Additional file 1 of MicroRNA signatures differentiate types, grades, and stages of breast invasive ductal carcinoma (IDC): miRNA-target interacting signaling pathways
Creators: name: Verma, Vinod Kumar
name: Beevi, Syed Sultan
name: Nair, Rekha A.
name: Kumar, Aviral
name: Kiran, Ravi
name: Alexander, Liza Esther
name: Dinesh Kumar, Lekha
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Information Systems not elsewhere classified
subject: Cancer
subject: Plant Biology
subject: Computational Biology

description: Additional file 1: Supplementary Figure S1. The morphological assessment of human breast cancer tissues of distinct grades was conducted. A. Illustrates a hyperchromatic nucleolus. B. Grade 1 IDC showcases numerous tubules, mild pleomorphic nuclei, and minimal mitotic activity. C. Demonstrates grade 2 IDC characterized by a reduced number of tubules, along with moderate pleomorphism and mitotic activity. D. Grade 3 reveals the absence of tubules, accompanied by high pleomorphism and mitotic activity. Supplementary Figure S2. Comparative analysis of common miRNA expression on TLDA and LNA array platform based on different grades, stages and estrogen receptors expression. A. Heat map expression of common miRNAs in TLDA and LNA Array across grade 2 and grade 3 along with their adjacent normal samples. Color scale shows 0 to -3 (Blue) log10 RQ (low expression) while 0 to +3 (Red) log10 RQ (high expression). B. Heat map shows the common miRNAs expression signature in ER+ve, ER-ve, Grade 2 (GR2), Grade 3(GR3), Grade 2 Stage I (GR2-Stg1), Grade 2 Stage II (GR2-Stg1I), Grade 2 Stage III (GR2-Stg1II), Grade 3 Stage I (GR3-Stg1), Grade 3 Stage II (GR3-Stg1I), Grade 3 Stage III (GR3-Stg1II). Color scale shows 0 to -3 (red) log10 RQ (low expression) while 0 to +3(green) log10 RQ (high expression). Supplementary Figure S3. Correlation of specific miRNAs clusters in grade 2 and grade 3 of human breast cancer. Correlation coefficient (R2) of miRNA cluster miR-19b-20a in grade 2 and grade 3 (0.971 &amp; 0.971), let-7-c-99a in grade 2 and grade 3 (0.862 &amp;0.955), miR-19a-19b in grade 2 and grade 3 (0.942 &amp; 0.970), miR-145-2143 in grade 2 and grade 3 (0.812 &amp; 0.912) all indicating a strong positive correlation among the subsets. Absolute correlation coefficient (R2) is equal to 1. Supplementary Figure S4. Individual assay validation of highly significant and common miRNAs. A. List of highly significant (p-values &lt; 0.005) miRNAs up/down regulated in each ER+ve, ER-ve, GR2, GR3, stage I, stage II and stage III for individual assay validation. B. Hsa-miR-190b, hsa-miR-224 and hsa-miR-452 in ER+ve and hsa-miR-126*, hsa-miR-9 and hsa-miR-137 in ER-ve were validate using individual taqman probes and compared with respective miRs TLDA expression. C. Grade 2 specific miRs like hsa-miR-26a-2* and hsa-miR-129-3p, while Grade 3 hsa-miR-767-5p and hsa-miR-147b individual assay in comparison with TLDA. Individual miRNAs expression represented as green bar (individual assay) and compared with yellow bar (TLDA) from stage I, II and III of grade 2 and Grade 2. D. Individual miRNAs (miR-155, miR-182, miR-429, and miR-141) expression from Stage II and Stage III of grade 2 and grade 3 were performed using individual Taqman probes and compared with respective miRNAs TLDA. Log10RQ 1 is 10 fold of expression. Supplementary Figure S5. Significant miRNAs in Grade 2 and Grade 3: A. List of 18 significant (p values 0.002 to 9.09E-06) miRNAs in grade 2 and non-significant (p values 0.063 to 0.99) in grade 3. The expression is represented by heat map with the scale (-3 to +3) where pink colour shows up regulation and red down regulation. The respective miRNAs (red dots) and their specific targets (blue dots) interaction are displayed using clustal analysis tool. C. Grade 3 ten significant (p values 0.01 to 8.52E-07) miRNAs vs non-significant (p values 0.1 to 0.97) in grade 3 along with heat map representation of expression pattern with the scale (-3 to +3) where green shows up regulation and red shows down regulation. miRNA (red node)-target (blue node) interaction analysis by clustal tool using R –program. Supplementary Figure S6. Significant miRNAs in stage I, II and III of Grade 2: A. Stage I significant miRNAs (6) with p values between 0.003 to 0.006 and non-significant stage II and III with p values between 0.092 to 0.99). The expression is represented by heat map with the scale (-3 to +3) where green colour shows up regulation and red down regulation. The respective miRNAs (red dots) and their specific targets (blue dots) interaction are displayed using cluster analysis tool. B. Stage II nineteen significant (p values 0.01 to 0.0005) miRNAs vs non-significant ( p values 0.1 to 0.97) in stage I and Stage III along with heat map representation of expression pattern with the scale (-3 to +3) where pink shows up regulation and red shows down regulation. The respective miRNA (red node)-target (blue node) interaction analysis by clustal tool. C. Stage III eight significant (p values 0.01 to 0.0003) miRNAs vs non-significant ( p values 0.1 to 0.97) in stage I and Stage II along with heat map representation of expression pattern with the scale (-3 to +3) where green shows up regulation and red shows down regulation. The respective miRNA (red node)-target (blue node) interaction analysis by clustal tool using R –program. Supplementary Figure S7. Significant miRNAs in stage I, II and III of Grade 3: A. Stage I significant miRNAs (18) with p values between 0.001 to 6.98E-05 and non-significant stage II and III with p values between 0.1 to 0.97). The expression is represented by heat map with the scale (-3 to +3) where green colour shows up regulation and red down regulation. The respective miRNAs (red dots) and their specific targets (blue dots) interaction are displayed using clustal analysis tool, B. Stage II six significant (p values 0.003 to 0.0008) miRNAs vs non-significant (p values 0.061 to 0.59) in stage I and stage III along with heat map representation of expression pattern with the scale (-3 to +3) where green shows up regulation and red shows down regulation. The respective miRNA (red node)-target (blue node) interaction analysis by clustal tool. C. Stage III four significant (p values 0.001 to 0.0008) miRNAs vs non-significant ( p values 0.1 to 0.99) in stage I and stage II along with heat map representation of expression pattern with the scale (-3 to +3) where green shows up regulation and red shows down regulation. The respective miRNA (red node)-target (blue node) interaction analysis by clustal tool using R –program. Supplementary Figure S8. Significant miRNAs in ER+ve and ER-ve, A. List of 17 significant (p values 0.0003 to 2.4E-14) miRNAs in ER+ve and non-significant (p values 0.12 to 0.98) in ER-ve. The expression is represented by heat map with the scale (-3 to +3) where green colour shows up regulation and red down regulation. The respective miRNAs (red dots) and their specific targets (blue dots) interaction are displayed using cluster analysis tool. B. ER-ve, nineteen significant (p values 0.01 to 2.59E-05) miRNAs vs non-significant ( p values 0.1 to 0.97) in ER+ve along with heat map representation of expression pattern with the scale (-3 to +3) where green shows up regulation and red shows down regulation. miRNA (red node)-target (blue node) interaction analysis by clustal tool using R –program. Supplementary Figure S9. MicroRNA-target interaction network and pathway enrichment analysis by GeneGO Metacore web computational tool. Highly significant and individually validated miRNAs were analysed for its specific targets involving in various oncogenic pathways A. MAPKinase, B. Notch signaling, C. VEGF signaling and D. Wnt signaling. Different coloured connecting lines between miRNAs and targets shows gene association, gene interaction, coexistence and expressions. Different coloured shape nodes represents enzymes transcription factors, receptors, ligands etc., where miRNA are represented as spiral single strand nucleotides. Supplementary Figure S10. In vitro validation of putative targets of significant miRNAs using miRNA mimics in MDA-MB 231 cells. Transfection of miRNA mimics (miR-432, miR-659, miR-105, miR-921, miR-662 and miR-21, scrambled as a control) and immunofluorescence of targets A. p53, B. p-β-catenin and C. APC. DAPI used to stain the nucleus, Phycoerythrin (PE-543) labelled with secondary antibody. The confocal image (DAPI channel, PE channel and Merged) was captured at 63X objective at 10μm scale. Supplementary Figure S11. In vitro validation of putative targets of significant miRNAs using miRNA mimics in MCF7 cells. Transfection of miRNA mimics (miR-921, miR-105, miR-432, miR-662 miR-659, miR-200c and miR-21, scrambled as a control) and immunoblotting for the targets A. p53, B.APC, PTEN, CASP3 and c-Myc C. PTEN, and D. BCL2. Beta-actin used as endogenous control and were used to normalize the target expression in densitometry plots. Band intensity was measured in triplicate and plotted with error bars. Supplementary Figure S12. Significant miRNAs from grade 2 (Stage I &amp; II) and grade 3 (Stage I &amp; II) target validation using antibody array. Antibody array of multiple targets of A. grade 2 stage I, B. grade 2 stage III, C. grade 3 Stage I, and D. grade 3 stage III. Protein samples from adjacent tissue (non cancerous) were used as normal control. Color range depicts the up regulation (+0.4 red color) and down regulation (-0.4 blue color). The yellow color indicates no expression.