Long non-coding RNA GRASLND enhances chondrogenesis via suppression of the interferon type II signaling pathway
- Department of Orthopaedic Surgery, Washington University, United States
- Shriners Hospitals for Children, United States
- Department of Cell Biology, Duke University, United States
- Center of Regenerative Medicine, Washington University, United States
- Department of Biomedical Engineering, Vanderbilt University, United States
Figures
Figure 1 with 2 supplements
GRASLND is important and specifically upregulated in MSC chondrogenesis.
(A) Expression pattern of GRASLND in chondrogenesis (GSE109503; Huynh et al., 2018b). Log2TPM: log transformed value of transcripts per million (TPM). (B) Effect of GRASLND knockdown on …
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Figure 1—source data 1
shRNA target sequences (5′ – sequence – 3′).
- https://cdn.elifesciences.org/articles/49558/elife-49558-fig1-data1-v2.xlsx
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Figure 1—source data 2
qRT-PCR sequencing primers (5′ – sequence – 3′).
- https://cdn.elifesciences.org/articles/49558/elife-49558-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Identification of lncRNA candidates.
LncRNAs whose expression patterns are correlated to crucial markers are of interest. (A) Expression patterns of previously identified MSC markers (left), chondrogenic markers (middle), and SOX …
Figure 1—figure supplement 2
Functional validation of identified lncRNA candidates.
Left: expression pattern of candidates during chondrogenesis (GSE109503). Log2TPM, log-transformed value of transcripts per million (TPM). Middle: efficiency of designed target shRNAs. Individual …
Figure 2
GRASLND is localized to the cytoplasm.
(A) RNA in situ hybridization of MSC-derived pellets at different time points during chondrogenesis. GAPDH and GRASLND probes were hybridized on separate slides. Top three panels, scale bar = 20 µm; …
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Figure 2—source data 1
Cloning primer sequences (5′ – sequence – 3′).
- https://cdn.elifesciences.org/articles/49558/elife-49558-fig2-data1-v2.xlsx
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Figure 2—source data 2
GRASLND probe set sequences (5′ – sequence – 3′).
- https://cdn.elifesciences.org/articles/49558/elife-49558-fig2-data2-v2.xlsx
Figure 3 with 1 supplement
GRASLND relationship to RNF144A and SOX9.
(A) GRASLND genomic location and conservation across different species. Data were retrieved from UCSC Genome Browser. (B) Knockdown of GRASLND and expression of RNF144A (n = 4). (C) Overexpression GR…
Figure 3—figure supplement 1
Full bands of western blot from Figure 3D.
Lanes indicate biological replicates.
Figure 4 with 2 supplements
GRASLND enhances chondrogenesis.
(A) Experimental timeline. (B, C) Biochemical analyses of day 21 MSC pellets (n = 4). Welch’s t-test. (D, E) qRT-PCR analyses of day 21 MSC pellets (n = 5 in panel [D]; n = 6 in panel [E]). Welch’s …
Figure 4—figure supplement 1
Effect of GRASLND overexpression across time points and tested doses.
(A) Overview of designed lentiviral backbone. GRASLND is driven under a doxycycline-inducible promoter, and poly-adenylated with BGHpA signal. (B, C) Relative expression of GRASLND under different …
Figure 4—figure supplement 2
Synthetic guide RNA screening for efficient activation of endogenous GRASLND.
Dunnett’s test compared to ‘No Guide’ control. ns, not significant. **, p<0.01; ***, p<0.001.
Figure 5 with 2 supplements
GRASLND suppresses interferon type II signaling.
(A) Top 20 up- and down-regulated genes in GRASLND KD pellets compared to scrambled controls. (B) Gene ontology analysis of affected pathways. (C) Upregulated targets and related gene ontology terms …
Figure 5—figure supplement 1
Relationship between GRASLND and IFN.
(A) (B) IFN signal was upregulated in OA patients. Samples are indicated as disease state followed by patients’ age (GSE57218). (C) IFN was upregulated while GRASLND was downregulated in damaged …
Figure 5—figure supplement 2
Full bands of RNA pull-down followed by western blot.
Figure 6
GRASLND enhances chondrogenesis in adipose-derived stem cells.
(A) Biochemical analyses (n = 5). (B) qRT-PCR analyses (n = 6). (C) Representative histological images of day 21 ASC pellets. COLII IHC, Collagen type II immunohistochemistry; hOC, Human …
Author response image 1
No: Without IL-1.
Yes: with IL-1. n=4 Early: P0 and P1. Late: P4 and P5. n=5.
Tables
Table 1
Long non-coding RNA candidates shortlist.
| Gene symbol | Gene name | ENSEMBL gene ID | Relationship to MSC chondrogenesis | Relationship to SOX9 |
|---|---|---|---|---|
| LOXL1-AS1 | LOXL1 antisense RNA 1 | ENSG00000261801 | Correlated with MSC marker expression | Downregulated upon SOX9 overexpression |
| MIR4435-2HG gene synonym: MIR4435-1HG | MIR4435-2 host gene | ENSG00000172965 | Correlated with MSC marker expression | Downregulated upon SOX9 overexpression |
| HMGA2-AS1 gene synonym: RP11-366L20.2 | HMGA2 antisense RNA 1 | ENSG00000197301 | Correlated with MSC marker expression | Upregulated upon SOX9 overexpression |
| RNF144A-AS1 Referred to as GRASLND in this manuscript | RNF144A antisense RNA 1 | ENSG00000228203 | Correlated with chondrogenic marker expression | Upregulated upon SOX9 overexpression |
Table 2
Top 5 enriched Cis-BP motifs and associated transcription factors for upregulated genes upon GRASLND knockdown.
| Transcription factor | Cis-BP motif* | Number of genes with enriched motifs/number of upregulated genes |
|---|---|---|
| STAT2 |  | 212/817 |
| IRF2 |  | 189/817 |
| IRF1 |  | 220/817 |
| IRF1 |  | 153/817 |
| STAT1 |  | 262/817 |
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*Cis-BP: Catalogue of Inferred Sequence Preferences of DNA-Binding Proteins (Weirauch et al., 2014). Curated position weight matrices were retrieved from http://motifcollections.aertslab.org.
Additional files
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Supplementary file 1
RNA-seq databases on MSC chondrogenesis (GSE109503) (Huynh et al., 2018b) and SOX9 overexpression in ASCs (GSE69110) (Ohba et al., 2015).
- https://cdn.elifesciences.org/articles/49558/elife-49558-supp1-v2.xlsx
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Supplementary file 2
Differentially expressed genes with GRASLND knockdown as compared to scrambled control.
Data is expressed as log2FoldChange in KD compared to scrambled control (a positive log2FC means that the gene is upregulated in KD samples. A negative log2FC means that the gene is downregulated in KD samples).
- https://cdn.elifesciences.org/articles/49558/elife-49558-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/49558/elife-49558-transrepform-v2.docx
