Rikki Brown

Altered Iron Metabolism and Impact in Cancer Biology, Metastasis, and Immunology

Frontiers in Oncology

Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

BMC biotechnology, Jan 16, 2018

microRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant express... more microRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods. These included either: phenol: chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction (column-based; miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II). We then evaluated each extraction method for the quality and quantity of RNA recovered, and the expression of miRNAs and target genes. We identified differences between e...

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Evaluation of MicroRNA Delivery In Vivo

MicroRNA and Cancer

MicroRNAs (miRNAs) are a family of short noncoding RNA molecules that fine-tune expression of mRN... more MicroRNAs (miRNAs) are a family of short noncoding RNA molecules that fine-tune expression of mRNAs. Often their altered expression is associated with a number of diseases, including cancer. Given that miRNAs target multiple genes and &amp;amp;amp;amp;quot;difficult to drug&amp;amp;amp;amp;quot; oncogenes, they present attractive candidates to manipulate as an anti-cancer strategy. MicroRNA-7 (miR-7) is a tumor suppressor miRNA that has been shown to target oncogenes overexpressed in cancers, such as the epidermal growth factor receptor (EGFR) and the nuclear factor-κ B subunit, RelA. Here, we describe methods for evaluating systemic delivery of miR-7 using a lipid nanoparticle formulation in an animal model. The microRNA is delivered three times, over 1 week and tissues collected 24 h after the last injection. RNA and protein are extracted from snap frozen tissues and processed to detect miRNA distribution and subsequent assessment of downstream targets and signaling mediators, respectively. Importantly, variability in efficiency of miRNA delivery will be observed between organs of the same animal and also between animals. Additionally, delivering the microRNA to organs other than the liver, particularly the brain, remains challenging. Furthermore, large variation in miRNA targets is seen both within tissues and across tissues depending on the lysis buffer used for protein extraction. Therefore, analyzing protein expression is dependent upon the method used for isolation and requires optimization for each individual application. Together, these methods will provide a foundation for those planning on assessing the efficacy of delivery of a miRNA in vivo.

A miR-7/GAS6/TYRO3 axis regulates the growth and invasiveness of sorafenib-resistant cells in human hepatocellular carcinoma

Hepatology (Baltimore, Md.), Jan 18, 2017

Sorafenib remains the only approved drug for treating patients with advanced hepatocellular carci... more Sorafenib remains the only approved drug for treating patients with advanced hepatocellular carcinoma (HCC). However, the therapeutic effect of sorafenib is transient and patients invariably develop sorafenib resistance (SR). Recently, TYRO3, a member of the TAM family of receptor tyrosine kinases, was identified as being aberrantly expressed in a significant proportion of HCC, however its role in SR is unknown. In this study, we generated two functionally distinct sorafenib resistant human Huh-7 HCC cell lines, in order to identify new mechanisms to abrogate acquired SR, as well as new potential therapeutic targets in HCC. Initially, we investigated the effects of a microRNA, miR-7, in both in vitro and in vivo preclinical models of human HCC and identified miR-7 as a potent tumour suppressor of human HCC. We identified TYRO3 as a new functional target of miR-7, which regulates proliferation, migration and invasion of Huh-7 cells via the PI3-Kinase/AKT pathway and is markedly eleva...

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microRNA-7-5p inhibits melanoma cell proliferation and metastasis by suppressing RelA/NF-κB

Oncotarget, Jan 17, 2016

microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and i... more microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway. While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma. Here, we investigated the biological and clinical significance of miR-7-5p in melanoma. We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest. Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo. We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis. Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7-5p suppresses NF-κB activ...

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