C. Courtney2, J. Hu3, J. Xu3, K. Liechty3, R. C. Burns2, T. Westmoreland2 2Nemours Children’s Hospital; University Of Central FL,General Surgery,Orlando, FL, USA 3Children’s Hospital Colorado,General Surgery,Aurora, CO, USA
Introduction: MYCN amplification portends a poor prognosis in neuroblastoma patients and is associated with increased tumor growth, but MYCN amplification does not always equate to increased MYCN gene expression. Regulation of growth occurs at many levels, including proliferation and the genes to regulate this process. Recent novel regulators of proliferation have been identified and include long noncoding RNAs (lncRNA) and micro RNAs (miRNA). Specifically, the lncRNA growth arrest-specific 5 (GAS5) and its negative regulator miRNA-21 have been implicated in the regulation of proliferation during tumor growth. We hypothesized that lncRNA GAS5 and miRNA-21 would be differentially expressed in MYCN amplified and MYCN non-amplified neuroblastoma cell lines.
Methods: To test this hypothesis, total cellular RNA was isolated from the human neuroblastoma cell lines SK-N-AS (MYCN non-amplified) and IMR-32 (MYCN amplified). The expression of the lncRNA GAS5 and miRNA 21 were measured using real-time reverse transcriptase polymerase chain reaction (QPCR). The proliferation rates of both cell lines were also quantified using the MTT cell proliferation assay and compared to gene expression. In addition, miRNA 21 was ectopically expressed in a lentivirus and knocked down utilizing an antagomiR. Expression of miRNA 21 and lncRNA GAS5 was measured using QPCR.
Results: The expression of lncRNA GAS5 is increased in the IMR-32 (MYCN amplified) as compared to the SK-N-AS (MYCN non-amplified). miRNA 21 expression demonstrates an inverse relationship to lncRNA GAS 5 in these cell lines. In the IMR-32 cell line, ectopic expression of miRNA 21 resulted in a decreased expression of lncRNA GAS5. The inverse was also true. When the antagomiR for miRNA 21 was transfected into the IMR-32 cell line, the lncRNA GAS5 expression increased. Proliferation rates were decreased in the IMR-32 cell line as compared to the SK-N-AS cell line.
Conclusion: miRNA 21 and lncRNA GAS5 are differentially expressed in neuroblastoma cell lines with and without MYCN amplification. The inverse correlation of miRNA 21 and lncRNA GAS5 demonstrated in our neuroblastoma study has also been described in other tumors. It is known that accumulation of lncRNA GAS5 results in decreased levels of apoptosis-related genes, cIAP2 and SGK1. The inactivation of these genes results in reduced apoptosis. Furthermore, reduced proliferation, as shown in our study, has been associated with reduced apoptosis. This work gives mechanistic insight to the regulation of apoptosis in MYCN amplified neuroblastoma.