Fig 1: SNORA37 promotes gastric cancer progression via facilitating CMTR1-ELAVL1 interplay. a-c Representative images (a and b) and quantification (c) of soft agar and matrigel invasion assays indicating the in vitro growth and invasion of HGC-27 and AGS cells stably transfected with scramble shRNA (sh-Scb) or sh-SNORA37 #1, and those co-transfected with CMTR1 or ELAVL1 (n = 5). d In vivo imaging, growth curve, and weight at the endpoints of xenograft tumors formed by subcutaneous injection of HGC-27 cells stably transfected with sh-Scb or sh-SNORA37 #1, and those co-transfected with CMTR1 or ELAVL1 into dorsal flanks of nude mice (n = 5 for each group). e and f Representative images (e) and quantification (f) of immunohistochemical staining showing the intertumoral expression of CD31 and Ki-67 (brown, arrowheads) within subcutaneous xenograft tumors of nude mice formed by HGC-27 cells stably transfected with sh-Scb or sh-SNORA37 #1, and those co-transfected with CMTR1 or ELAVL1 (n = 5 for each group). g and h Hematoxylin & eosin (HE) staining (g), representative images (h, left panel) and quantification (h, middle panel) of lung metastatic colonization (arrowheads) and Kaplan–Meier curves (h, right panel) of nude mice treated with tail vein injection of HGC-27 cells stably transfected with sh-Scb or sh-SNORA37 #1, and those co-transfected with CMTR1 or ELAVL1 into tail vein of nude mice (n = 5 for each group). One-way analysis of variance (ANOVA) was used to compare the difference in c, d, f, and h. Log-rank test was used for survival comparison in h. *P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SEM (error bars) and representative of three independent experiments in a-c
Fig 2: SNORA37 facilitates CMTR1-ELAVL1 interplay in alternative splicing of CD44. a Western blot assay showing the expression of CMTR1 and ELAVL1 in MKN-45 and AGS cells stably transfected with empty vector (mock), SNORA37, scramble shRNA (sh-Scb), sh-SNORA37 #1, or sh-SNORA37 #2. b Co-IP and western blot assays indicating the interaction of CMTR1 with ELAVL1 in AGS cells stably transfected with sh-Scb, sh-SNORA37 #1, or sh-SNORA37 #2. c BiFC assay showing the interaction between CMTR1 and ELAVL1 (arrowheads) within HGC-27 cells co-transfected with pBiFC-VN173-CMTR1 and pBiFC-VC155-ELAVL1, and those stably transfected with sh-Scb or sh-SNORA37 #1, with nuclei staining by DAPI. d Western blot assay indicating the expression of CMTR1 or ELAVL1 in subcellular fractions of MKN-45 and AGS cells stably transfected with mock, SNORA37, sh-Scb, sh-SNORA37 #1, or sh-SNORA37 #2. e Western blot assay showing the levels of CMTR1 and ELAVL1 in subcellular fractions of AGS cells stably transfected with sh-Scb or sh-SNORA37 #1, and those co-transfected with mock or CMTR1. f Cross-linking RIP assay indicating the interaction of ELAVL1 with CD44 pre-mRNA containing alternative splicing sites around exon 10 in AGS cells stably transfected with sh-Scb or sh-SNORA37 #1, and those co-transfected with mock or CMTR1. g and h RT-PCR (g) and western blot (h) assays showing the alternative splicing and expression of CD44v6 and CD44s in AGS cells stably transfected with sh-Scb or sh-SNORA37 #1, and those co-transfected with mock, CMTR1, or ELAVL1. Data are shown as representative of three independent experiments in a-h
Fig 3: CMTR1 regulates the expression of a group of genes involved in the cell cycle.A RNA-seq was conducted on RKO cells transfected with control siRNA (si-Ctrl) or CMTR1 siRNA (si-CMTR1). A pie chart was used to show genes positively and negatively regulated by CMTR1. B, C The expression levels of genes positively and negatively regulated by CMTR1 are displayed in a heatmap and a box plot. D Hallmark gene set analysis revealed the gene term with the greatest enrichment of genes positively regulated by CMTR1. E–G Graphical presentation of the effects of CMTR1 on representative cell cycle-related genes, such as CDKN1A, CDK6, and CCND1, as determined by RNA-seq. H–Q Expression of CDKN1A, CDK6, and CCND1, measured by RT‒qPCR and western blotting in CMTR1 knockdown and CMTR1-overexpressing cells. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 4: SNORA37/CMTR1/ELAVL1 axis is associated with poor prognosis of gastric cancer patients. a Western blot assay showing the expression of CMTR1 and ELAVL1 in gastric cancer (GC) and corresponding normal (CN) epithelial tissues. b Kaplan–Meier curves indicating overall survival (OS) and first progression (FP) survival of gastric cancer cases with low or high levels of CMTR1 (cutoff values = 8.96 and 8.82, respectively) or ELAVL1 (cutoff values = 8.33 and 8.18, respectively). c The mechanism of SNORA37/CMTR1/ELAVL1 axis-mediated cancer progression: As an ELAVL1-facilitated H/ACA box snoRNA derived from host gene MBD2, SNORA37 directly binds to CMTR1 to promote its interaction with ELAVL1, resulting in nuclear retention and activity of ELAVL1 in regulating alternative splicing of CD44. SNORA37 exerts oncogenic roles in gastric cancer progression via facilitating CMTR1-ELAVL1 interaction. Log-rank test for survival comparison in b. Data are shown as representative of three independent experiments in a
Fig 5: CMTR1 knockdown enhances the efficacy of PD1 blockade immunotherapy in colorectal cancer.A Schematic diagram showing the time point of anti-PD1 antibody treatment and analysis of tissues from C57BL/6 J mice implanted with MC38 cells. B, C After the knockdown of CMTR1 expression using shRNA, the expression of CMTR1 was measured by RT‒qPCR and western blotting. D, E Representative pictures of MC38 cell-derived tumors in C57BL/6 J mice, and tumor weights indicating the effects of CMTR1 knockdown or in combination with PD1. F, G Immunohistochemical staining and semi-quantitative analysis of CD8 positive T cells in tumors from mice indicating the effects of CMTR1 knockdown alone or in combination with PD1 blockade therapy. Scale bar: 50 µm. H, I Immunofluorescent staining and quantification of ki67 in tumors from mice upon CMTR1 knockdown or in combination with PD1. Scale bar: 50 µm. J–M Relative mRNA levels of inflammatory mediators in tumors from mice treated as indicated. *P < 0.05, **P < 0.01, and ***P < 0.001.
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