Fig 1: DHX36 binds to Gnai2 mRNA via the 5’ UTR rG4.a Sequence of the WT or mutant (Mut) Gnai2 mRNA 5' UTR. Green color denotes the coding sequence. All GGGs are highlighted in red. Mutated Gs are highlighted in blue. b Top: Genomic snapshot of DHX36 CLIP-seq track showing its binding at Gnai2 mRNA. Bottom: Snapshot of RNA-seq tracks showing Gnai2 mRNA levels in freshly isolated SCs, SCs cultured for 24 or 72 h. c GNAI2 protein levels in SCs cultured for the indicated time points were examined by western blot. d WT or Mut Gnai2 5’ UTR RNAs were treated with 150 mM Li+ or K+ together with 1 µM ThT and excited at 425 nm. Plot of the intensity of Fluorescent spectrum (FLS) with the wavelength from 440 to 700 nm. Red line: WT RNA treated with K+; blue line: WT RNA treated with Li+; green line: Mut RNA treated with K+; orange line: Mut RNA treated with Li+. e WT or Mut Gnai2 5’ UTR RNAs were treated with 150 mM Li+, 150 mM K+, or 150 mM K+ plus 2 µM PDS. Reverse transcriptase was stalled at the rG4 formation site to form reverse stalling site (RTS). The cDNAs generated by the above reverse transcription were run on a Dideoxy sequencing gel. Left: In WT Gnai2 5’ UTR RNA, two major RTS (highlighted as RTS #1 and #2) sites were identified. Right: no RTSs were detected in Mut Gnai2 5’ UTR RNA. f RNA pull-down was performed with in vitro transcribed and biotin-labeled WT or Mut Gnai2 5’ UTR RNAs and the retrieved DHX36 protein was detected by western blot. HNRNPL and DHX9 were used as negative controls. g C2C12 myoblast cells were treated with Ctrl (same volume of DMSO in the culture medium as cPDS) or 5 µM cPDS for 24 h and RNA immunoprecipitation (RIP) was performed to detect retrieved Gnai2 transcripts. 18s and Sam transcripts were used as negative controls. Data represent the average of three independent experiments ± s.d. (g). Source data are provided as a Source Data file.
Fig 2: SNHG1 Sponged miR-556-5p to Up-regulate GNAI2. (A) Subcellular fraction assay revealed the subcellular location of SNHG1 in basal HUVECs and ox-LDL-induced HUVECs. (B) qRT-PCR was applied to examine expression of four candidate miRNAs in ox-LDL-induced HUVECs. **P < 0.01 versus the control group. Student t test. (C). RIP assay was applied to detect relative enrichment of SNHG1 and miR-556-5p in Ago2 and IgG group. **P < 0.01 versus the anti-IgG group. Student t test. (D) qRT-PCR verified the overexpression efficiency of miR-556-5p. **P < 0.01 versus the NC-mimics group. Student t test. (E) Binding sequences of wild type/mutant SNHG1 and miR-556-5p were predicted from “starBase”. (F) RNA pull-down assay examined relative enrichment of SNHG1 pulled down by biotin-labeled wild type/mutant miR-556-5p. **P < 0.01 versus the Bio-NC group. Student t test. (G) Luciferase activity of wild and mutant SNHG1 by overexpression of miR-556-5p was examined in luciferase reporter assay. **P < 0.01 versus the NC-mimics group. Student t test. (H) qRT-PCR examined GNAI2 expression in HUVECs. **P < 0.01 versus the control group. Student t test. (I) RIP assay detected relative enrichment of GNAI2 and miR-556-5p in Ago2 and IgG group. **P < 0.01 versus the anti-IgG group. Student t test. (J) Binding sites of wild type/mutant GNAI2 and miR-556-5p were predicted from “starBase” database. (K) Luciferase activity of wild and mutant GNAI2 by overexpression of miR-556-5p was examined in luciferase reporter assay. **P < 0.01 versus the NC-mimics group. Student t test. (L) RNA pull-down assay examined relative enrichment of GNAI2 pulled down by biotin-labeled wild type/mutant miR-556-5p. **P < 0.01 versus the Bio-NC and Bio-GNAI2-mut group. Student t test. (M) qRT-PCR detected GNAI2 expression in HUVECs by indicated transfections. **P < 0.01 of the 2nd group versus the 1st group and 3rd group versus 2nd group. One-way ANOVA followed with the Tukey post hoc test. Data obtained from more than three repeated experiments were shown as mean ± SD. ** indicated P < 0.01 meant data were statistically significant.
Fig 3: SNHG1 regulated ox-LDL-induced cell injury and inflammatory response via down-regulating miR-556-5p and up-regulating GNAI2. (A, B) CCK-8 and MTT assay respectively determined cell viability of ox-LDL-induced HUVECs under indicated transfections. One-way ANOVA followed with the Tukey post hoc test. (C)–(G). Cell apoptosis rate (C), caspase-3/8/9 activity (D), LDH release and protein expression (E), IL-6 secretion and protein expression (F), IL-1ß secretion and protein expression (G) in ox-LDL-induced HUVECs in four different groups were determined. Four groups in the experiments in this figure: pcDNA3.1-NC, pcDNA3.1-SNHG1 (**P < 0.01 versus the 1st group), pcDNA3.1-SNHG1+miR-556-5p-mimics (**P < 0.01 versus the 2nd group), pcDNA3.1-SNHG1+sh-GNAI2 (*P < 0.05 versus the 2nd group). One-way ANOVA followed with the Tukey post hoc test. Data obtained from more than three repeated experiments were shown as mean ± SD. * indicated P < 0.05 and ** indicated P < 0.01 meant data were statistically significant.
Fig 4: Dhx36 loss exerts a potential effect on total mRNA abundance.a–d Scatterplot showing the total mRNA abundance (log10(FPKM-total)) values of all, 5’ UTR, 3’ UTR or CDS DHX36-bound mRNAs in Ctrl vs KO; the number of up- (KO vs Ctrl =1.5 fold), down-(=0.66 fold) or unchanged mRNAs are shown. e Kernel density estimates showing log2(?FPKM-total) values of mRNAs with DHX36 binding only in 5’ UTRs, 3’ UTRs, or CDS. ?FPKM-total: the mRNA level alteration in KO vs Ctrl. f–h Prediction of rG4 formation in the binding sites of 5’ UTR, 3’ UTR, and CDS-bound mRNAs was conducted and the number of sites possessing each subtype is shown in up- or downregulated mRNAs. i 5’ UTR region predicted folding energies of rG4 and dsRNA structures in up and downregulated mRNAs with 5’ UTR binding, n = 116 (upregulated mRNAs), 1075 (background mRNAs), 22 (downregulated mRNAs). Data represent mean values ± s.d. j The number of 3’ UTR-bound mRNAs possessing ARE sites is shown. k Predicted folding energies of dsRNAs in up- or downregulated mRNAs with 3’ UTR ARE binding, n = 9 (upregulated mRNAs), 2499 (background mRNAs), 18 (downregulated mRNAs). Data represent mean values ± s.d. l Left: Scatterplot illustrating TE (y axis) and mRNA (x axis) alterations in KO vs Ctrl with a threshold of ±0.585 (dashed line) for 7300 genes with FPKM-total values larger than 1 in either Ctrl or KO condition. Blue dots: TE-up genes with DHX36 binding; yellow dots: TE-down genes with DHX36 binding. Gray dots: the genes with no significant TE change or no DHX36 binding. Right: A total of five bound genes (yellow dots) including Ubq1n1, Nfix, Gnai2, Fzd2, and Zdhhc16 (red font) showed constant mRNA and decreased TE levels in KO vs Ctrl using a more stringent threshold (log2(?TE) <-1, ?TE<0.5). Gray dots: 197 genes with significant TE decrease and constant mRNA levels but with no DHX36 binding. m DHX36 binding regions and ?TE values are shown for the five genes in panel l and Gnai2 is highlighted. One-tailed Mann–Whitney test was used to calculate the statistical significance between folding energy values in panels i and k with P values shown in the figure.
Fig 5: SNHG1 inhibited ox-LDL-induced cell injury and inflammatory response via up-regulating GNAI2 and PCBP1. (A, B) CCK-8 and MTT assay determined cell viability of indicated ox-LDL-induced cells. (C, G). Cell apoptosis rate (C), caspase-3/8/9 activity (D), LDH release and protein expression (E), IL-6 secretion and protein expression (F), IL-1ß secretion and protein expression (G) in ox-LDL-induced HUVECs in indicated groups. Four groups in the experiments: pcDNA3.1-NC, pcDNA3.1-SNHG1 (**P < 0.01 versus the 1st group), pcDNA3.1-SNHG1+sh-PCBP1 (*P < 0.05 versus the 2nd group), pcDNA3.1-SNHG1+sh-GNAI2/PCBP1 (**P < 0.01 versus the 2nd group). One-way ANOVA followed by Tukey post hoc test. Data obtained from more than three repeated experiments were shown as mean ± SD. * indicated P < 0.05 and ** indicated P < 0.01 meant data were statistically significant.
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