Keratinocyte-specific long non-coding RNA NR037661 controls alternative splicing of ANGPTL4 to induce keratinocyte differentiation

Figures

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  Fig. 1. Induction of NR037661 Expression During Epidermal Differentiation. Primary human keratinocytes were isolated as detailed in the Materials and Methods section and plated onto dishes. Prior to reaching confluence, these cells were induced to differentiate by supplementing the culture medium with 1.8 mM CaCl2. Cells were collected at specified time points for Western blot analysis. (A) The differentiation process was assessed via Western blot using involucrin, KRT10, and TG1 as markers. GAPDH served as a loading control. The presented plot is representative of experiments conducted in triplicate. The histograms depict the quantitative mRNA levels of involucrin, KRT10, and TG1 (fold change over control), presented as the mean ± SD from three independent experiments. (B) A mean-centered, hierarchical clustering analysis was performed on annotated non-coding RNAs that exhibited alterations (greater than twofold change) in undifferentiated cells (day 0) and during various days of calcium-induced differentiation in vitro. (C) Relative quantification of NR037661 expression was conducted during calcium-induced in vitro differentiation of human epidermal keratinocytes (HEKn) at 1, 3, and 7…

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  Fig. 2. Impact ofNR037661on epidermal differentiation and role of SRRM2 in NR037661-mediated keratinocyte differentiation. (A) Keratinocytes were infected with adenoviral NR037661-specific shRNA or a non-targeting shRNA control (shRNA-NC). Twenty-four hours post-infection, the cells were treated with 1.8 mM CaCl2 for 72 h. The levels of NR037661 were analyzed using qRT-PCR. The data presented are the mean ± S.D. of three independent experiments. (B) Inhibition of keratinocyte differentiation by NR037661 knockdown in response to calcium stimulation. Keratinocytes were treated as outlined in Figure (A). The levels of involucrin, a marker of keratinocyte differentiation, were analyzed using immunoblotting (n = 3). (C) Silver-Stained SDS-PAGE gel analysis of proteins immunoprecipitated from keratinocyte cell extracts using NR037661 and its Antisense RNA. The arrow indicates the region of the gel that was excised for mass spectrometry determination using the liquid chromatography-dual mass spectrometry (LC-MS/MS) method. (D) A Western blot assay of SRRM2 protein levels in keratinocytes following pull-down assays using antisense NR037661 and sense NR037661 is presented. (E) Keratinocytes were…

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  Fig. 3. NR037661 modulates aberrant RNA splicing. (A) A heatmap illustrates the gene expression profile of human epidermal keratinocytes (HEKn) silenced by either control or NR037661-specific shRNA, analyzed by gene microarray after treatment with 1.8 mM CaCl2 for 72 h. The data represents three technical replicates using cells derived from three human donors. Significant changes (p < 0.05, Student's t-test, absolute fold change >1.5) are highlighted. (B) A volcano plot visualizes the fold changes of differentially expressed genes from (A), with up-regulated genes shown in red and down-regulated genes in green. (C) Gene Ontology (GO) term analysis was performed for genes dyregulated upon NR037661 depletion, as identified in (A). (D) Quantification of the various alternative splicing (AS) events regulated by NR037661, including skipped exons (SE), alternative 3′ splicing sites (A3SS), alternative 5′ splicing sites (A5SS), retained introns (RI), and mutually exclusive exons (MXE). (E) A combinatorial analysis was conducted to identify genes exhibiting altered SE events (false discovery rate <0.1) upon NR037661 silencing, genes displaying altered expression levels (fold change >1.5, p <…

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  Fig. 4. NR037661 inhibits aberrant alternative splicing (AS) of ANGPTL4. (A) Real-time PCR analysis was conducted to assess the relative expression level of IL6R after knockdown of NR037661 in human keratinocytes (HKEn). The data are presented as the mean ± standard deviation (S.D.) of three independent experiments. (B, C) Real-time PCR (B) and Western blot (C) analyses were performed to evaluate the relative expression level of ANGPTL4 after knockdown of NR037661 in HKEn. The data are displayed as the mean ± S.D. of three independent experiments. (D) A schematic diagram illustrates the isoforms of ANGPTL4, specifically highlighting the presence or absence of exon 4 (ANGPTL4 exon 4+ and ANGPTL4 exon 4-). (E) Sashimi plots visualize the alternatively spliced transcripts of ANGPTL4 in human keratinocytes (HKEn) transfected with control or NR037661 shRNAs under calcium-induced differentiating conditions. The numbers correspond to the number of reads supporting the indicated exon-exon junctions, normalized to the total expression of the transcript. The DNA sequence is displayed in the track below the Sashimi plot. Ψ represents the inclusion level of the exon. (F) The percentage of ANGPTL4…

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  Fig. 5. NR037661 inhibits aberrant alternative splicing (AS) of ANGPTL4 via binding to SRRM2. (A,B) Keratinocytes were infected with siRNA targeting SRRM2 or NC. (A) Western blot assay was conducted to analyze the relative expression level of ANGPTL4 (n = 3). (B) The percentage of ANGPTL4 exon 4+ (transcript containing exon 4, mRNA) was assessed by semi-quantitative PCR in human keratinocytes (HKEn). (C) Binding between SRRM2 and ANGPTL4 pre-mRNA detected by RNA Immunoprecipitation (RIP) Assays. Real-time PCR analysis of anti-SRRM2 RIP assays in HKEn revealed the binding between SRRM2 and ANGPTL4 mRNA. Agarose electrophoresis was used to visualize the RIP assays in control HKEn. (D) Interaction between SRRM2 Protein and ANGPTL4 pre-mRNA After Knockdown of NR037661 detected by RIP Assay. RIP assays with anti-SRRM2 antibodies were conducted to detect the interaction between SRRM2 protein and ANGPTL4 pre-mRNA following the knockdown of NR037661. Agarose electrophoresis was used to visualize the RIP assays in control or NR037661-knockdown HKEn.

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  Fig. 6. ANGPTL4 is involved in NR037661-mediated keratinocyte differentiation. (A) Keratinocytes were induced to differentiate by supplementing the culture medium with 1.8 mM CaCl2. Cells were harvested at the specified time points for Western blot analysis. The Western blot assay was conducted to assess the relative expression level of ANGPTL4 (n = 3). (B) Effect of NR037661 and ANGPTL4 on involucrin expression during keratinocyte differentiation. Keratinocytes were infected with adenoviral vectors encoding NR037661-specific shRNA or a non-targeting shRNA control (shRNA-NC), Twenty-four hours post-infection, the cells were treated with 1.8 mM CaCl2 for 72 h, with or without the addition of recombinant ANGPTL4 (rec. ANGPTL4, 6 mg/ml). Western blot analysis was then performed to evaluate the relative expression level of involucrin (n = 3). (C) NR037661 expression was analyzed by qRT-PCR in psoriatic lesions samples and paired normal skin tissues (n = 23). (D) NR037661 specifically interacts with the splicing factor Serine/arginine repetitive matrix protein 2 (SRRM2), facilitating its nuclear localization. This interaction modulates the alternative splicing (AS) of ANGPTL4 mRNA, ultimately…