Decreased RBM7 expression was associated with poor prognosis of breast cancer.

(A-B) The correlation between RBM7 mRNA expression and overall survival (OS) and disease-free-survival (DFS) of breast cancer patients (n=1980) based on the METABRIC dataset. The samples were divided into four equal parts, containing lower quartile Q1, median quartile Q2, upper quartile Q3 and higher quartile Q4 according to the expression of RBM7. (C) RBM7 mRNA level was analyzed in primary breast carcinoma (n=1097) and normal tissues (n=114) from the UALCAN dataset. (D) RBM7 expression level was analyzed in breast invasive carcinoma based on nodal metastasis status from TCGA dataset. BRCA samples were classified into N0 (No regional lymph node metastasis) (n=515), and metastases in axillary lymph node (N1-N3) (n=565). (E) Representative images of IHC staining with RBM7 expression in high (n=32) and low (n=58) clinical stages in breast cancer tissue microarray. Scale bars = 300 μm (top) or 30 μm (bottom). (F) Representative images of IHC staining with RBM7 expression in Triple-Negative Breast Cancer (n=119) and para-carcinoma tissue (n=20) in our tissue microarray. Scale bars = 300 μm (top) and 30 μm (bottom). (G) Quantitative analysis of RBM7 expression by IHC staining in primary breast cancer (n=12) and unpaired metastatic cancer (n=9). (H-I) Representative immunostaining images of RBM7 in 3 paired primary breast cancer and lymphatic metastasis. Scale bars = 100 μm. The p value was obtained by log-rank test (A, B), and unpaired Student’s T test (C, D, E, F, G, I). Data are presented as means ± SD.

RBM7 negatively regulated breast cancer metastatic potential.

(A) Gene Ontology analysis showed the significantly affected biological process upon RBM7 knockdown in MDA-MB-231 cells. (B) Functional association network of RBM7-regulated targets. The genes were analyzed using the STRING database, and subgroups are marked according to their function. (C-E) 4T1 cells without or with RBM7 knockdown were injected into tail vein of immunodeficient NYG mice to establish a lung metastasis model. And spontaneous pulmonary metastases were assessed after about 2 weeks. (C)The white arrow indicates lung lesions macroscopically. (D) H&E stained lung sections were quantified for the number of spontaneous metastatic lesions from NYG mice (n=6, 5 and 9). Data are mean ± SD. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. (E) Representative images of H&E stained lung sections are shown. Scale bars=7 mm (top) and 300 μm (bottom). (F-I) The metastatic ability of RBM7 for breast cancer cells was evaluated by Transwell assay. Data are mean ± SD from three random fields. P values were determined by unpaired Student’s T test (I) or one-way ANOVA with Dunnett’s multiple comparisons test (G). (J) Representative images of the tube formation of HUVEC cells, treated with conditional medium, which cultured RBM7-KD cells and control cells for 48h and collected. Scale bars: 100 μm. Quantification of master junction was by imageJ software.

Global identification of alternative splicing events regulated by RBM7.

(A-E) RNA-seq was performed on RBM7-knockdown MDA-MB-231 cells or control cells. And changes in splicing events were analyzed. (A) Volcano plot illustrating alternative splicing events regulated by RBM7. (B) Quantification of the different AS events affected by RBM7. (C) The relative fraction of each AS event affected positively or negatively by RBM7. (D) Examples of alternative exons affected by RBM7. Alternative splicing of MFGE8 was chosen to represent a decrease of PSI, and numbers of exon junction reads were indicated. (E) The hit top enriched motifs identified in the differentially spliced genes regulated via RBM7. (F) RBM7-regulated exon skipping events were identified by semiquantitative RT-PCR using RBM7-konckdown or control MDA-MB-231 cells. The mean ± SEM from 3 experiments was plotted. P values of percent-spliced-in (PSI) values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. (G) Gene Ontology analysis showed that the significant splicing events affected biological process upon RNM7 knockdown in MDA-MB-231 cells. (H) KEGG pathway enrichment of the RBM7-mediated AS targets.

RBM7 knockdown promoted MFGE8 exon 7 skipping via binding to its pre-mRNA.

(A) Venn diagram of 3509 RBM7-binding genes from the RIP-seq (based on data set GSE144075), 43 genes in RBM7-sh1 cells compared with control cells, and 43 genes in RBM7-sh2 cells compared with control cells. The gene lists of the last two groups were from RNA sequencing with top 50 AS events. The gene lists on the right were shown according to the P value. (B) Schematics of human MFGE8 pre-mRNA (NM_005928.4). And MFGE8-long isoform included the exon 7 (hereafter referred to as MFGE8-L), while MFGE8-short isoform skipped the exon (hereafter referred to as MFGE8-S). The arrow indicates the direction of transcription. Primers were designed on the upstream and downstream exons of exon 7, and RT-PCR was performed to identify MFGE8 splicing event using RBM7-KD cells or control MDA-MB-231 cells. The PCR products were subjected to sanger sequencing. The base peak diagram of sanger sequencing showed the splicing junction sites (filled with gray) of upstream and downstream exon 7. (C) Different breast cancer cells lines with stable low-expression of RBM7 or control were constructed. Alternative splicing events of MFGE8 regulated by RBM7 were examined by semi-quantitative RT-PCR. The level of RBM7 protein was detected by Western Blotting assay in various cell lines. The mean ± SD of PSIs from three independent repeated experiments were plotted. P value was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (D) As schematic diagram, an exon of the orange box is a variable exon as shown in the diagram. P1 and P2 were the putative binding regions and primers were designed here. Binding of MFGE8 pre-mRNA with RBM7 was examined by RNA-immunoprecipitation (RIP) in HEK293T cells expressing Flag-RBM7 or Flag-empty plasmid. (E) The diagram shows ASOs targeting the indicated region. MCF7 and MDA-MB-231 cells were transfected with MFGE8-ASO for 48h. And then MFGE8 splicing events were measured by RT-PCR. (F) Schematics of human MFGE8 mini splicing reporter gene and the reporter contained 3 exons and two mini-introns. RBM7 stable knockdown cells or control cells were transfected with mini reporter gene, and RNA was collected 24 hours of transfection. The splicing changes of exogenous MFGE8 were detected by RT-PCR with specific primers on the reporter gene. The level of RBM7 protein was detected in MCF7 cells by Western blotting assay. P value was calculated by one-way ANOVA with Dunnett’s multiple comparisons test from three repeated experiments.

MFGE8-L inhibited breast cancer cell motility and invasion.

(A) Effects of MFGE8 two isoforms on breast cancer cell migration and invasion were evaluated by Transwell assays. (B) The subcellular localizations of MFGE8 isoforms. MDA-MB-231 cells were transfected with Flag-tagged MFGE8-L or MFGE8-S, and visualized with immunofluorescence assay. Scale bar = 10 μm. The localization of MFGE8-L and MFGE8-S was quantified with foci, and the percent of cells were plotted (about 80 cells were captured and quantified in both samples). (C) Transcriptome sequencing was performed using MDA-MB-231 cells of overexpressing MFGE8-L and MFGE8-S. Venn diagram of 782 MFGE8-L down-regulated genes, 647 genes from MFGE8-S up-regulated genes compared with MFGE8-L. (D) Gene ontology analysis and KEGG pathway analysis of genes up-regulated by MFGE-S compared with MFGE8-L. (E) The expression of MFGE8 and p-STAT1(Tyr701) in HCC1937 cells overexpressing respectively MFGE8-L and MFGE8-S was detected by Western Blotting assay.

RBM7 knockdown enhanced aggressiveness of breast cancer relying on MFGE8 splicing switch to the short variant and NF-κB pathway activation.

(A-B) Breast cancer cells MDA-MB-231 (A) and MCF7 (B) were transfected with shRBM7 or control and re-transfection Flag-MFGE8-L vector. Then these cells were subjected to Transwell migration assay or invasion assay. Cells migrating through Transwell membrane with matrigel or not were imaged and representative images are shown (right) along with quantification (left). (C) HCC1937 and BT-549 cells stably expressing MFGE8-L, MFGE8-S and control were generated. The expression of metastasis-related inflammatory factors was measured by real-time PCR assay. (D) NF-κB signaling pathway was enrichment via KEGG analysis in RBM7 knockdown cells. (E) Western blotting showed the expression of NF-κB-associated proteins in RBM7-depleted or control MDA-MB-231 and BT-549 breast cancer cell lines. (F) RBM7-depleted MDA-MB-231 cells were treated with or without NF-κB inhibitor PDTC 10 μm for 48h. Then Western blotting tested the expression of RBM7, NF-κB -p65 and phosphorylated NF-κB p65. (G) Representative images of the tube formation of HUVEC cells, treated with conditional medium with 10 μm NF-κB inhibitor PDTC. Scale bars: 100 μm. Quantification of junctions was by imageJ software.

Splicing shift of MFGE8 toward exon7 exclusion was negatively correlated with RBM7 expression in patients with breast cancer.

(A) MFGE8_exon7 expression level was analyzed in breast carcinoma from the TCGA dataset. (B-C) MFGE8 alternative splicing events were quantified in 6 pairs of primary BRCA tissues and adjacent normal tissues using RT-PCR (B). Semi-quantitative RT-PCR was performed using 3 pairs of primary breast cancer tissues and lymph node metastasis of breast cancer tissues (C). (D) Correlation of RBM7 with the MFGE8 exon7 expression levels was analyzed with Pearson’s correlation coefficient. (E) The correlation between MFGE8-S and overall survival (OS) of breast cancer patients based on the TCGA dataset. (F) The mechanistic model of this study. Schematic illustration of the roles of RBM7-MFGE8 splicing switch axis in metastasis of breast cancer.

The low expression of RBM7 in breast cancer was positively correlated with the poor survival of patients.

(A)The correlation between RBM7 expression and overall survival of breast cancer patients based on data from the Kaplan–Meier Plotter cohort. (B-C) METABRIC analysis of the correlation between RBM7 expression and overall survival (OS) and disease-free survival (DFS) of breast cancer patients based on the METABRIC dataset. The p value was obtained by log-rank test (A, B, C). (D) RBM7 mRNA level was analyzed in breast invasive carcinoma based on nodal metastasis status from the UALCAN dataset. The p-value was calculated by unpaired Student’s t-test by comparing lymph node metastasis of breast cancer tissues with normal tissue.

RBM7 inhibited breast cancer cell migration and invasion.

(A) The mouse cell line with RBM7 knockdown was constructed. And the expression of RBM7 was detected by real-time quantitative PCR. (B) The migration and invasion ability of murine RBM7 was detected by Transwell assay using RBM7-KD 4T1 cells. (C) The metastatic capability of RBM7 on 4T1 cells was evaluated by wound healing assay. (D) Western blotting showed the expression of RBM7 in MDA-MB-231 cells and MCF7 cells of RBM7-depleted. (E) The metastatic capability of RBM7 on MDA-MB-231 cells was evaluated by wound healing assay. (F) Western blotting showed the expression of RBM7 in various breast cancer cell lines of RBM7 over-expression. (G) The metastatic ability of RBM7 for BT-549 cells and HCC1937 cells of promoted expression of RBM7 was evaluated by Transwell assay. Data are mean ± SD from three random fields. P values were determined by unpaired Student’s T test (G). P value was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (A, B, C, E).

RBM7 could regulate alternative splicing in breast cancer cells.

(A) Representative image of MDA-MB-231 and HCC1937 cells stained for RBM7. Scale bar=10 μm. (B) Semi-quantitative RT-PCR was performed to validate the AS events in MAP7D1, and HNRNPC in MDA-MB-231 cells with RBM7 knockdown compared with controls. P value was calculated by one-way ANOVA with Dunnett’s multiple comparisons test.

MFGE8-L suppressed breast cancer cells migration and invasion.

(A) The protein expression of endogenous MFGE8 was determined by western blot in MDA-MB-231 cells knocking down or over-expression MFGE8. SE denotes long exposure and LE denotes long exposure. (B-C) Transwell assay with Matrigel or not was performed using MFGE8 promoted MDA-MB-231 cells or MFGE8 decreased expression cells. P value was calculated by unpaired Student’s T test. (D) Western blot showed the extrinsic expression of MFGE8-L isoform and MFGE8-S isoform in MDA-MB-231 and HCC1937 transfected respectively Flag-MFGE8-L, Flag-MFGE8-S and empty vectors. The red arrow indicates the location of the protein. (E) MDA-MB-231 cell line expressing MFGE8-L, MFGE8-S or a vector control were analyzed by wound healing assay. Percent of wound closure was measured in triplicate experiments, with mean ± SD plotted (p values were calculated by one-way ANOVA with Tukey’s multiple comparisons test).

MFGE8-L/S isoforms had no effect on NF-κB pathway in breast cancer cells.

(A) MCF7 and MDA-MB-231 cells stably expressing shRBM7, shRBM7/MFGE8 and control were generated. The expression of RBM7 and MFGE8 was measured by real-time PCR assay. The mean ± SD of relative mRNA levels from triplicate experiments are plotted. (B) HCC1937 and BT-549 cells stably expressing MFGE8-L, MFGE8-S and control were generated. The expression of MFGE8 was measured by real-time PCR assay. (C) Representative images of the tube formation of HUVEC cells, treated with conditional medium, which cultured MFGE8-L, MFGE8-S stably transfected cells and control cells for 48h and collected. Scale bars: 100 μm. Quantification of junctions was by imageJ software. (D) Western blotting showed the expression of indicated proteins in RBM7-promoted or control breast cancer cells. (E) Western blotting showed the indicated antibodies in up-expression of MFGE8 different isoforms in breast cancer cells. (F) RBM7-depleted MDA-MB-231 cells were treated with or without NF-κB inhibitor PDTC 10 μm for 48h. Then Transwell assays were performed to assess cell migration ability. p values were calculated by one-way ANOVA with Tukey’s multiple comparisons test (C, F).

MFGE8 exon7 skipping is enhanced in a variety of cancers.

(A) MFGE8_exon7 skipping event was analyzed in various cancers according to TCGA SpliceSeq dataset. The p-value was calculated by unpaired Student’s t-test.