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Polysome profiling of mRNA translation in neuroblastoma cells in response to asparagine/alanine supplement

ABSTRACT: Neuroblastoma, the most common extracranial solid malignancy in infants and young children, originates from aberrant neural crest cells and accounts for 10-15% of childhood cancer deaths. However, the underlying mechanisms regulating neuroblastoma progression remain to be determined. To investigate the mechanisms regulating tumorigenesis and aggressiveness, we employed the Illumina HiSeq X Ten as a discovery platform to analyze the polysome profiling of mRNA translation in neuroblastoma cells in response to asparagine/alanine (Asn/Ala) supplement. The results showed that 618 and 566 translating mRNAs were respectively elevated or reduced in SK-N-BE(2) after Asn/Ala treatment. Furthermore, we validated the polysome profiling results by western blot with high identity. Overall, our results provided fundamental information about the mRNA translation changes in response to Asn/Ala treatment in human neuroblastoma cells, and these findings will help us understand the pathogenesis of neuroblastoma progression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE294552 | GEO | 2025/05/30

REPOSITORIES: GEO

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Project description:Osteolytic lesions with suppression of osteoblastic (OB) differentiation are hallmarks of multiple myeloma (MM). Most recently, Gln availability has been found to be essential to sustain bone mass formation in murine models. On the other hand, MM cells are Gln-addicted and consume huge amounts of the amino acid, leading to a partial Gln depletion in the bone marrow (BM) plasma. We hypothesize that MM-dependent Gln depletion contributes to the defective OB differentiation characteristic of MM. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors and MM patients indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease.

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Polysome profiling of mRNA translation in neuroblastoma cells in response to asparagine/alanine supplement

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