Small non-coding RNAs control normal development and differentiation in the embryo. These regulatory molecules play a key role in the development of human diseases and are used often today for researching new treatments for different pathologies. In this study, CaCo2 colorectal adenocarcinoma cells were initially epigenetically reprogrammed and transformed into CD4+ cells with nano-sized complexes of amphiphilic poly-(N-vinylpyrrolidone) (PVP) with miRNA-152 and piRNA-30074. The transformation of cells was confirmed by morphological and genetic changes in the dynamic of reprogramming. CD4+ lymphocytes marker was detected using immunofluorescence. Amphiphilic poly-(N-vinylpyrrolidone)/small non-coding RNAs complexes were investigated for transfection efficiency and duration of transfection of CaCo2 colorectal adenocarcinoma cells using fluorescence.

• Klíčová slova
• AGO2, argonaute 2, Amphiphilic poly-(N-vinylpyrrolidone), BACH1, BTB domain and CNC homolog 1, CD, cluster of differentiation, CaCo2 colorectal adenocarcinoma, DICER1, ribonuclease III, DNMT1, DNA methyltransferase 1, DTT, dithyothreitol, ERK1/2, extracellular signal regulated kinase ½, FGF2, fibroblast growth factor 2, GITR3A, glucocorticoid-induced TNFR-related protein, H3K9me3, tri-methyl lysine 9 of histone H3, HILI, human piwi, HMOX1, heme oxygenase 1, HOXA10, homebox A10, ICOS1B, inducible T-cell co-stimulator, IL, interleukin, KIR1DL2, CD158b, expressed on natural killer cells and a subset of T cells, MKI-67, marker of proliferation ki-67, OCT4, octamer-binding transcription factor 4, PIWIL1, piwi-like protein 1, PNVP, poly-(N-vinylpyrrolidone), Polymer carriers, RB1, retinoblastoma 1, Reprogramming, SncRNAs, small non-coding RNAs, TE, transposon elements, TGFBR2, transforming growth factor beta receptor 2, TNFRS6B, TNF receptor superfamily 6B, TSS, transcriptional start sites, VMAF, musculoaponeurotic fibrosarcoma, Wnt-1, wingless type MMTV integration site family, member 1, iPS, induced pluripotent stem cells, mTOR, mechanistic target of rapamycin, miR, micro-RNA, miRNA-152, piR, piwi-interacting RNA, P-element induced wimpy testis interacting RNA, piRNA-30074,
• Publikační typ
• časopisecké články MeSH

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with β-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and β-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

• Klíčová slova
• RT-qPCR, human liver, mRNA, miRNA, precision-cut liver slices, reference gene,
• MeSH
• beta-2-mikroglobulin genetika   metabolismus   MeSH
• beta-naftoflavon farmakologie   MeSH
• cytochrom P-450 CYP1A2 genetika   metabolismus   MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• dimethylsulfoxid farmakologie   MeSH
• dospělí MeSH
• játra účinky léků   metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce normy   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• referenční standardy MeSH
• rifampin farmakologie   MeSH
• senioři MeSH
• stanovení celkové genové exprese normy   MeSH
• systém (enzymů) cytochromů P-450 farmakologie   MeSH
• transkriptom MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• B2M protein, human MeSH Prohlížeč
• beta-2-mikroglobulin MeSH
• beta-naftoflavon MeSH
• CYP1A2 protein, human MeSH Prohlížeč
• CYP3A4 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP1A2 MeSH
• cytochrom P-450 CYP3A MeSH
• dimethylsulfoxid MeSH
• messenger RNA MeSH
• mikro RNA MeSH
• proteiny 14-3-3 MeSH
• rifampin MeSH
• systém (enzymů) cytochromů P-450 MeSH
• YWHAZ protein, human MeSH Prohlížeč

Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes. The inhibition (Inh) of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the overexpression (OvExp) and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFβ, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the Inh group and downregulated in the OvExp group of oocytes. Moreover, IGF1R was significantly upregulated in the Inh oocyte group compared to the control one and IGFBP6 was downregulated in the Inh oocyte group compared to the other groups. Blastocysts developed from the OvExp oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. In conclusion, our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

• Klíčová slova
• blastocyst rate, miR-152, oocyte, porcine,
• Publikační typ
• časopisecké články MeSH