**3.9 Effect of miR-665 and siRNAs on histone acetylation**

Our results show that miR-665 and siRNA-HADC8, decreased total HDAC activity and decrease HDAC 8 protein, therefore, we measured the acetylation of histones in the cell extracts and the results were compared among all treatments. MiR-665 transfected cells show increases in the acetylation of histones Ac-H2B by 25%, Ac-H3 by 40% and Ac-H4 by 50% compared to negative control miRNA transfected cells (**Figure 6A**). miR-665 acetylates predominantly H3 and H4 histones.

Likewise, Si RNA-HDAC8 treated cells also show increases in the acetylation of histones Ac-H2B by 38%, acetylation of Ac-H3 by 58% and show higher acetylation of Ac-H4 by 2-fold (200%) compared to negative control siRNA treated cells

#### **Figure 5.**

*siRNA effects on neuroblastoma cells.siRNA specific for HDAC8 (siRNA-HDAC8) or c-MYC (siRNA-c-MYC, a mixture of 4 siRNAs) were used to confirm the effects of miR665 in neuroblastoma cells. (A) siRNA effect on cell proliferation. Neuroblastoma cells were transfected with 50 nM siRNA-HDAC8, 100 nM siRNA-c-MYC, or both siRNAs together. Cell viability was measured via MTS assay. SEM bars represent the standard deviation from two independent experiments with three biological replicates each. \*P < 0.005, \*\*P < 0.001, \*\*\*P = 6.8x10–5. (B) Cell extracts from negative control siRNA- or siRNA-HDAC8-treated cells were used to quantify HDAC8 levels via ELISA. HDAC8 was down regulated in HDAC8-siRNA-transfected cells \*P < 0.04. (C) Caspase 3 activity was quantified in cell extracts via Casp-3 kit. Caspase 3 activity increased in siRNA-HDAC8- and siRNA-c-MYC-transfected cells. SEM bars represent the standard deviation from two independent experiments with two biological replicates each. \*P < 0.01, \*\*P < 0.004. (figures were printed from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

(**Figure 6B**). siRNA-HDAC8 acetylated predominately AC-H4 and correlate with the results of miR-665.

#### **3.10 miR-665 targets c-MYC and HDAC8**

Taken together, our results indicate that miR-665 targets c-MYC and HDAC8, decreasing their expression, increasing histone acetylation, and modulating expression of cell proliferation related genes. We propose a model (**Figure 7**)

**111**

cell proliferation [10].

*N.Prashad Vol 9, 33186–33201, 2018).*

**Figure 7.**

**Figure 6.**

**cells in vitro**

siRNA-MYC + siRNA- HDAC8.

inhibition of neuroblastoma cell proliferation.

*Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells…*

*Histone acetylation. Cell extracts from negative control miRNA-, miR-665-, negative control siRNA-, or siRNA-HDAC8 treated cells were used to quantitate histone acetylation via ELISA. Data represent standard deviations from two independent experiments. Negative control miRNA (white) and miR-665 transfection increased histone acetylation (black) (A) \*P < 0.01, \*\*P < 0.01, \*\*\*P < 0.04. Negative control siRNA (white) and siRNA-HDAC8 increased histone acetylation (black) (B) \*P < 0.008, \*\*P < 0.04, \*\*\*P < 0.001.*

illustrating suppressor miR-665 involvement in the inhibition of neuroblastoma

*Proposed model illustrating how suppressor miR-665 targets c-MYC and HDAC8 to inhibit neuroblastoma cell proliferation and maintain cellular homeostasis. (figures were printed from published article in "Oncotarget",* 

**3.11 Effects of combination of siRNA-HDAC 8 ± siRNA-MYC on neuroblastoma** 

On the basis of these results, we hypothesized that neuroblastoma tumor xenograft growth in mice can be inhibited when treated with the combination of

When cells were treated with the combination of siRNA HDAC 8 + siRNA-MYC, cell proliferation was inhibited by 86% [10]. Therefore, HDAC 8 and MYC are critical targets and effective blockade of both targets is required to ensure a maximum

*DOI: http://dx.doi.org/10.5772/intechopen.96021*

*Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells… DOI: http://dx.doi.org/10.5772/intechopen.96021*

#### **Figure 6.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

(**Figure 6B**). siRNA-HDAC8 acetylated predominately AC-H4 and correlate with

*siRNA effects on neuroblastoma cells.siRNA specific for HDAC8 (siRNA-HDAC8) or c-MYC (siRNA-c-MYC, a mixture of 4 siRNAs) were used to confirm the effects of miR665 in neuroblastoma cells. (A) siRNA effect on cell proliferation. Neuroblastoma cells were transfected with 50 nM siRNA-HDAC8, 100 nM siRNA-c-MYC, or both siRNAs together. Cell viability was measured via MTS assay. SEM bars represent the standard deviation from two independent experiments with three biological replicates each. \*P < 0.005, \*\*P < 0.001, \*\*\*P = 6.8x10–5. (B) Cell extracts from negative control siRNA- or siRNA-HDAC8-treated cells were used to quantify HDAC8 levels via ELISA. HDAC8 was down regulated in HDAC8-siRNA-transfected cells \*P < 0.04. (C) Caspase 3 activity was quantified in cell extracts via Casp-3 kit. Caspase 3 activity increased in siRNA-HDAC8- and siRNA-c-MYC-transfected cells. SEM bars represent the standard deviation from two independent experiments with two biological replicates each. \*P < 0.01, \*\*P < 0.004. (figures were printed* 

Taken together, our results indicate that miR-665 targets c-MYC and HDAC8,

decreasing their expression, increasing histone acetylation, and modulating expression of cell proliferation related genes. We propose a model (**Figure 7**)

**110**

**Figure 5.**

the results of miR-665.

**3.10 miR-665 targets c-MYC and HDAC8**

*from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

*Histone acetylation. Cell extracts from negative control miRNA-, miR-665-, negative control siRNA-, or siRNA-HDAC8 treated cells were used to quantitate histone acetylation via ELISA. Data represent standard deviations from two independent experiments. Negative control miRNA (white) and miR-665 transfection increased histone acetylation (black) (A) \*P < 0.01, \*\*P < 0.01, \*\*\*P < 0.04. Negative control siRNA (white) and siRNA-HDAC8 increased histone acetylation (black) (B) \*P < 0.008, \*\*P < 0.04, \*\*\*P < 0.001.*

#### **Figure 7.**

*Proposed model illustrating how suppressor miR-665 targets c-MYC and HDAC8 to inhibit neuroblastoma cell proliferation and maintain cellular homeostasis. (figures were printed from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

illustrating suppressor miR-665 involvement in the inhibition of neuroblastoma cell proliferation [10].

#### **3.11 Effects of combination of siRNA-HDAC 8 ± siRNA-MYC on neuroblastoma cells in vitro**

When cells were treated with the combination of siRNA HDAC 8 + siRNA-MYC, cell proliferation was inhibited by 86% [10]. Therefore, HDAC 8 and MYC are critical targets and effective blockade of both targets is required to ensure a maximum inhibition of neuroblastoma cell proliferation.

On the basis of these results, we hypothesized that neuroblastoma tumor xenograft growth in mice can be inhibited when treated with the combination of siRNA-MYC + siRNA- HDAC8.
