**3.2 miR-665 targets HDAC 8, c MYC and MYCN**

Computational algorithm prediction site TargetScan and miRanda (microRNA. org) predicts mRNA targets for Mirna*. miranda* predicted hsa-miR-665 targets 3' UTR of HDAC 8 and the sequence alignment is presented in **Figure 2A***. miranda*, also predicts that hsa-miR-665 targets MYCN 3' UTR and the sequence alignment is presented in **Figure 2B.**

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MYC (**Figure 2C**).

**Figure 1.**

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

MYC is overexpressed in 30% of all human cancers and frequently predicts for a poor clinical outcome, and deregulated expression of MYC is a hallmark feature of cancer [3] miRanda and Targetscan did not include 3' UTR of C MYC as miR-665 target. Therefore, Complimentary sequences between miR-665 and 3' UTR of C MYC were compared at online pairwise sequence alignment site www.ebi.ac.uk. Results show two complementary binding sites for miR-665 in the 3' UTR of c

*miR-665 effects on cell proliferation (A) cells treated with 100 nM negative control miRNA and miR-665 (B) for 72 hr. were prepared for cell cycle distribution analysis. Propidium iodide stained cells were analyzed by FLOW cytometry (C) Several concentration of miR-665 effect on cell viability (D) STDEV was used for +/*− *standard error bar; data is from 2 independent experiments with 3 biological replicates for each experiment was used. (figures were printed from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

First we measured total HDAC activity in the cell extracts prepared from negative control miRNA and miR-665 transfected cells in the presence of acetylated HDAC substrate Ac-Lys (Ac)-p NA and deacetylated end product was measured colorimetrically. HDAC 8 and c MYC proteins were measured with antibody in ELISA. The results show that total Pan HDAC activity was decreased by 40%, and HDAC 8 and c MYC proteins were decreased by 40% in miR-665 transfected cells

Next, we tested whether HDAC 8, c MYC and MYCN genes are the direct target of miR-665. In these experiments HepG2 cells were used because miR-665 does not affect the growth of these cells (our unpublished results). Luciferase reporter

compared to negative control miRNA treated cells (**Figure 3A–C**).

*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 1.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

extracts for ELISA.

**2.13 Statistical analysis**

statistically significant.

**3.1 miR-665 inhibits cell proliferation**

**3. Results**

**2.12 Tumor extract preparation**

Antisense Sequence: UUUUUGAGGAUCACUACCUtg.

(#13778075) were purchased from Life Technologies/Ambion.

using Pierce's BCA Assay as per the manufacturer's instructions.

Negative control #2 siRNA (#4390846), and Lipofectamine RNAi Max

A total of 3 nmol Negative control siRNA or 3 nmol combinations of siRNA-MYC + siRNA-HDAC8 were mixed with Lipofectamine RNAi max (Liposome) in DMEM media without fetal bovine serum and without antibiotic. siRNA complexed with Lipofectamine in a volume of 30 ul was delivered into tumors by intratumoral injection every third day. Tumors were measured every second day with a caliper and mice were weighed every third day. Tumor volume was calculator with a formula, V = Length X width2/2. Experiment was stopped when the control tumors treated with negative control siRNA reached a tumor burden volume of 1200 mm3. Mice were euthanized by CO2 inhalation 2 days after last treatment with siRNA. Tumors were removed and weighed. Tumors were frozen in liquid nitrogen and stored at -80o C freezers until used for preparation of tumor

Tumors treated with NC-siRNA or with combined siRNA-HDAC8 + siRNA-MYC were cut into small pieces and homogenized in assay buffer in a glass homogenizer. Assay buffer as described by Khandelia et al. [16], 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

Error bars represent standard error of the mean (SEM) from 2 to 3 biological replicates from 3 to 5 independent experiments. P-values were calculated using T.Test (2 tailed, 3 samples, unequal variance) and p < 0.05 was considered

NB cells transfected with miR-665 show changes in cell morphology, lost the normal spindle shape and cells grew in clumps without processes compared to cells transfected with negative control miRNA **(Figure 1A** and **B**). Cell cycle analysis results show that miR-665 treated cells show an increase of 16% of cells in G1 phase of cell cycle and the cell number decreased by 18% in S phase compared to negative control miRNA transfected cells. miR-665 treatment did not affect the cells in G2 phase (**Figure 1C**). Cell viability decreased proportionally with the increasing

Computational algorithm prediction site TargetScan and miRanda (microRNA. org) predicts mRNA targets for Mirna*. miranda* predicted hsa-miR-665 targets 3' UTR of HDAC 8 and the sequence alignment is presented in **Figure 2A***. miranda*, also predicts that hsa-miR-665 targets MYCN 3' UTR and the sequence alignment is

concentration of miR-665, represented by black bars (**Figure 1D**)**.**

**3.2 miR-665 targets HDAC 8, c MYC and MYCN**

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presented in **Figure 2B.**

*miR-665 effects on cell proliferation (A) cells treated with 100 nM negative control miRNA and miR-665 (B) for 72 hr. were prepared for cell cycle distribution analysis. Propidium iodide stained cells were analyzed by FLOW cytometry (C) Several concentration of miR-665 effect on cell viability (D) STDEV was used for +/*− *standard error bar; data is from 2 independent experiments with 3 biological replicates for each experiment was used. (figures were printed from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

MYC is overexpressed in 30% of all human cancers and frequently predicts for a poor clinical outcome, and deregulated expression of MYC is a hallmark feature of cancer [3] miRanda and Targetscan did not include 3' UTR of C MYC as miR-665 target. Therefore, Complimentary sequences between miR-665 and 3' UTR of C MYC were compared at online pairwise sequence alignment site www.ebi.ac.uk. Results show two complementary binding sites for miR-665 in the 3' UTR of c MYC (**Figure 2C**).

First we measured total HDAC activity in the cell extracts prepared from negative control miRNA and miR-665 transfected cells in the presence of acetylated HDAC substrate Ac-Lys (Ac)-p NA and deacetylated end product was measured colorimetrically. HDAC 8 and c MYC proteins were measured with antibody in ELISA. The results show that total Pan HDAC activity was decreased by 40%, and HDAC 8 and c MYC proteins were decreased by 40% in miR-665 transfected cells compared to negative control miRNA treated cells (**Figure 3A–C**).

Next, we tested whether HDAC 8, c MYC and MYCN genes are the direct target of miR-665. In these experiments HepG2 cells were used because miR-665 does not affect the growth of these cells (our unpublished results). Luciferase reporter

#### **Figure 2.**

*Predicted binding sites for miR-665 in targets HDAC 8, c MYC and MYCN 3' UTR. Computational prediction site miRanda (microRNA.org) predicted hsa-miR-665 targets 3' UTR of HDAC 8 and the sequence alignment is presented in (*A). miranda*, also predicts that hsa-miR-665 targets MYCN 3' UTR and the sequence alignment is presented in* (B). miranda *and Targetscan did not include 3' UTR of C MYC as miR-665 target. Therefore, complimentary sequences between miR-665 and 3' UTR of C MYC were compared at online pairwise sequence alignment site www.ebi.ac.uk. Sequence alignment is presented in (C). (D) miR-665 targets were validated by co transfection of 100 ng luciferase expression plasmids with 3'-UTR and 100 nM negative control miRNA and miR-665 into HepG2 cells. Empty vector without 3' UTR was used as a control. After 48 hr., luciferase activity was measured and normalized luciferase activity is presented. Data is presented from 2 independent experiments with 3 biological replicates were used. STDEV was used for +/*− *standard error bar. (figures were printed from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

plasmids with 3' UTR were cotransfected with negative control miRNA and miR-665 in HepG2 cells and after 48 hr. luciferase activity were measured in cell extracts. Results show a 40% decrease in luciferase activity of HDAC 8 3' UTR, a 51% decrease in luciferase activity of c MYC 3' UTR and a 50% decrease in luciferase activity of MYCN 3'UTR from the cells co transfected with miR-665 compared to the co transfection with negative control miRNA (**Figure 2D**). These results validate that m RNAs of HDAC 8, c MYC and MYCN are the direct targets of suppressor miR-665.

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**Figure 3.**

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

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

**3.3 MiR-665 induced activation of caspase 3**

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

HDAC inhibitors induce the caspase 3-dependent apoptosis [8] Suppressor miR-34a increased the activation of caspase 3 and caused caspase dependent apoptosis in neuroblastoma cells [17, 18]. Caspase 3 is a critical part of apoptosis, and is required for the DNA fragmentation and for the typical morphological changes of cells undergoing apoptosis. We investigated the effect of miR-665 on the activation of caspase 3 and activity was measured by the hydrolysis of the peptide substrate attached to p-nitroanilid. Caspase 3 activity was measured in cell extracts prepared from cells transfected with negative control miRNA and miR-665. Results show that caspase 3 activity was increased by 2.5-fold in miR-665 transfected cells compared to negative control miRNA (**Figure 3D**). Specificity of the caspase 3 was determined by the addition of caspase 3 inhibitor TSA before the addition of substrate in the assay. The results show that inhibitor binds to caspase 3 and inhibited 90% of miR-665 activated caspase 3 activity **(Figure 3D)**. These

*miR-665 effect on target HDAC 8, c MYC and MYCN cell extracts from100nM negative control miRNA and miR-665 transfected cells were used for the quantitation of; (A) total HDAC activity, (B) HDAC8 protein by ELISA, (C) c MYC protein by ELISA and (D) caspase 3 activity and specificity of caspase 3 enzyme activity was determined in the presence of inhibitor. Data is presented from 2 independent experiments with 3 biological replicates were used. STDEV was used for +/*− *standard error bar. (figures were printed from* 

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

#### **Figure 3.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

plasmids with 3' UTR were cotransfected with negative control miRNA and miR-665 in HepG2 cells and after 48 hr. luciferase activity were measured in cell extracts.

*Predicted binding sites for miR-665 in targets HDAC 8, c MYC and MYCN 3' UTR. Computational prediction site miRanda (microRNA.org) predicted hsa-miR-665 targets 3' UTR of HDAC 8 and the sequence alignment is presented in (*A). miranda*, also predicts that hsa-miR-665 targets MYCN 3' UTR and the sequence alignment is presented in* (B). miranda *and Targetscan did not include 3' UTR of C MYC as miR-665 target. Therefore, complimentary sequences between miR-665 and 3' UTR of C MYC were compared at online pairwise sequence alignment site www.ebi.ac.uk. Sequence alignment is presented in (C). (D) miR-665 targets were validated by co transfection of 100 ng luciferase expression plasmids with 3'-UTR and 100 nM negative control miRNA and miR-665 into HepG2 cells. Empty vector without 3' UTR was used as a control. After 48 hr., luciferase activity was measured and normalized luciferase activity is presented. Data is presented from 2 independent experiments with 3 biological replicates were used. STDEV was used for +/*− *standard error bar.* 

Results show a 40% decrease in luciferase activity of HDAC 8 3' UTR, a 51% decrease in luciferase activity of c MYC 3' UTR and a 50% decrease in luciferase activity of MYCN 3'UTR from the cells co transfected with miR-665 compared to the co transfection with negative control miRNA (**Figure 2D**). These results validate that m RNAs of HDAC 8, c MYC and MYCN are the direct targets of suppressor

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

**106**

miR-665.

**Figure 2.**

*miR-665 effect on target HDAC 8, c MYC and MYCN cell extracts from100nM negative control miRNA and miR-665 transfected cells were used for the quantitation of; (A) total HDAC activity, (B) HDAC8 protein by ELISA, (C) c MYC protein by ELISA and (D) caspase 3 activity and specificity of caspase 3 enzyme activity was determined in the presence of inhibitor. Data is presented from 2 independent experiments with 3 biological replicates were used. STDEV was used for +/*− *standard error bar. (figures were printed from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

#### **3.3 MiR-665 induced activation of caspase 3**

HDAC inhibitors induce the caspase 3-dependent apoptosis [8] Suppressor miR-34a increased the activation of caspase 3 and caused caspase dependent apoptosis in neuroblastoma cells [17, 18]. Caspase 3 is a critical part of apoptosis, and is required for the DNA fragmentation and for the typical morphological changes of cells undergoing apoptosis. We investigated the effect of miR-665 on the activation of caspase 3 and activity was measured by the hydrolysis of the peptide substrate attached to p-nitroanilid. Caspase 3 activity was measured in cell extracts prepared from cells transfected with negative control miRNA and miR-665. Results show that caspase 3 activity was increased by 2.5-fold in miR-665 transfected cells compared to negative control miRNA (**Figure 3D**). Specificity of the caspase 3 was determined by the addition of caspase 3 inhibitor TSA before the addition of substrate in the assay. The results show that inhibitor binds to caspase 3 and inhibited 90% of miR-665 activated caspase 3 activity **(Figure 3D)**. These

results show that mimic miR-665 activated caspase 3 in neuroblastoma cells, suggesting that miR-665 can inhibit cell growth and reduced viable cells by caspase 3 dependent apoptosis.

## **3.4 miR-665 levels following transfection**

miR-665 levles were quantitated in neuroblastoma cells transfected with negative control miRNA and miR 665 using real time qPCR. Mouse neuroblastoma cells have very low levels of endogenous miR-665 **(Figure 4A)**; however, miR-665 expression increased 848-fold in cells transfected with mimic miR-665 compared to cells transfected with the negative control miRNA, cel miR-67 **(Figure 4A** and **B**). miRNA levels reportedly increased by over 1000-fold in cells transfected with miR 200a [19]. Our results strongly indicate that miR-665 upregulation decreased MYC and HDAC8 expression, thus inhibiting proliferation and inducing apoptosis in mouse neuroblastoma cells.

#### **Figure 4.**

*Quantitation of miR-665 in transfected cells. miR-665 was quantitated via real-time qPCR normalized to the U6 gene from three biological replicates 48 h after transfection with negative control miRNA (cel-miR-67) or miR-665. From left, lane 1 and 14 (M), show DNA molecular weight ladder (A) lanes 2–7 (NC-1–NC3 and 665–1–665-3) show the U6 gene. Lanes 8–10 (NC-1–NC3) show miR-665 levels from cells transfected with negative control miRNA. Lanes 11–13 (665–1–665-3) show miR-665 levels from cells transfected with miR-665. The miR-665 fold increase in miR-665-transfected cells was quantitated using the 2-*ΔΔ*Ct method (B) miR665 levels are shown in cells transfected with negative control miRNA (black bar) and in miR-665-transfected cells (white). Error bars were calculated from the standard deviation from three biological replicates. \*P < 0.54x10–6. (figures were printed from published article in "Oncotarget", N.Prashad Vol 9, 33186–33201, 2018).*

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histones.

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

NB cells were transfected with negative control siRNA, siRNA-HDAC 8, siRNA-c MYC and the combination of siRNA-HDAC 8 + siRNA-c MYC. After 48 hr. of growth, cell viability was determined with CellTiter assay (Promega). SiRNA-HDAC8 inhibited 42% and siRNA-c MYC inhibited 55% of cell proliferation, however, the combination of both siRNAs inhibited 86% of the growth of the cells (**Figure 5A**). Therefore, the combination of siRNA-HDAC8 plus siRNA-c MYC was more targeted towards mRNA of HDAC8 and c MYC and caused more effective apoptosis and loss of cells. These results show that HDAC 8 and MYC are critical targets and inhibition of both targets is required for the inhibition of

HDAC 8 and c MYC proteins were quantitated by antibody in ELISA in cell extracts prepared from the cells transfected with negative control siRNA and siRNA-HDAC 8. The results show that siRNA-HDAC 8 transfection inhibited 40% of HDAC 8 proteins (**Figure 5B**) as well as inhibited 35% of MYC protein. Inhibition of MYC may be indirect effect of HDAC 8 inhibition. HDAC8 inhibition increases acetylation of histones and alters gene expression, thus decreasing MYC expression. Therefore, miR-665 represses the expression of c MYC both at the transcription and at the post transcription levels. siRNA-HDAC 8 and siRNA-c MYC

Caspase 3 activity was measured in the cell extracts prepared from cells transfected with negative control siRNA, siRNA-HDAC8 and siRNA-c MYC. The results show that siRNA-HADC8 increased the activity of caspase 3 by 1.8-fold and siRNA –c MYC increased the activity of caspase 3 by 2.5-fold compared to negative control siRNA (**Figure 5C**). Therefore, the results of siRNA effects substantiate the

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

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

miRNA targets hundreds of m RNAs and suppresses their expression, however, siRNA targets a specific m RNA. In these experiments, siRNA for HDAC 8 (siRNA-HDAC 8) and siRNA for MYC (siRNA-c MYC) were used to substantiate the effects

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

of miR-665 on neuroblastoma cells.

neuroblastoma.

**3.6 SiRNA effect on cell proliferation**

**3.7 SiRNA effect on HDAC 8 and C MYC**

**3.8 siRNA activates caspase 3**

substantiated the effects of miR-665 on neuroblastoma cells.

effects of miR-665 on the activation of caspase 3.

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

**3.5 siRNA effect on mouse neuroblastoma cells**
