**1. Introduction**

Neuroblastoma is the most frequently diagnosed extracranial solid tumor in children. About 90% of cases occur in children less than 5 years old and it is rare in adults. Of cancer deaths in children, about 15% are due to neuroblastoma [1]. Chances of long-term survival, however, are less than 40% despite aggressive treatment [2].

MYC is an oncogenic transcription factor that is overexpressed in many types of cancer. MYC has been shown to directly upregulate a protumorigenic group of miRNAs and represses several suppressor miRNAs, thus contributing to tumorigenesis [3]. For example, MYC overexpression can upregulate the oncogenic miR-17-92 cluster, that are directly activated in lymphoma [4], and can also repress several suppressor miRNAs [3]. The MYC gene is amplified in various human cancers, including in lung carcinoma, breast carcinoma, and colon carcinoma [5].

Histone deacetylases affect gene expression by altering the histone acetylation status, and that as a consequence, HDAC overexpression contribute to tumorigenesis by affecting the expression of key mRNAs and miRNAs. HDACs are overexpressed in most cancers, leading to histone deacetylation, inhibition of growth- suppressive genes, and increased cell proliferation [6]. HDAC8 overexpression correlates with advanced neuroblastoma in patient tumor samples, and HDAC8 inhibition reduced cell proliferation and induced neuroblastoma cell differentiation [7]. HDAC inhibitors reduced the proliferation and induced the apoptosis of neuroblastoma cells in vitro and in vivo in mice [8, 9].

Given that both MYC and HDACs play an important role in the maintenance of the normal cellular physiological functions and that their overexpression is linked to neuroblastoma tumorigenesis, we asked whether the levels of both MYC and HDAC8 should be reduced to obtain significant inhibition of cell proliferation.

Our results demonstrate that miR-665 targets c-MYC and HDAC8 m RNA, miR-665 treatment also increased the percentage of cells in G1 phase and reduced the percentage of cells in S phase of the cell cycle. This is the first report to show that miR-665 is a suppressor miRNA directly targeting the 3'-UTR of c-MYC and HDAC8 in neuroblastoma [10].

We investigated the effects of small interfering RNAs (siRNAs) targeting HDAC8 and MYC in murine neuroblastoma cells. RNA interference is a process of posttranscriptional gene silencing in which a double stranded RNA inhibits gene expression in a sequence-dependent manner via degradation of the corresponding mRNA. siRNAs can be used as potent and specific tools for gene knockdown. Several laboratories have reported siRNA targeting of gene expression in cancer cells and the inhibition of cell proliferation *in vitro* and tumor growth *in vivo* [11–14].

We reported that *in vitro*, single-agent siRNA HDAC8 or siRNA-MYC inhibited cell proliferation by 40–50%; however, treatment with the combination of siRNA MYC + siRNA-HDAC8 inhibited cell proliferation by 86% [10]. To further confirm these findings in an animal model, we set out to verify if tumor growth can be inhibited in a neuroblastoma xenograft mouse model when tumors are treated with a combination of siRNA-MYC and siRNA HDAC8. Our findings from this study show that the tumor growth was reduced by 80% following intratumoral delivery of a combination of siRNAs targeting both MYC and HDAC8 simultaneously [15].

### **2. Materials and methods**

#### **2.1 Reagents**

Cell culture media, DMEM with high glucose (D6429), essential and nonessential amino acids (M5550, m7145), Bt2c AMP (D0627), the colorimetric Caspase 3 kit (Code CASP-3-C), and propidium iodide (P4170) were purchased from Sigma Aldrich, St. Louis. Fetal bovine serum (FBS) was purchased from Phenix Research Products, Candler, NC, USA. BD-Falcon tissue culture 96-well plates (353072) were purchased from BD Biosciences. The RNA extraction miR-Neasy kit (Cat No. 217084) was purchased from Qiagen, Germantown, MD, USA.

**101**

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

The MTS Cell. Titer 96 Aqueous One Solution (Cat # G3580) cell proliferation assay was purchased from Promega Biotechnology, Madison,WI, USA. The HDAC Kit (#K331–100) was purchased from BioVision, Inc. Co rning. 96-well EIA/RIA plates (CLS3369) were used for ELISA. Antibodies for HDAC 8, H-145 (sc11405), C MYC, C-19 (SC-786), acetylated Histones, Ac- H2B, Lys 5/12/15/20 (SC-8652), Ac-H3, lys9 (sc-8655), Ac-H4, lys16 (sc-8662), and siRNA for c-MYC (pool of 4 different siRNA duplexes, sc-29227) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Negative control #2 siRNA (#4390846), siRNA-HDAC 8 (S88696) and Lipofectamine RNAi Max (#13778075) were purchased from Life Technologies/ Ambion/ Invitrogen. Negative control miRNA Cel-miR-67 (#CN-001000) sequences based on *C. elegans* miRNA, mimic hsa-miR-665 (#C 301246–01), and transfection reagent Dharmafect Duo (#T2010–01) were purchased from Dharmacon. Luciferase expression plasmids with the 3'-UTR for HDAC8

(#S804229), C-MYC (#S804638), MYCN (Product No S807230), or empty vector without 3'-UTR (#S890005), and the LightSwitch luciferase assay kit (#32031,

Mouse neuroblastoma cholinergic clonal cells (S20) were obtained from Dr. Marshall Nirenberg of The US National Institutes of Health (NIH). Cells were grown in monolayers in DMEM supplemented with essential and nonessential amino acids,

After 48–72 h, cell viability was measured colorimetrically using the MTS Cell Titer 96 Aqueous One Solution. Samples were incubated at 37°C for 3–4 h and samples were read at 490 nm in a plate reader according to the manufacturer's instructions.

trypsinized, treated with 75% ethanol and 100ug/ml RNAse A, and then stained with propodium iodide (PI). Untreated and (20,000 cells/ sample) were analyzed for cell cycle distribution via flow cytometry at the Core lab of Children's Cancer

The effects of miR-665 or siRNA on cell proliferation were determined using reverse transfection. First, 100 nM negative control miRNA, miR-665, negative control siRNA, C-MYC siRNA, or HDAC8 siRNA was mixed with Lipofectamine

miRNA effects on the cell cycle were assessed using reverse transfection of cells with 100 nM negative control miRNA or miR-665 mimic plus Lipofectamine RNAimax. The

96-well plates. After 48–72 h, cell viability was measured using MTS Cell Titer 96 Aqueous One Solution and incubated at 37°C for 3–4 h. Samples were read at

After 48 h, cells were trypsinized, treated with 75% ethanol and 100ug/ml RNAse A, and then stained with PI. For cell cycle analysis, 20,000 cells/sample were analyzed via flow cytometry in the Core lab of Children's Cancer Center Hospital, Houston, TX.

Cell extracts were prepared from untreated,, and miRNA transfected cells for target assays. miRNA-transfected cells were reverse transfected with 100 nM

cells per well and

cells were plated in T25 flasks. After 48 h, cells were

cells, which were then plated in

cells, which were then plated in a T25 flask.

penicillin/streptomycin, and 10% FBS at 37°C with 5% CO2 and humidity.

Neuroblastoma cells were plated in 96-well plates at 12x103

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

LS010) were purchased from Active Motif, CA, USA.

**2.2 Cells and cell culture**

For cell cycle analysis, 1x106

Center Hospital, Houston, TX, USA.

RNAimax. This mixture was added to 12x103

transfection mixture was added to 1x106

**2.4 Whole cell extracts**

490 nm according to manufacturer's instructions.

**2.3 Transfections**

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

The MTS Cell. Titer 96 Aqueous One Solution (Cat # G3580) cell proliferation assay was purchased from Promega Biotechnology, Madison,WI, USA. The HDAC Kit (#K331–100) was purchased from BioVision, Inc. Co rning. 96-well EIA/RIA plates (CLS3369) were used for ELISA. Antibodies for HDAC 8, H-145 (sc11405), C MYC, C-19 (SC-786), acetylated Histones, Ac- H2B, Lys 5/12/15/20 (SC-8652), Ac-H3, lys9 (sc-8655), Ac-H4, lys16 (sc-8662), and siRNA for c-MYC (pool of 4 different siRNA duplexes, sc-29227) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Negative control #2 siRNA (#4390846), siRNA-HDAC 8 (S88696) and Lipofectamine RNAi Max (#13778075) were purchased from Life Technologies/ Ambion/ Invitrogen. Negative control miRNA Cel-miR-67 (#CN-001000) sequences based on *C. elegans* miRNA, mimic hsa-miR-665 (#C 301246–01), and transfection reagent Dharmafect Duo (#T2010–01) were purchased from Dharmacon. Luciferase expression plasmids with the 3'-UTR for HDAC8 (#S804229), C-MYC (#S804638), MYCN (Product No S807230), or empty vector without 3'-UTR (#S890005), and the LightSwitch luciferase assay kit (#32031, LS010) were purchased from Active Motif, CA, USA.

#### **2.2 Cells and cell culture**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

neuroblastoma cells in vitro and in vivo in mice [8, 9].

HDAC8 in neuroblastoma [10].

**2. Materials and methods**

**2.1 Reagents**

MYC is an oncogenic transcription factor that is overexpressed in many types of cancer. MYC has been shown to directly upregulate a protumorigenic group of miRNAs and represses several suppressor miRNAs, thus contributing to tumorigenesis [3]. For example, MYC overexpression can upregulate the oncogenic miR-17-92 cluster, that are directly activated in lymphoma [4], and can also repress several suppressor miRNAs [3]. The MYC gene is amplified in various human cancers, including in lung carcinoma, breast carcinoma, and colon carcinoma [5].

Histone deacetylases affect gene expression by altering the histone acetylation status, and that as a consequence, HDAC overexpression contribute to tumorigenesis by affecting the expression of key mRNAs and miRNAs. HDACs are overexpressed in most cancers, leading to histone deacetylation, inhibition of growth- suppressive genes, and increased cell proliferation [6]. HDAC8 overexpression correlates with advanced neuroblastoma in patient tumor samples, and HDAC8 inhibition reduced cell proliferation and induced neuroblastoma cell differentiation [7]. HDAC inhibitors reduced the proliferation and induced the apoptosis of

Given that both MYC and HDACs play an important role in the maintenance of the normal cellular physiological functions and that their overexpression is linked to neuroblastoma tumorigenesis, we asked whether the levels of both MYC and HDAC8 should be reduced to obtain significant inhibition of cell proliferation. Our results demonstrate that miR-665 targets c-MYC and HDAC8 m RNA, miR-665 treatment also increased the percentage of cells in G1 phase and reduced the percentage of cells in S phase of the cell cycle. This is the first report to show that miR-665 is a suppressor miRNA directly targeting the 3'-UTR of c-MYC and

We investigated the effects of small interfering RNAs (siRNAs) targeting HDAC8 and MYC in murine neuroblastoma cells. RNA interference is a process of posttranscriptional gene silencing in which a double stranded RNA inhibits gene expression in a sequence-dependent manner via degradation of the corresponding mRNA. siRNAs can be used as potent and specific tools for gene knockdown. Several laboratories have reported siRNA targeting of gene expression in cancer cells and the

We reported that *in vitro*, single-agent siRNA HDAC8 or siRNA-MYC inhibited cell proliferation by 40–50%; however, treatment with the combination of siRNA MYC + siRNA-HDAC8 inhibited cell proliferation by 86% [10]. To further confirm these findings in an animal model, we set out to verify if tumor growth can be inhibited in a neuroblastoma xenograft mouse model when tumors are treated with a combination of siRNA-MYC and siRNA HDAC8. Our findings from this study show that the tumor growth was reduced by 80% following intratumoral delivery of a combination of siRNAs targeting both MYC and HDAC8 simultaneously [15].

Cell culture media, DMEM with high glucose (D6429), essential and nonessential amino acids (M5550, m7145), Bt2c AMP (D0627), the colorimetric Caspase 3 kit (Code CASP-3-C), and propidium iodide (P4170) were purchased from Sigma Aldrich, St. Louis. Fetal bovine serum (FBS) was purchased from Phenix Research Products, Candler, NC, USA. BD-Falcon tissue culture 96-well plates (353072) were purchased from BD Biosciences. The RNA extraction miR-Neasy kit (Cat No. 217084) was purchased from Qiagen, Germantown, MD, USA.

inhibition of cell proliferation *in vitro* and tumor growth *in vivo* [11–14].

**100**

Mouse neuroblastoma cholinergic clonal cells (S20) were obtained from Dr. Marshall Nirenberg of The US National Institutes of Health (NIH). Cells were grown in monolayers in DMEM supplemented with essential and nonessential amino acids, penicillin/streptomycin, and 10% FBS at 37°C with 5% CO2 and humidity.

Neuroblastoma cells were plated in 96-well plates at 12x103 cells per well and After 48–72 h, cell viability was measured colorimetrically using the MTS Cell Titer 96 Aqueous One Solution. Samples were incubated at 37°C for 3–4 h and samples were read at 490 nm in a plate reader according to the manufacturer's instructions.

For cell cycle analysis, 1x106 cells were plated in T25 flasks. After 48 h, cells were trypsinized, treated with 75% ethanol and 100ug/ml RNAse A, and then stained with propodium iodide (PI). Untreated and (20,000 cells/ sample) were analyzed for cell cycle distribution via flow cytometry at the Core lab of Children's Cancer Center Hospital, Houston, TX, USA.

#### **2.3 Transfections**

The effects of miR-665 or siRNA on cell proliferation were determined using reverse transfection. First, 100 nM negative control miRNA, miR-665, negative control siRNA, C-MYC siRNA, or HDAC8 siRNA was mixed with Lipofectamine RNAimax. This mixture was added to 12x103 cells, which were then plated in 96-well plates. After 48–72 h, cell viability was measured using MTS Cell Titer 96 Aqueous One Solution and incubated at 37°C for 3–4 h. Samples were read at 490 nm according to manufacturer's instructions.

miRNA effects on the cell cycle were assessed using reverse transfection of cells with 100 nM negative control miRNA or miR-665 mimic plus Lipofectamine RNAimax. The transfection mixture was added to 1x106 cells, which were then plated in a T25 flask. After 48 h, cells were trypsinized, treated with 75% ethanol and 100ug/ml RNAse A, and then stained with PI. For cell cycle analysis, 20,000 cells/sample were analyzed via flow cytometry in the Core lab of Children's Cancer Center Hospital, Houston, TX.

#### **2.4 Whole cell extracts**

Cell extracts were prepared from untreated,, and miRNA transfected cells for target assays. miRNA-transfected cells were reverse transfected with 100 nM negative control miRNA, miR-665, negative control siRNA, c-MYC siRNA, or HDAC8 siRNA plus Lipofectamine RNAimax. Transfected cells were plated in T25 flasks. After 48–72 h, cell extracts were prepared in assay buffer as described by Khandelia, *et al.* [16]. Assay buffer consisted of 20 mM Tris–HCL pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Nonidet P40, and protease inhibitor cocktail from Sigma (P8340). Protein concentrations were determined using Pierce's BCA Assay as per the manufacturer's instructions.

miR-665 inhibit cell growth compared to untreated cells and cells treated with negative control miRNA. Assays were normalized using equal concentrations of protein (50–100 ug) from untreated, negative control miRNA-, and miR-665 treated cells in assessing total HDAC and Caspase 3 activity, and HDAC8 and c-MYC levels via ELISA.
