Journal: Nature Communications

Article Title: MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing

doi: 10.1038/s41467-020-14437-3

Figure Lengend Snippet: a Heat map of the global mRNA expression profile in non-tumor tissues ( n = 220) and hepatocellular carcinoma (HCC) tissues ( n = 225) in GSE14520 dataset. The levels of MTR4 are indicated with a white line. b Box plot showing the relative mRNA levels of MTR4 in HCC tissues ( n = 225) and non-tumor tissues ( n = 220) in GSE14520 (GPL3921) dataset. The significance of the difference was assessed by two-tailed, unpaired t -test. p value is indicated. Centre is median within box, bound of the box spans the interquartile range, and whiskers visualize 5 and 95% of the data points. c , d Chips of HCC samples ( n = 108) and adjacent non-tumor (ANT) samples ( n = 108) were stained with anti-MTR4 antibody and the intensity of staining was scanned and scored. Representative immunohistochemistry (IHC) images are shown. Two-tailed, unpaired t -test. Data are presented as mean value ± s.d. p value is indicated. Scale bar = 200 µm. e The MTR4 mRNA levels in HCC samples ( n = 77) were compared with the corresponding ANT samples ( n = 77). Eighty-three percent (64/77) of HCC samples have higher MTR4 mRNA levels than ANT. f The MTR4 levels were inversely correlated with the postoperative recurrence-free survival (RFS) of HCC patients. RFS of the patients with high MTR4 mRNA levels ( n = 12) is significantly lower than those with lower MTR4 ( n = 50). The difference in survival rates was assessed with the log-rank test (Mantel Cox). p value is indicated. g The knockdown of MTR4 in HCC cells PLC/PRF/5 was confirmed by western blotting. CTL, HCC cells expressing scramble shRNA. Tubulin was used as an internal control. Consistent data were obtained from two independent experiments. h Proliferation of MTR4 KD and control cells was analyzed with CCK8 assay. n = 3 biologically independent experiments. Difference between two groups was calculated by two-way ANOVA, followed by Bonferroni post-tests. Data are presented as mean value ± s.d. p value is indicated. i Colony formation assay of the control and MTR4 KD cells. Difference between two groups was calculated by two-tailed, unpaired t -test. Data are presented as mean value ± s.d. p value is indicated. n = 3 biologically independent samples. j Inducible knockdown of MTR4 in PLC/PRF/5 cells (iMTR4 cells) was confirmed by western blotting after the treatment with 1 µg/ml doxycycline (Doxy) for 4 days. Consistent data were obtained from two independent experiments. k , l The volumes ( k ) and weight ( l ) of tumors formed by PLC/PRF/5 cells expressing scramble shRNA (iSC) or iMTR4 cells in NSG mice were measured after daily i.p. injection of Doxy (20 mg kg −1 body weight) or mock treatment for 8 days. Individual tumor volumes were measured every day after doxy treatment. Repeated measures two-way ANOVA, followed by Turkey’s post-tests. p value is indicated. Repeated measures two-way ANOVA, followed by Bonferroni post-tests. Data are presented as mean value ± s.d. p value is indicated. n = 6 independent samples for each group. At the end of the treatment, the weight of all tumors in each group was compared. Mann–Whitney test. p value is indicated. n = 6 independent samples for each group. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Myc antibody (13987; Cell Signaling), rabbit polyclonal anti-MTR4 antibody (ab70551; Abcam), anti-Glut1 antibody (ab150299; Abcam), mouse monoclonal anti-α-tubulin antibody (T5168; Sigma-Aldrich), anti-rabbit IgG, HRP-linked antibody (7074S; Cell Signaling), anti-mouse IgG antibody, HRP-linked antibody (7076S; Cell Signaling).

Techniques: Expressing, Two Tailed Test, Staining, Immunohistochemistry, Knockdown, Western Blot, shRNA, Control, CCK-8 Assay, Colony Assay, Injection, MANN-WHITNEY

Journal: Nature Communications

Article Title: MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing

doi: 10.1038/s41467-020-14437-3

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of recurrence-free survival for MTR4 ( n = 62).

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Myc antibody (13987; Cell Signaling), rabbit polyclonal anti-MTR4 antibody (ab70551; Abcam), anti-Glut1 antibody (ab150299; Abcam), mouse monoclonal anti-α-tubulin antibody (T5168; Sigma-Aldrich), anti-rabbit IgG, HRP-linked antibody (7074S; Cell Signaling), anti-mouse IgG antibody, HRP-linked antibody (7076S; Cell Signaling).

Techniques: Expressing

Journal: Nature Communications

Article Title: MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing

doi: 10.1038/s41467-020-14437-3

Figure Lengend Snippet: a Global gene expression analysis of iMTR4 cells before and after Doxy treatment identified differential expressed genes (DEGs), which were enriched in various pathways with pathway analysis and visualized as the overlap graph. b The mRNA levels of glycolytic genes in iSC and iMTR4 cells before and after Doxy treatment. Two-tailed, paired t -test. Data are presented as mean value ± s.d. p value is indicated. n.s., non-significant. n = 3 biologically independent samples. c , d Extracellular acidification rate (ECAR) in control cells and MTR4 KD cells with either MMC (5 µg/ml) or DMSO as a solvent control in response to glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM). n = 3 independent experiments. Data are presented as mean value ± s.d., two-way ANOVA with a Tukey’s multiple comparison test. p value is indicated. e , f Oxygen consumption rate (OCR) in control cells and MTR4 KD cells with either MMC or DMSO as a solvent control in response to oligomycin (1 μM), fluorocarbonyl cyanide phenylhydrazone (FCCP, 1.5 μM), rotenone/antimycin A (Rot/AA, 0.5 μM). n = 3 independent experiments for each group. Data are presented as mean value ± s.d., two-way ANOVA with Tukey’s multiple comparison test. p value is indicated. Res., respiration; Cap, capability. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Myc antibody (13987; Cell Signaling), rabbit polyclonal anti-MTR4 antibody (ab70551; Abcam), anti-Glut1 antibody (ab150299; Abcam), mouse monoclonal anti-α-tubulin antibody (T5168; Sigma-Aldrich), anti-rabbit IgG, HRP-linked antibody (7074S; Cell Signaling), anti-mouse IgG antibody, HRP-linked antibody (7076S; Cell Signaling).

Techniques: Gene Expression, Two Tailed Test, Control, Solvent, Comparison

Journal: Nature Communications

Article Title: MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing

doi: 10.1038/s41467-020-14437-3

Figure Lengend Snippet: a Ectopic expression of GLUT1 in control and MTR4 KD cells was analyzed for MTR4 and GLUT1 by western blotting. Representative data from two independent experiments are shown. b , c ECAR in control cells and MTR4 KD cells transfected with either empty vector (EV) or plasmids expressing GLUT1 (GLUT1 OE) in response to glucose, oligomycin, and 2-DG. Data are presented as mean value ± s.d., two-way ANOVA with a Tukey’s multiple comparison test. n = 3 independent experiments for each group. d The proliferation of indicated cells were analyzed with CCK8 assay. Data are presented as mean value ± s.d. Two-way ANOVA with a Tukey’s multiple comparison test. n = 3 independent biological samples. e The weight of tumors formed by indicated cells in NSG mice was measured and compared. Data are presented as mean value ± s.d. One-way ANOVA, followed by Bonferroni post-tests. n = 8 independent biological samples for each group. p value is indicated. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Myc antibody (13987; Cell Signaling), rabbit polyclonal anti-MTR4 antibody (ab70551; Abcam), anti-Glut1 antibody (ab150299; Abcam), mouse monoclonal anti-α-tubulin antibody (T5168; Sigma-Aldrich), anti-rabbit IgG, HRP-linked antibody (7074S; Cell Signaling), anti-mouse IgG antibody, HRP-linked antibody (7076S; Cell Signaling).

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Comparison, CCK-8 Assay

Journal: Nature Communications

Article Title: MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing

doi: 10.1038/s41467-020-14437-3

Figure Lengend Snippet: a MTR4 binding motifs, UAAAAAA(U)AA(C)A(U)AA (which is the conventional MTR4 binding region, poly(A) sequences) and CCAG(C/U/G)C, are defined by analyzing sequence of 3301 peaks from RIP-seq with motif analysis tool, MEME-Chip. b , d A schematic of PKM gene ( b ) or GLUT1 gene ( d ) displaying potential binding motifs for MTR4 and two RNA alternative splicing factors PTBP1 and hnRNPA. Numbers indicate the locations of primers for PCR. c , e RIP analysis of the binding of MTR4 to the predicted MTR4 binding motifs of PKM gene ( c ) or GLUT1 gene. e Due to the lack of anti-MTR4 antibodies that can be used for immunoprecipitation, PLC/PRF5 cells expressing HA-tagged MTR4 were subjected to immunoprecipitated with anti-HA antibody or IgG. Eluted RNA was analyzed by RT-qPCR and display as % of input. n = 2 independent experiments. Data are presented as mean values. Numbers indicate the locations of primers for PCR ( f , g ) Relative mRNA levels of PKM2/PKM1 ( f ) or GLUT1b/GLUT1a ( g ) in MTR4 KD cells versus control cells. Dotted line indicates 1. Data are presented as mean values. n = 2 independent experiments. h , i Alternative splicing (AS) of minigenes (mini.) of PKM ( h ) or GLUT1 ( i ) genomic DNA containing exons undergoing AS in MTR4 KD or control cells. Primers for PCR analysis are indicated by arrows. Data are presented as mean value ± s.d. Two-tailed, unpaired t -test. p value is indicated. n = 3 independent experiments. j , k RIP analysis of MTR4 binding to the WT and MTR4 binding motif mutant PKM minigene ( j ) or GLUT1 minigene ( k ). PLC/PRF5 cells expressing HA-tagged MTR4 in combination with vector expressing indicated minigenes were subjected to immunoprecipitation with anti-HA antibody or IgG. Eluted RNA was analyzed by RT-qPCR and display as fold changes over IgG. Data are presented as mean values. n = 2 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Myc antibody (13987; Cell Signaling), rabbit polyclonal anti-MTR4 antibody (ab70551; Abcam), anti-Glut1 antibody (ab150299; Abcam), mouse monoclonal anti-α-tubulin antibody (T5168; Sigma-Aldrich), anti-rabbit IgG, HRP-linked antibody (7074S; Cell Signaling), anti-mouse IgG antibody, HRP-linked antibody (7076S; Cell Signaling).

Techniques: Binding Assay, Sequencing, Alternative Splicing, Immunoprecipitation, Expressing, Quantitative RT-PCR, Control, Two Tailed Test, Mutagenesis, Plasmid Preparation

Journal: Nature Communications

Article Title: MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing

doi: 10.1038/s41467-020-14437-3

Figure Lengend Snippet: a AS of WT and two MTR4 binding motif mutant (MT) including poly(A) MT in GLUT1 minigene in PLC/PRF5 cells. Data are presented as mean value ± s.d. Two-tailed, unpaired t -test. p value is indicated. n = 3 independent experiments. b AS of WT and two MTR4 binding motif mutant (MT) including poly(A) MT in PKM minigene in PLC/PRF5 cells. Data are presented as mean value ± s.d. One-way ANOVA with a Dunnett’s multiple comparison test. p value is indicated. n.s., non-significant. n = 3 independent experiments. c Impact of PTBP1 knockdown on AS of PKM minigene in PLC/PRF/5 cells with or without MTR4 overexpression. The levels of PKM1 and PKM2 isoforms were analyzed using primers described in ( b ). Representative data from two independent experiments are shown. d The co-immunoprecipitation analysis of the endogenous MTR4, hnRNPA1, and PTBP1 in PLC/PRF/5 cells in the absence or presence of RNase. The cell lysate was subjected to immunoprecipitation (IP) with indicated antibodies. The presence of various proteins in the immunoprecipitate was examined by western blotting using indicated antibodies. Because IP grade anti-MTR4 antibody is not commercially available, we did not perform the MTR4 IP. Representative data from two independent experiments are shown. e RIP analysis of the binding of PTBP1 to the WT and MTR4 binding motif mutant PKM minigene in PLC/PRF5 cells. Data are presented as mean values. n = 2 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Myc antibody (13987; Cell Signaling), rabbit polyclonal anti-MTR4 antibody (ab70551; Abcam), anti-Glut1 antibody (ab150299; Abcam), mouse monoclonal anti-α-tubulin antibody (T5168; Sigma-Aldrich), anti-rabbit IgG, HRP-linked antibody (7074S; Cell Signaling), anti-mouse IgG antibody, HRP-linked antibody (7076S; Cell Signaling).

Techniques: Binding Assay, Mutagenesis, Two Tailed Test, Comparison, Knockdown, Over Expression, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: MTR4 drives liver tumorigenesis by promoting cancer metabolic switch through alternative splicing

doi: 10.1038/s41467-020-14437-3

Figure Lengend Snippet: a The analysis of Chip-seq data of c-Myc in human cancer cell lines available in the ENCODE database predicted that c-Myc binds to the promoter region of MTR4. b ChIP analysis of the binding of c-Myc to the predicted c-Myc binding sites within the MTR4 promoter. A schematic of MTR4 regulatory region (−0.7 to +1.3 kb from TSS) was shown. Arrow and black rectangle represent the transcriptional start site (TSS) and exon1, respectively. * indicates the canonical E box. Green bar indicates CpG Islands. Numbers indicates the regions for PCR. Chromatin DNA fragments were immunoprecipitated with anti-c-Myc antibody or IgG. Data are presented as mean values. n = 2 independent experiments. c The mRNA levels of c-Myc and MTR4 were detected by q-RT-PCR and normalized by actin . Data are presented as mean values. n = 2 biologically independent samples. d The protein levels of c-Myc and MTR4 after the knockdown of c-Myc in HCC cells. Representative data from two independent experiments are shown. e , f The protein levels of c-Myc and MTR4 in HCC patient samples were analyzed by western blotting ( e ) and their correlation analyzed ( f ). The protein samples were run on multiple gels with one common sample run on all gels as a quantitative control. Two-tailed Pearson correlation test. p value and pearson’s correlation coefficient r are indicated. n = 68 HCC patient samples. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Myc antibody (13987; Cell Signaling), rabbit polyclonal anti-MTR4 antibody (ab70551; Abcam), anti-Glut1 antibody (ab150299; Abcam), mouse monoclonal anti-α-tubulin antibody (T5168; Sigma-Aldrich), anti-rabbit IgG, HRP-linked antibody (7074S; Cell Signaling), anti-mouse IgG antibody, HRP-linked antibody (7076S; Cell Signaling).

Techniques: ChIP-sequencing, Binding Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Knockdown, Western Blot, Control, Two Tailed Test