Abstract 4175: Characterization of hsa-miR-135b in human neuroblastoma cell line SK-N-BE(2).

Abstract

Abstract Background: MicroRNAs (miRs) are an abundant class of small (∼22 nucleotides), non-coding, single-strand RNAs (ncRNA) that negatively regulate gene expression at a post-transcriptional level. miRs are thought to act by inhibiting the translation of messenger RNAs (mRNAs) into protein in addition to promoting mRNA degradation. These regulatory ncRNAs play an important role in the control of many biological processes and their deregulation has been implicated in a variety of pathological conditions, including many cancers. Emerging evidence supports the concept that in many cancer types a pool of cancer cells responsible for cancer maintenance and long-term remission failure exhibit stem-like properties. Using a high throughput miR microfluidic card assay qRT-PCR approach, we identified 26 miRs (23 up-regulated and 3 down regulated) as stem-like associated miRs in six pediatric cancer cell lines, including neuroblastoma cell line SK-N-BE(2). hsa-miR-135b was one of the miRs that was up-regulated. The purpose of this study was to characterize the functional consequences of hsa-miR-135b silencing in the SK-N-BE(2) cell line. Methods: hsa-miR-135b expression was knocked down (KD) using Lipofectamine® transfection reagent and a stable cell line was generated in the presence of puromycin. Cell cycle distribution as well as the effect of inhibition on the ability to form neurospheres (read-out for self-renewal) was determined. To investigate the effect on anchorage-independent growth, the ability of the KD cells to form colonies in soft agar was measured. Cell capacity for invasion was tested using basement membrane matrix invasion culture system. Western blot analysis was performed on the components of the Wnt/****B-catenin signaling pathways. Results: Our results indicated that inhibition of hsa-miR-135b impaired sphere formation (read-out for self renewal) as well as growth of colonies on a semi-solid culture media. No significant difference in cell cycle distribution was observed between control and hsa-miR-135b KD cells. Conclusion: Our findings suggest that in our model system, hsa-miR-135b likely plays a role in the maintenance of cancer stem cells and tumor progression. Therefore, it is a promising therapeutic target in neuroblastoma patients. Citation Format: Chelsea A. Castillo, Judy C. Chang, Jaclyn Y. Hung. Characterization of hsa-miR-135b in human neuroblastoma cell line SK-N-BE(2). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2013-4175