Layered double hydroxides (LDHs) as efficient and safe carriers for miRNA inhibitors: In vitro and in vivo assessment of biocompatibility.

Layered double hydroxides (LDHs) have been employed as nano-sized carriers for therapeutic/bio-active molecules, including small interfering RNAs (siRNAs). However, the potential of LDHs nanoparticles for an efficient and safe antisense oligonucleotide (AMO) delivery still requires studies. In this research, we have tested the suitability of a Mg-Al-LDH-based nanocarrier loaded with a miRNA-196b-5p inhibitor. LDHs (and LDH-Oligo complex) were synthesized by the coprecipitation method followed by physicochemical characterization as hydrodynamic size, surface charge, crystallinity, and chemical groups. Thymic endothelial cell line (tEnd.1) were transfected with LDH-Oligo and were evaluated for i. cell viability by MTT, trypan blue, and propidium iodide assays; ii. transfection efficiency by flow cytometry, and iii. depletion of miRNA-196b-5p by RT-qPCR. In addition, Drosophila melanogaster larvae were fed LDHs and evaluated for: i. larval motility; ii. pupation rate; iii. larval-pupal transition; iv. lethality, and v. emergence rate. We demonstrated that LDHs nanoparticles are stable in aqueous solutions and exhibit a regular hexagonal shape. The LDH-AMO complex showed a transfection efficiency of 93.95 ± 2.15 % and induced a significant depletion of miRNA-196b-5p 48h after transfection. No cytotoxic effects were detected in tEnd.1 cells at concentrations up to 50 µg/ml, as well as in Drosophila exposed up to 500 µg of LDH. In conclusion, our data suggest that LDHs are biocompatible and efficient carriers for miRNA inhibitors and can be used as a viable and effective tool in functional miRNA inhibition assays.

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process.

Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p<0.05). We then investigated if these PMI-related changes in gene expression have the potential to adulterate or mask RT-qPCR results obtained with PILO-injected rats euthanized at acute or chronic phases. In the acute group, Npy transcript was significantly higher when compared with 0h-PMI rats, whereas Ppia transcript was lower than 6h-PMI group. When we used epileptic rats (chronic group), the RT-qPCR results showed higher Tnf-α only when compared to 6h-PMI group. In conclusion, our study demonstrates that PMI influences gene transcription and can mask changes in gene transcription seen during epileptogenesis in the PILO-SE model. Thus, to avoid erroneous conclusions, we strongly recommend that researchers account for changes in postmortem gene expression in their experimental design.

Identification of endogenous reference genes for the analysis of microRNA expression in the hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy.

Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.