**3.6 SiRNA effect on cell proliferation**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

**3.4 miR-665 levels following transfection**

dependent apoptosis.

mouse neuroblastoma cells.

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

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

*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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**Figure 4.**

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 neuroblastoma.
