Cancers, Vol. 15, Pages 128: MicroRNAs miR-584-5p and miR-425-3p Are Up-Regulated in Plasma of Colorectal Cancer (CRC) Patients: Targeting with Inhibitor Peptide Nucleic Acids Is Associated with Induction of Apoptosis in Colon Cancer Cell Lines

Data curation, J.G., R.C., R.G. and A.F.; formal analysis, J.G., R.C., R.G. and A.F.; funding acquisition, R.C., R.G. and A.F.; investigation, J.G., C.P., M.Z., L.G., A.M., A.R., M.F. and A.F.; methodology, J.G., C.P., L.G., A.R., M.Z. and A.M.; project administration, R.G. and A.F.; resources, R.C., R.G. and A.F.; supervision, R.G. and A.F.; validation, J.G., C.P., M.Z., L.G., A.M., A.R. and M.F.; writing—original draft, J.G., R.C., R.G. and A.F.; writing—review and editing, J.G., C.P., M.Z., L.G., A.M., R.C., R.G. and A.F. All authors have read and agreed to the published version of the manuscript.

Figure 1. Content of the miRNAs belonging to the “9-miRNA signature” in the plasma from CRC patients. (A) Pie chart reporting the number of CRC patients in which each indicated miRNA was found up-regulated (red), down-regulated (green), or to have no substantial variation detected (gray). (B) Summary of the “CRC-index” values of the “9-miRNA signature” compared to miR-221-3p. “CRC-index”: (number of patients displaying up-regulation–number of patients displaying down-regulation)/total number of patients analyzed. Up-regulated miRNAs are reported as white boxes, and down-regulated miRNAs as black boxes. Dotted box: miR-221-3p.

Figure 1. Content of the miRNAs belonging to the “9-miRNA signature” in the plasma from CRC patients. (A) Pie chart reporting the number of CRC patients in which each indicated miRNA was found up-regulated (red), down-regulated (green), or to have no substantial variation detected (gray). (B) Summary of the “CRC-index” values of the “9-miRNA signature” compared to miR-221-3p. “CRC-index”: (number of patients displaying up-regulation–number of patients displaying down-regulation)/total number of patients analyzed. Up-regulated miRNAs are reported as white boxes, and down-regulated miRNAs as black boxes. Dotted box: miR-221-3p.

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Figure 2. Effects of R8-PNA-a425 and R8-PNA-a584 on miRNA content. (A) Effects of anti-miRNA PNAs on their respective target miRNA, after performing 72 h of culturing of HT-29 cells. (B) Possible off-target effects were verified for both R8-PNA-a584 (white boxes) and R8-PNA-a425 (black boxes): miR-425-3p, miR-15b-5p, and miR-210-3p content was calculated for R8-PNA-a584-treated HT-29 cells, while miR-584-5p, miR-15b-5p, and miR-210-3p were quantified when R8-PNA-a425 was employed. (C) Specificity of R8-PNA-a15 was confirmed by quantifying miR-15b-5p, miR-425-3p, miR-584-5p, and miR-210-3p content in HT-29 R8-PNA-a15b-treated cells. (D) Effects on HT-29 cell growth of 4 and 8 µM R8-PNA-a584 (black boxes), R8-PNA-a425 (white boxes). (* p < 0.05, ** p < 0.01, ns: not significant, n = 3 independent experiments).

Figure 2. Effects of R8-PNA-a425 and R8-PNA-a584 on miRNA content. (A) Effects of anti-miRNA PNAs on their respective target miRNA, after performing 72 h of culturing of HT-29 cells. (B) Possible off-target effects were verified for both R8-PNA-a584 (white boxes) and R8-PNA-a425 (black boxes): miR-425-3p, miR-15b-5p, and miR-210-3p content was calculated for R8-PNA-a584-treated HT-29 cells, while miR-584-5p, miR-15b-5p, and miR-210-3p were quantified when R8-PNA-a425 was employed. (C) Specificity of R8-PNA-a15 was confirmed by quantifying miR-15b-5p, miR-425-3p, miR-584-5p, and miR-210-3p content in HT-29 R8-PNA-a15b-treated cells. (D) Effects on HT-29 cell growth of 4 and 8 µM R8-PNA-a584 (black boxes), R8-PNA-a425 (white boxes). (* p < 0.05, ** p < 0.01, ns: not significant, n = 3 independent experiments).

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Figure 3. Effects of R8-PNA-a425 and R8-PNA-a584 on apoptosis. (A) Effects of anti-miRNA PNAs on apoptosis induction in HT-29 cells treated for 72 h with 4 or 8 µM R8-PNA-a584 (black boxes) or R8-PNA-a425 (white boxes). (B) Representative plots of the apoptotic profile are reported. (C) Representative pictures showing the morphological appearance of HT-29-treated cells are reported. (* p < 0.05, ns: not significant; n = 3 independent experiments).

Figure 3. Effects of R8-PNA-a425 and R8-PNA-a584 on apoptosis. (A) Effects of anti-miRNA PNAs on apoptosis induction in HT-29 cells treated for 72 h with 4 or 8 µM R8-PNA-a584 (black boxes) or R8-PNA-a425 (white boxes). (B) Representative plots of the apoptotic profile are reported. (C) Representative pictures showing the morphological appearance of HT-29-treated cells are reported. (* p < 0.05, ns: not significant; n = 3 independent experiments).

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Figure 4. Effects of PNA combinations on apoptosis induction. (A,D) Effects of combinations of anti-miRNA PNAs on apoptosis induction in HT-29 (A) and LoVo (D) cells treated for 72 h with a 4 µM concentration of each PNA, as determined using Annexin V assay. Singularly administrated PNA (light gray boxes), combinations of two PNAs (dark gray boxes), triple-PNA combination (black box). The apoptotic profile was determined using Annexin V assay. (B,E) Effects of anti-miRNA PNA combinations on apoptosis induction in HT-29 (B) or LoVo (E) cells treated for 72 h with a 4 µM concentration of each PNA, as determined using Caspase 3/7 assay. Singularly administrated PNA (light gray boxes), combinations of two PNAs (dark gray boxes), triple-PNA combination (black box). (C,F) Representative plots obtained by Annexin V (upper line) and Caspase 3/7 (lower line) assays in HT-29 (C) or LoVo (F) cells. (* p < 0.05, ** p < 0.01, double-PNA treatment was compared with singularly administrated PNA, while triple-PNA treatment was compared with double-PNA combinations. n = 3 independent experiments).

Figure 4. Effects of PNA combinations on apoptosis induction. (A,D) Effects of combinations of anti-miRNA PNAs on apoptosis induction in HT-29 (A) and LoVo (D) cells treated for 72 h with a 4 µM concentration of each PNA, as determined using Annexin V assay. Singularly administrated PNA (light gray boxes), combinations of two PNAs (dark gray boxes), triple-PNA combination (black box). The apoptotic profile was determined using Annexin V assay. (B,E) Effects of anti-miRNA PNA combinations on apoptosis induction in HT-29 (B) or LoVo (E) cells treated for 72 h with a 4 µM concentration of each PNA, as determined using Caspase 3/7 assay. Singularly administrated PNA (light gray boxes), combinations of two PNAs (dark gray boxes), triple-PNA combination (black box). (C,F) Representative plots obtained by Annexin V (upper line) and Caspase 3/7 (lower line) assays in HT-29 (C) or LoVo (F) cells. (* p < 0.05, ** p < 0.01, double-PNA treatment was compared with singularly administrated PNA, while triple-PNA treatment was compared with double-PNA combinations. n = 3 independent experiments).

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Figure 5. Effects of anti-miRNA PNAs combined with sulforaphane (SFN) on apoptosis induction. (A,E) Effects of anti-miRNA PNA against miR-584 (R8-PNA-a584) combined with active SFN on apoptosis induction in HT-29 (A) and LoVo (E) cells treated for 72 h. Apoptosis profile was determined using Annexin V assay. (B,F) Representative Annexin V assay plots. (C,G) Effects of anti-miRNA PNA against miR-425 (R8-PNA-a425) combined with active SFN concentration on apoptosis induction in HT-29 (C) and LoVo (G) cells. Apoptosis profile was determined using Annexin V assay. (D,H) Representative plots obtained by Annexin V assays. (* p < 0.05, ** p < 0.01, ns: not significant, n = 3 independent experiments).

Figure 5. Effects of anti-miRNA PNAs combined with sulforaphane (SFN) on apoptosis induction. (A,E) Effects of anti-miRNA PNA against miR-584 (R8-PNA-a584) combined with active SFN on apoptosis induction in HT-29 (A) and LoVo (E) cells treated for 72 h. Apoptosis profile was determined using Annexin V assay. (B,F) Representative Annexin V assay plots. (C,G) Effects of anti-miRNA PNA against miR-425 (R8-PNA-a425) combined with active SFN concentration on apoptosis induction in HT-29 (C) and LoVo (G) cells. Apoptosis profile was determined using Annexin V assay. (D,H) Representative plots obtained by Annexin V assays. (* p < 0.05, ** p < 0.01, ns: not significant, n = 3 independent experiments).

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Table 1. Complete list of the employed miRNA-TaqMan assays.

Table 1. Complete list of the employed miRNA-TaqMan assays.

miRNA NameAssay IDhsa-miR-15b-5p000390hsa-miR-210-3p000512hsa-miR-425-3p002302hsa-miR-584-5p001624hsa-snRNA U6001973hsa-let-7c-5p000379

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