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Therapeutic implications of activation of the host gene (Dleu2) promoter for miR-15a/16-1 in chronic lymphocytic leukemia.

Kasar S, Underbayev C, Yuan Y, Hanlon M, Aly S, Khan H, Chang V, Batish M, Gavrilova T, Badiane F, Degheidy H, Marti G, Raveche E - Oncogene (2013)

Bottom Line: Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1.CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells.With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs).

View Article: PubMed Central - PubMed

Affiliation: 1] New Jersey Medical School, Rutgers, Newark, NJ, USA [2].

ABSTRACT
Genetic lesions and other regulatory events lead to silencing of the 13q14 locus in a majority of chronic lymphocytic leukemia (CLL) patients. This locus encodes a pair of critical proapoptotic microRNAs, miR-15a/16-1. Decreased levels of miR-15a/16-1 are critical for the increased survival exhibited by CLL cells. Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1. In this paper, we have identified additional molecular mechanisms regulating miR-15a/16-1 levels and have shown that the transcription factor BSAP (B-cell-specific activator protein) directly interacts with Dleu2, the host gene containing the miR-15a/16-1 loci, and by negative regulation of the Dleu2 promoter, results in repression of miR-15a/16-1 expression. CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells. With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs). BSAP knockdown led to an increase in the expression of miR-15a/16-1 and an increase in apoptosis, and a cell cycle arrest in both the cell line and patient PBMCs. Moreover, using Dleu2 promoter analysis by chromatin immunoprecipitation assay, we have shown that BSAP directly interacts with the Dleu2 promoter. Derepression of the Dleu2 promoter via inhibition of histone deacetylation combined with BSAP knockdown increased miR-15a/16-1 expression, and also increased malignant B-cell death. In summary, therapy targeting enhanced host gene Dleu2 transcription may augment CLL therapy.

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BSAP Knockdown Leads to Increased miR-15a/16-1 and Cell Cycle Arrest in vitro3ug siRNA BSAP or non-targeting siRNA (Neg.CTRL) was transiently transfected into the murine NZB CLL cell line LNC and analyzed at the indicated time-points. A) Representative histogram overlay of cells transfected with either siRNA BSAP (black dotted line) or with Neg.CTRL (Grey filled) and stained with BSAP-PE antibody. B) The BSAP MFI ± SEM at indicated time-points post transfection with siRNA BSAP (Black bar) or with Neg.CTRL (Grey Bar). n=3, *p<0.05, C) Average miR-15a levels 24hr post-transfection, n=6, *p<0.05 D) Percentage of cells in the G1 and S phase of the cell cycle at 36hr post-transfection. n=7, *p<0.01. E) Percent change in cells in G1 phase and S phase, 36hr post-transfection. Percent Change = [(%G1 or %S in siRNA BSAP group − %G1 or %S in Neg.CTRL group)/(%G1 or %S in Neg.CTRL group)]*100. n=7, *p<0.01, large effect size. F) Decrease in the CyclinD1 MFI in the siRNA BSAP group in relation to the Neg.CTRL group, 24hr post transfection. n=3. Statistics employed: Two-tailed paired Student’s t test.
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Figure 5: BSAP Knockdown Leads to Increased miR-15a/16-1 and Cell Cycle Arrest in vitro3ug siRNA BSAP or non-targeting siRNA (Neg.CTRL) was transiently transfected into the murine NZB CLL cell line LNC and analyzed at the indicated time-points. A) Representative histogram overlay of cells transfected with either siRNA BSAP (black dotted line) or with Neg.CTRL (Grey filled) and stained with BSAP-PE antibody. B) The BSAP MFI ± SEM at indicated time-points post transfection with siRNA BSAP (Black bar) or with Neg.CTRL (Grey Bar). n=3, *p<0.05, C) Average miR-15a levels 24hr post-transfection, n=6, *p<0.05 D) Percentage of cells in the G1 and S phase of the cell cycle at 36hr post-transfection. n=7, *p<0.01. E) Percent change in cells in G1 phase and S phase, 36hr post-transfection. Percent Change = [(%G1 or %S in siRNA BSAP group − %G1 or %S in Neg.CTRL group)/(%G1 or %S in Neg.CTRL group)]*100. n=7, *p<0.01, large effect size. F) Decrease in the CyclinD1 MFI in the siRNA BSAP group in relation to the Neg.CTRL group, 24hr post transfection. n=3. Statistics employed: Two-tailed paired Student’s t test.

Mentions: The NZB derived CLL cell line LNC was transfected with either 3ug non-targeting negative control siRNA (Neg.CTRL) or with 3ug siRNA BSAP (siRNA-BSAP) using Amaxa Nucleofection, in order to transiently knockdown BSAP. The expression of BSAP was reduced as early as 12hr post transfection and the suppression began to diminish after 48hr. Flow cytometric analysis via histogram overlays of BSAP levels on a per cell basis showed a decrease in BSAP in siRNA treated cells compared to Neg.CTRL treated cells as early as 12hr post-transfection (Fig. 5A). The average BSAP MFI at different time-points post-transfection is shown in Fig. 5B and was found to be lowest at 24hr post siRNA-BSAP treatment when compared to the Neg.CTRL at that time-point (p<0.05). BSAP protein was reduced by 53% at 24hr as compared to the Neg.CTRL. The miR-15a levels were measured in cells with successful knocked down BSAP to determine the potential causal relationship between BSAP and miR-15a/16-1 expression in our system. miR-15a level was measured using TaqMan MicroRNA Assays and an approximately two-fold increase in miR-15a levels was observed in the siRNA-BSAP treated cells at 24hr (p<0.05) (Fig. 5C). Interestingly, the increase in miR-15a expression corresponded with the peak reduction in BSAP suggesting a strong influence of BSAP on the expression of the microRNA. LNC cells mimic aggressive CLL and are very rapidly dividing. We and others have shown that an increase in miR-15a/16-1 leads to cell cycle arrest and reduced proliferation [30–32]. Initially, we have shown that reducing BSAP levels results in an increase in miR-15a/16-1 expression. To test the biological significance of this, the increased miR-15a/16-1 should result in decreased downstream targets like cyclin D leading to cell cycle arrest. As compared to the Neg.CTRL treated cells which have baseline level of BSAP and miR-15a/16-1, the siRNA-BSAP treated cells exhibited a significant increase in percentage of cells in G1 and a reduction in the percentage of cells in S phase (Fig. 5D and 5E, p<0.05). Moreover, we also observed a decrease in the expression of Cyclin D1 in the siRNA-BSAP treated cells as compared to the Neg.CTRL treated cells (Fig. 5F) indicating that the cell cycle arrest is a direct consequence of increased miR-15a/16-1 since it has been shown to be a key regulator of Cyclin D1.


Therapeutic implications of activation of the host gene (Dleu2) promoter for miR-15a/16-1 in chronic lymphocytic leukemia.

Kasar S, Underbayev C, Yuan Y, Hanlon M, Aly S, Khan H, Chang V, Batish M, Gavrilova T, Badiane F, Degheidy H, Marti G, Raveche E - Oncogene (2013)

BSAP Knockdown Leads to Increased miR-15a/16-1 and Cell Cycle Arrest in vitro3ug siRNA BSAP or non-targeting siRNA (Neg.CTRL) was transiently transfected into the murine NZB CLL cell line LNC and analyzed at the indicated time-points. A) Representative histogram overlay of cells transfected with either siRNA BSAP (black dotted line) or with Neg.CTRL (Grey filled) and stained with BSAP-PE antibody. B) The BSAP MFI ± SEM at indicated time-points post transfection with siRNA BSAP (Black bar) or with Neg.CTRL (Grey Bar). n=3, *p<0.05, C) Average miR-15a levels 24hr post-transfection, n=6, *p<0.05 D) Percentage of cells in the G1 and S phase of the cell cycle at 36hr post-transfection. n=7, *p<0.01. E) Percent change in cells in G1 phase and S phase, 36hr post-transfection. Percent Change = [(%G1 or %S in siRNA BSAP group − %G1 or %S in Neg.CTRL group)/(%G1 or %S in Neg.CTRL group)]*100. n=7, *p<0.01, large effect size. F) Decrease in the CyclinD1 MFI in the siRNA BSAP group in relation to the Neg.CTRL group, 24hr post transfection. n=3. Statistics employed: Two-tailed paired Student’s t test.
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Figure 5: BSAP Knockdown Leads to Increased miR-15a/16-1 and Cell Cycle Arrest in vitro3ug siRNA BSAP or non-targeting siRNA (Neg.CTRL) was transiently transfected into the murine NZB CLL cell line LNC and analyzed at the indicated time-points. A) Representative histogram overlay of cells transfected with either siRNA BSAP (black dotted line) or with Neg.CTRL (Grey filled) and stained with BSAP-PE antibody. B) The BSAP MFI ± SEM at indicated time-points post transfection with siRNA BSAP (Black bar) or with Neg.CTRL (Grey Bar). n=3, *p<0.05, C) Average miR-15a levels 24hr post-transfection, n=6, *p<0.05 D) Percentage of cells in the G1 and S phase of the cell cycle at 36hr post-transfection. n=7, *p<0.01. E) Percent change in cells in G1 phase and S phase, 36hr post-transfection. Percent Change = [(%G1 or %S in siRNA BSAP group − %G1 or %S in Neg.CTRL group)/(%G1 or %S in Neg.CTRL group)]*100. n=7, *p<0.01, large effect size. F) Decrease in the CyclinD1 MFI in the siRNA BSAP group in relation to the Neg.CTRL group, 24hr post transfection. n=3. Statistics employed: Two-tailed paired Student’s t test.
Mentions: The NZB derived CLL cell line LNC was transfected with either 3ug non-targeting negative control siRNA (Neg.CTRL) or with 3ug siRNA BSAP (siRNA-BSAP) using Amaxa Nucleofection, in order to transiently knockdown BSAP. The expression of BSAP was reduced as early as 12hr post transfection and the suppression began to diminish after 48hr. Flow cytometric analysis via histogram overlays of BSAP levels on a per cell basis showed a decrease in BSAP in siRNA treated cells compared to Neg.CTRL treated cells as early as 12hr post-transfection (Fig. 5A). The average BSAP MFI at different time-points post-transfection is shown in Fig. 5B and was found to be lowest at 24hr post siRNA-BSAP treatment when compared to the Neg.CTRL at that time-point (p<0.05). BSAP protein was reduced by 53% at 24hr as compared to the Neg.CTRL. The miR-15a levels were measured in cells with successful knocked down BSAP to determine the potential causal relationship between BSAP and miR-15a/16-1 expression in our system. miR-15a level was measured using TaqMan MicroRNA Assays and an approximately two-fold increase in miR-15a levels was observed in the siRNA-BSAP treated cells at 24hr (p<0.05) (Fig. 5C). Interestingly, the increase in miR-15a expression corresponded with the peak reduction in BSAP suggesting a strong influence of BSAP on the expression of the microRNA. LNC cells mimic aggressive CLL and are very rapidly dividing. We and others have shown that an increase in miR-15a/16-1 leads to cell cycle arrest and reduced proliferation [30–32]. Initially, we have shown that reducing BSAP levels results in an increase in miR-15a/16-1 expression. To test the biological significance of this, the increased miR-15a/16-1 should result in decreased downstream targets like cyclin D leading to cell cycle arrest. As compared to the Neg.CTRL treated cells which have baseline level of BSAP and miR-15a/16-1, the siRNA-BSAP treated cells exhibited a significant increase in percentage of cells in G1 and a reduction in the percentage of cells in S phase (Fig. 5D and 5E, p<0.05). Moreover, we also observed a decrease in the expression of Cyclin D1 in the siRNA-BSAP treated cells as compared to the Neg.CTRL treated cells (Fig. 5F) indicating that the cell cycle arrest is a direct consequence of increased miR-15a/16-1 since it has been shown to be a key regulator of Cyclin D1.

Bottom Line: Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1.CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells.With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs).

View Article: PubMed Central - PubMed

Affiliation: 1] New Jersey Medical School, Rutgers, Newark, NJ, USA [2].

ABSTRACT
Genetic lesions and other regulatory events lead to silencing of the 13q14 locus in a majority of chronic lymphocytic leukemia (CLL) patients. This locus encodes a pair of critical proapoptotic microRNAs, miR-15a/16-1. Decreased levels of miR-15a/16-1 are critical for the increased survival exhibited by CLL cells. Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1. In this paper, we have identified additional molecular mechanisms regulating miR-15a/16-1 levels and have shown that the transcription factor BSAP (B-cell-specific activator protein) directly interacts with Dleu2, the host gene containing the miR-15a/16-1 loci, and by negative regulation of the Dleu2 promoter, results in repression of miR-15a/16-1 expression. CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells. With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs). BSAP knockdown led to an increase in the expression of miR-15a/16-1 and an increase in apoptosis, and a cell cycle arrest in both the cell line and patient PBMCs. Moreover, using Dleu2 promoter analysis by chromatin immunoprecipitation assay, we have shown that BSAP directly interacts with the Dleu2 promoter. Derepression of the Dleu2 promoter via inhibition of histone deacetylation combined with BSAP knockdown increased miR-15a/16-1 expression, and also increased malignant B-cell death. In summary, therapy targeting enhanced host gene Dleu2 transcription may augment CLL therapy.

Show MeSH
Related in: MedlinePlus