoforms ( v1 and v5 ). Rnu1 RNA and Malat1 lncRNA are negative controls. 10% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments). Immunoblotting reflects the recovery of Ezh2, Jarid2 and Suz12 proteins using the corresponding antibodies. ( B ) In vitro-transcribed, biotinylated Meg3 RNA isoforms retrieved Ezh2. ( C ) Ezh2 interacts with Jarid2 in ESC~MNs, but knockdown of Meg3 impairs this interaction. The abundance of Jarid2 is shown on the right (N.S.: not significant; error bars represent SD, n = 3 independent experiments; ** p-value<0.01 by Student’s t -test). ( D and E ) The design of inducible ‘Tet-On’ ESC lines expressing Meg3 v1 or Meg3 5 under the doxycycline (Dox)-regulated promoter. In the presence of Dox, the reverse tetracycline-controlled transactivator (rtTA) is recruited to the TRE (tetracycline response element), thereby initiating robust transcription of Meg3 v1 or Meg3 v5 , respectively. ( F ) Overexpression of Meg3 v1 or Meg3 v5 does not alter the protein levels of Ezh2 or Jarid2. ( G ) Meg3 v5 but not Meg3 v1 stimulates more Ezh2 and Jarid2 interaction. The abundances of Jarid2 are shown on the right (FC: fold-change; N.S.: not significant; error bars represent SD, n = 3 independent experiments; * p-value<0.05, ** p-value<0.01 by Student’s t -test).

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Immunoprecipitation, Western Blot, In Vitro, Knockdown, Expressing, Over Expression

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A ) Genome-wide profiling of H3K27me3 by ChIP-seq and gene expression by Agilent microarray in control, IG-DMR matΔ , and Meg3 KD ESC~MNs. Venn diagram shows the number of genes that are up-regulated in both IG-DMR matΔ and Meg3 KD MNs while also displaying the reduction of H3K27me3 epigenetic landscape. Loss of Meg3 imprinted lncRNAs is related to the up-regulation of progenitor genes, as well as Hox genes. ( B and C ) Heatmaps illustrating the expression profiles of ESC~MNs in control scrambled and Meg3 KD lines. The color indicates the log 2 fold-change in signal intensity between ESCs and ESC~MNs. Genes in light grey are not represented in the microarrays. Loss of Meg3 imprinted lncRNAs results in ectopic expression of progenitor genes in ( B ), and a majority of caudal Hox genes ( Hox8~13 ) in ( C ), with concomitant down-regulation of H3K27me3 levels.

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Genome Wide, ChIP-sequencing, Gene Expression, Microarray, Control, Expressing

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A ) Expression of paternally-expressed protein coding genes ( Dlk1 and Dio3 ) and maternally-expressed lncRNAs ( Meg3 , Rian, and Mirg ) within the imprinted Dlk1-Dio3 locus in ESC~MNs, as revealed by qPCR (FC: fold-change; N.S.: not significant; error bars represent SD, n = 3 independent experiments; * p-value<0.05, ** p-value<0.01 by Student’s t -test). ( B ) The correlation plots and associated R 2 values are indicated by comparing H3K27me3 of MN samples from two independent controls and Meg3 KD lines. ( C ) Left panel: The correlation plots and associated R 2 values indicate that the two Meg3 KD lines infected by independent shRNAs are almost identical. Middle and right panels: MA plot of control versus Meg3 KD pMNs (middle) and MNs (right). At the postmitotic stage (day 7), Meg3 KD leads to up-regulation of the neural progenitor genes Pax6 , Dbx1 , Neurog2, Irx3 and caudal Hox genes ( Hox8~13 ), as well as down-regulation of rostral Hox genes ( Hox1~5 ). Two biological replicate experiments were used to generate the data. The generic MN marker Hb9 is unaffected, whereas Meg3 and Mirg are the most down-regulated genes after Meg3 KD. ( D ) Dysregulated genes are grouped by gene ontology (GO). ( E ) Loss of Meg3 leads to ectopic expression of most caudal Hox genes ( Hox8~13 ), with concomitant down-regulation of rostral Hox genes ( Hox1~5 ), as verified by qPCR. A third shRNA (I6) was further assessed and Hox expression was normalized against Gapdh expression levels (FC: fold-change; error bars represent SD, n = 3; * p-value<0.05, ** p-value<0.01 by Student’s t -test).

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Expressing, Infection, Control, Marker, shRNA

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A and B ) Concordant enrichments of Ezh2 and H3K27me3 in progenitor genes ( A ) and caudal Hox genes ( B ).(C~E) ChIP-qPCR verifies compromised H3K27me3 occupancy of Ezh2 and Jarid2 for the Pax6 , Irx3 , and Hoxc8 loci upon Meg3 KD. 1% input was used to normalize the retrieval efficiency (error bars represent SD, n = 3 independent experiments; * p-value<0.05, ** p-value<0.01 by Student’s t -test).

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: ChIP-qPCR

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A ) Meg3 is absent from the spinal cords of IG-DMR matΔ embryos at E14.5, as revealed by in situ hybridization. ( B and C ) Immunostaining analysis of MN progenitors Nkx2.2 and Olig2 shows comparable numbers in wild type control and IG-DMR matΔ spinal cord sections at E10.5. Quantification of MN progenitors (number of positive cells per 15 μm brachial spinal cord ventral-half sections) in wild type control and IG-DMR matΔ embryos revealed no significant differences amongst different segments from the spinal cord (error bars represent SD, n = 3 embryos at E10.5; N.S.: not significant) ( D and E ) Immunostaining analysis of segmental MNs (Hoxc6 on at brachial, Hoxc9 on at thoracic, and Hoxd10 on at lumbar regions) shows comparable MN numbers for E12.5 wild type control and IG-DMR matΔ spinal cord sections. Quantification of segmental MNs (number of positive cells per 15 μm brachial spinal cord ventral-half sections) in wild type control and IG-DMR matΔ embryos revealed no significant differences amongst different segments from the spinal cord (error bars represent SD, n = 3 embryos at E12.5; N.S.: not significant). ( F and G ) Columnar axial MNs (Lhx3 on ) and limb-innervating MNs (Foxp1 on ) are unaffected in the IG-DMR matΔ embryos. Quantification of columnar MNs (number of positive cells per 15 μm brachial spinal cord ventral-half sections) in wild type control and IG-DMR matΔ embryos revealed no significant differences amongst different segments from the spinal cord (error bars represent SD, n = 3 embryos at E11.5; N.S.: not significant).

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: In Situ Hybridization, Immunostaining, Control

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet: ( A ) Illustration of the sgRNAs target sites and sequences for the sgRNA:Cas9-mediated Rian and Mirg deletions respectively. Deleted sequences shown in Rian Δ/Δ and Mirg Δ/Δ ESC lines. The PAM sites are displayed in green; the sgRNA target sequences are reflected in red. ( B ) Heatmaps representing the abundances of the paternal gene ( Dlk1 ) and maternal lncRNAs ( Meg3 , Rian , and Mirg ) in control, Meg3 KD, and KOs ( IG-DMR matΔ , Rian Δ/Δ , and Mirg Δ/Δ ) ESCs respectively. ( C ) Rostral Hox gene Hoxa5 is significantly down-regulated in the Meg3 KD line, whereas the expression of Hoxa5 is comparable between Rian Δ/Δ and Mirg Δ/Δ when compared to the controls. Conversely, the expressions of progenitor gene Pax6 , as well as caudal Hox gene Hoxc8 , are significantly up-regulated in the Meg3 KD line, while their expressions are restored between Rian Δ/Δ and Mirg Δ/Δ when compared to the controls. (FC: fold-change; error bars represent SD, n = 3~5, * p-value<0.05, ** p-value<0.01 by Student’s t -test). ( D ) Summary of the functions of lncRNAs from the imprinted Dlk1-Dio3 locus in ESC~MNs. Meg3 and other lncRNAs from the Dlk1-Dio3 locus are directly activated by MN-TFs (i.e., Lhx3 and Isl1) and RAR, leading to enrichment of Meg3 in the rostral segment of postmitotic MNs. One major function of Meg3 and other lncRNAs from the Dlk1-Dio3 locus is to stimulate Ezh2/Jarid2 interactions. Loss of these lncRNAs compromises the H3K27me3 epigenetic landscape and leads to aberrant expressions of progenitor and caudal Hox genes in postmitotic MNs. Our model illustrates that the lncRNAs of the imprinted Dlk1-Dio3 locus ( Meg3 particularly) play a critical role in maintaining postmitotic MN cell fate by repressing progenitor genes, and that they shape MN subtype identity by regulating Hox genes.

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Control, Expressing

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet:

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Labeling, Immunoprecipitation, Software

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet:

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: In Situ Hybridization

Journal: eLife

Article Title: Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity

doi: 10.7554/eLife.38080

Figure Lengend Snippet:

Article Snippet: The Meg3 HuSH-29 shRNA plasmids (Origene, cat. No. TG501330) and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs.

Techniques: Quantitative RT-PCR