lncRNA LL35 as a regulator of Foxa 2 expression in vitro and in vivo – a probable target for diagnostics and therapeutics
During last years significant role of non-coding RNAs (ncRNA) in the regulation of many biological processes, including transcription, translation and cell differentiation was demonstrated. There are examples of ncRNA participation in the development of some diseases, including cancer (Smekalova et al., 2016), that allows to consider some ncRNAs as potential targets for therapy and diagnostics.
It was demonstrated previously that DEANR1 ncRNA participates in the regulation of endotherm differentiation in human embryonic stem cells by cis-regulation of the Foxa2 transcription factor (W. Jiang et al., 2015). Foxa2 protein plays an important role in the glucose homeostasis in the liver, affects fatty acids catabolism under the stress conditions and regulates the biogenesis of high-density lipoproteins (M. Kanaki et al, 2017). Foxa2 factor is also a transcriptional activator for liver-specific albumin and transferrin genes. Also DEANR1 influences on the epithelial-mesenchymal cell transition, which occurs not only during embryonic development but also in fibrosis and during progression of cancer tumors (Y. Fan et al, 2016). We want to emphasize that expression level of the transcription factor Foxa2 in liver is higher than in other organs (K. Kaestner et al., 1994).
Using bioinformatic analysis we found a homologue of the ncRNA DEANR1 in mouse - LL35 ncRNA and preliminary confirmed its expression in the mouse liver. ncRNA LL35 - the mouse homologue of DEANR1 is located in the genome in 2,5 kB downstream of the transcription factor Foxa2. We want to emphasize that the location of ncRNAs associated with transcription factors is quite conservative in mammals. Based on the published data we assume that ncRNA LL35 may be involved in the transcription regulation of the Foxa2 factor in the liver and can affect metabolic processes and various liver diseases in mice.
In the beginning for the first time we are planning to perform the knockdown of LL35 ncRNA in the Hepa1-6 mouse hepatoma cell line by siRNAs. Efficient RNA interference allows reducing the expression level of the target RNA by 80-95% in vitro and in vivo. We will perform design, in vitro screening and selection of the siRNA with maximal inhibitory efficacy of ncRNA LL35 followed by determination of its potency (half inhibitory concentration IC50). The quantity of ncRNA LL35 will be determined by the qRT-PCR. Phenotypes of the cells after RNAi will be studied by microscopy and by proteome analysis. Then we will use the RNA interference in vivo to reduce the amount of LL35 dsRNA in the liver of C57BL / 6 mice. The targeted delivery of the siRNA selected during in vitro studies to the mouse liver will be carried out after formulation to the lipid nanoparticles (Zatsepin et al, 2016). The efficacy of ncRNA inhibition will be determined by qRT-PCR. Biochemical blood analysis and histological studies will be performed to determine changes in the liver metabolic processes.
The study of the potential regulator of transcription factor regulator Foxa2 can be valuable both for understanding of molecular mechanisms of transcription and development of novel therapeutics and diagnostics. During the project for the first time we plan to conduct the study of the role of ncRNA LL35 in vitro and in vivo. This will allow us to evaluate for the first time the therapeutic and diagnostic significance of LL35 in vivo. Successful confirmation that LL35 is a functional analog of DEANR1 will allow us to use RNAi knockdown in vivo model to perform further studies on the role of Foxa2 regulation in steatosis, liver fibrosis and hepatocellular carcinoma in mice models.