RESUMO
Neuroblastoma is the most common extra-cranial solid tumor diagnosed mostly in children below the age of five years and comprises of about 15 % of all paediatric cancer deaths. Tumor initiating cancer stem cells (CSCs) can be targeted for better treatment approaches. BASP1-AS1 is a long non coding (Lnc) RNA that is a divergent LncRNA for its coding gene brain abundant membrane attached signal protein 1 (BASP1). We had earlier demonstrated it to be expressed in foetus derived human neural progenitor cells (hNPCs), where it was a positive regulator of BASP1 and was critical for neural differentiation. In this study, we have investigated the role of BASP1-AS1 in CSCs derived from the human neuroblastoma cell line SH-SY5Y. We cultured SH-SY5Y cells on Poly-d-Lysine coated flasks in serum free media supplemented with growth factors, which led to the enrichment of CSCs as determined by marker expression. When grown on ultra-low attachment flasks, these cells formed CSCs enriched neurospheres. We examined the effects of BASP1-AS1 siRNA mediated knockdown on CSCs enriched SH-SY5Y cells and SH-SY5Y derived neurospheres. BASP1-AS1 knockdown decreased the levels of the corresponding gene BASP1 and the rate of cell proliferation of CSCs enriched cells along with low expression of Ki67. It also reduced the mRNA levels of stem cell and pluripotency gene markers (CD133, CD44, c-KIT, SOX2, OCT4 and NANOG), as also Wnt 2 and the Wnt pathway effector ß catenin. It also abrogated the formation of neurospheres in ultra-low attachment flasks. A similar effect on proliferation and stemness related properties was seen on BASP1 knockdown. BASP1-AS1 and its related pathways may provide a point of intervention for the CSCs population in neuroblastoma.
RESUMO
Neuroblastomas are pediatric tumors arising from undifferentiated cells of neural crest origin with stem cell-like characteristics. Dysregulation of Wnt/ß-catenin signaling has been shown to be linked to the development of various tumors. Activated Wnt signaling results in ß-catenin accumulation in the nucleus to support pro-neoplastic traits. DKK1, a secreted glycoprotein, is an inhibitor of Wnt signaling, and the addition of DKKI to the culture medium has been used to suppress the Wnt pathway. This study aimed to analyze the role of Dickopff-1 as a potential differentiating agent for the neuroblastoma cell line SH-SY5Y and neurospheres derived from it. The treatment of SH-5Y5Y derived neurospheres by DKK1 resulted in their disintegration and reduced proliferation markers like Ki67, PCNA. DKK1 treatment to the neurospheres also resulted in the loss of cancer stem cell markers like CD133, KIT and pluripotency markers like SOX2, OCT4, NANOG. DKK1 treatment caused reduction in mRNA expression of ß-catenin and TCF genes like TCF4, TCF12. When the SH-SY5Y cancer cells were grown under differentiating conditions, DKKI caused neuronal differentiation by itself, and in synergy with retinoic acid. This was verified by the expression of markers like MAPT, DCX, GAP43, ENO2 and also with changes in neurite length. We concluded that Wnt inhibition, as exemplified by DKK1 treatment, is therefore a possible differentiating condition and also suppresses the proliferative and cancer stemness related properties of SH-SY5Y neuroblastoma cells.