Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer
"> Figure 1
<p>(<b>A</b>) Overview of RNA-Seq data processing and of the computational and experimental approach used for the identification and validation of the new spliced transcripts; (<b>B</b>) Barplot with log<sub>2</sub> normalized expression values (FPKM) of semaphorin/Plexin/Neuropilin—and of the adhesion markers <span class="html-italic">CDH1</span> and <span class="html-italic">CD44</span>—according to RNA-Seq data.</p> "> Figure 2
<p>Schematic representation of <span class="html-italic">de novo</span> identified transcripts for adhesion-related genes and their differential expression by RT-PCR in breast cell lines (a representative image is shown for each gene). (<b>A</b>) <span class="html-italic">THBS1</span> new transcript; (<b>B</b>) <span class="html-italic">RAP1GAP</span> new transcript; (<b>C</b>) <span class="html-italic">LGALS1</span> new transcript; (<b>D</b>) <span class="html-italic">CD47</span> new transcript. For each new transcript, in the upper part is shown the genomic region encompassing the gene; red arrows indicate the direction of transcription. Below, the exon/intron structure of the gene is depicted. Red cross indicates the exons that are spliced out in the new transcript. The electropherogram and the nucleotide sequence above refer to the new splice junction identified. Images of PCR amplicons on agarose gel from three independent replicates experiments were acquired. For all panels, M is 100 bp DNA marker. The black arrows indicate the PCR band corresponding to the newly identified transcripts. Pixel density of each band (only for the newly identified mRNA isoforms) was quantified by ImageJ software and compared to the pixel density of the 100 bp DNA marker (at known concentration) to normalize data across replicates and cell lines. GAPDH gel bands are reported below to show that the starting amount of template was the same for all analyzed samples. Values are reported as AU = Arbitrary Units in the boxplots. Dark horizontal lines represent the mean, the box represents the 25th and 75th percentiles and the whiskers the 5th and 95th percentiles. Asterisks indicate significant <span class="html-italic">p</span> values (* <span class="html-italic">p</span> < 0.05).</p> "> Figure 3
<p>Schematic representation of <span class="html-italic">de novo</span> identified transcripts of motility-related genes and their expression by RT-PCR in breast cell lines (a representative image is shown for each gene). (<b>A</b>) <span class="html-italic">RHOA</span>; (<b>B</b>) <span class="html-italic">RHOD</span>; (<b>C</b>) <span class="html-italic">CASK</span>; (<b>D</b>) <span class="html-italic">JAG2</span>; (<b>E</b>) <span class="html-italic">CTTN</span> new transcripts. Details about the gel bands and their quantification are provided in <a href="#ijms-17-00121-f002" class="html-fig">Figure 2</a> legend. Asterisks indicate significant <span class="html-italic">p</span> values (* <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01).</p> "> Figure 4
<p>Schematic representation of <span class="html-italic">de novo</span> identified transcripts for (<b>A</b>) <span class="html-italic">SEMA3C</span> (<b>B</b>) <span class="html-italic">PLXNB1</span> genes. Nucleotide sequenceand their expression by RT-PCR in breast cell lines are also depicted. Details about the gel bands and their quantification are provided in <a href="#ijms-17-00121-f002" class="html-fig">Figure 2</a>’s legend. Asterisks indicate significant <span class="html-italic">p</span> values (** <span class="html-italic">p</span> < 0.01).</p> "> Figure 5
<p>(<b>A</b>) Scheme of the new <span class="html-italic">SEMA3F</span> transcript and expression by RT-PCR on MCF-7 cell line, showing the canonical (307 bp) and the new alternative transcript (149 bp) of <span class="html-italic">SEMA3F</span>; M is 100 bp DNA marker; (<b>B</b>) Boxplots with the relative expression of both annotated and new <span class="html-italic">SEMA3F</span> transcript in breast cell lines measured with qRT-PCR (<span class="html-italic">n</span> = 3); (<b>C</b>) On the <b>left</b>, immunoreactive bands—corresponding to the canonical (about 90 kDa) and the new isoform (about 70 kDa) of semaphorin 3F—detected by Western Blot on whole breast cell lines lysates. On the <b>right</b>, the densitometry results (AU = Arbitrary Units) by ImageJ of the Western Blot bands, after normalization with actin, are shown as bar graphs (<span class="html-italic">n</span> = 2); (<b>D</b>) A detail of the protein alignment of the canonical and the new semaphorin 3F protein isoform is shown. The dashed boxes represent the functional domains that are deleted in the novel protein sequence. Asterisks indicate significant <span class="html-italic">p</span> values (* <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001).</p> "> Figure 6
<p>Semaphorin 3F expression in BC tumor biopsies (<span class="html-italic">n</span> = 18). (<b>A</b>,<b>B</b>) qRT-PCR analysis of the new (LN626688) and the annotated (NM_004186.3) <span class="html-italic">SEMA3F</span> transcripts, respectively. Samples have been ordered according to tumor grade (G2, G3); # indicates Triple Negative biopsies; asterisks (*) indicate the sample with the lowest absolute expression, used as reference; (<b>C</b>) Relative ratio of Δ<span class="html-italic">C</span><sub>t</sub> values between skipping and canonical mRNA isoforms of <span class="html-italic">SEMA3F</span> across breast cancer subtypes. TNM: pT1 (<span class="html-italic">n</span> = 7); pT2 (<span class="html-italic">n</span> = 9); pT3 (<span class="html-italic">n</span> = 2). Tumor grade: G2 (<span class="html-italic">n</span> = 11); G3 (<span class="html-italic">n</span> = 7). ER<sup>+</sup>/PR<sup>+</sup> (<span class="html-italic">n</span> = 15); TN (triple negative samples) (<span class="html-italic">n</span> = 3); (<b>D</b>) Six representative images by immunohistochemistry assay on FFPE slices from breast cancer specimens are shown; N-terminal anti-Sema3F (that recognizes both the canonical and the new splicing variant) primary antibody was used. Scale Bar (in red) value = 33.76 µm.</p> ">
Abstract
:1. Introduction
2. Results
2.1. MCF-7 Transcriptome and Alternative Splicing Profile
Gene Name | Positions | ID | Skipped Exons | ORF Change | Affected Domain(s) | Protein Length |
---|---|---|---|---|---|---|
THBS1 | 15q14 | LN607833 | 10 | Del | TSP1 | 1112 |
LGALS1 | 22q13.1 | LN607840 | 3 | Frameshift | β-galactoside binding | 39 |
RAP1GAP | 1p36.12 | LN607839 | 24 | Ins | N/A | 821 |
CD47 | 3q13.12 | LN680437 | 8–10 | Frameshift | Cytoplasmic C-term | 293 |
RHOA | 3p21.31 | LN607834 | 3–4 | Del | GTP binding | 109 |
RHOD | 11q13.2 | LN607835 | 4 | Del | GTP binding | 165 |
CASK | Xp11.4 | LN607837 | 19–20 | Del | Linker between PDZ and SH3 | 886 |
CTTN | 11q13.3 | LN607836 | 10–11 | Del | Cortactin | 476 |
JAG2 | 14q32.33 | LN607838 | 24 | Del | VWFC | 1194 |
SEMA3C | 7q21.11 | LN626689 | 15 | Frameshift | Type 1 Ig-like and R/K rich | 514 |
SEMA3F | 3p21.31 | LN626688 | 16 | Frameshift | Type 1 Ig-like and R/K rich | 571 |
PLXNB1 | 3p21.31 | LN626690 | 32 | Frameshift | Cytoplasmic C-term | 1876 |
2.2. New Transcripts of Adhesion-Related Proteins
2.3. New Transcripts of Motility-Related Proteins
2.4. New Semaphorins and Plexins Isoforms
2.5. A New Semaphorin 3F Isoform Expressed in Breast Cancer
3. Discussion
4. Experimental Section
4.1. Cell Lines and Tissue Samples
4.2. RNA Isolation and RNA-Sequencing
4.3. In Silico Analysis
4.4. RT-PCR, Cloning and Sequencing
4.5. qRT PCR
4.6. Western Blot and Immunohistochemistry
4.7. Statistics
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Aversa, R.; Sorrentino, A.; Esposito, R.; Ambrosio, M.R.; Amato, A.; Zambelli, A.; Ciccodicola, A.; D’Apice, L.; Costa, V. Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer. Int. J. Mol. Sci. 2016, 17, 121. https://doi.org/10.3390/ijms17010121
Aversa R, Sorrentino A, Esposito R, Ambrosio MR, Amato A, Zambelli A, Ciccodicola A, D’Apice L, Costa V. Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer. International Journal of Molecular Sciences. 2016; 17(1):121. https://doi.org/10.3390/ijms17010121
Chicago/Turabian StyleAversa, Rosanna, Anna Sorrentino, Roberta Esposito, Maria Rosaria Ambrosio, Angela Amato, Alberto Zambelli, Alfredo Ciccodicola, Luciana D’Apice, and Valerio Costa. 2016. "Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer" International Journal of Molecular Sciences 17, no. 1: 121. https://doi.org/10.3390/ijms17010121