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Droplet Digital PCR Quantification of Selected Intracellular and Extracellular microRNAs Reveals Changes in Their Expression Pattern during Porcine In Vitro Adipogenesis
Extracellular miRNAs have attracted considerable interest because of their role in intercellular communication, as well as because of their potential use as diagnostic and prognostic biomarkers for many diseases. It has been shown that miRNAs secreted by adipose tissue can contribute to the pathophysiology of obesity. Detailed knowledge of the expression of intracellular and extracellular microRNAs in adipocytes is thus urgently required. The system of in vitro differentiation of mesenchymal stem cells (MSCs) into adipocytes offers a good model for such an analysis. The aim of this study was to quantify eight intracellular and extracellular miRNAs (miR-21a, miR-26b, miR-30a, miR-92a, miR-146a, miR-148a, miR-199, and miR-383a) during porcine in vitro adipogenesis using droplet digital PCR (ddPCR), a highly sensitive method. It was found that only some miRNAs associated with the inflammatory process (miR-21a, miR-92a) were highly expressed in differentiated adipocytes and were also secreted by cells. All miRNAs associated with adipocyte differentiation were highly abundant in both the studied cells and in the cell culture medium. Those miRNAs showed a characteristic expression profile with upregulation during differentiation.
Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing
Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5′-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.
The Effects of Peruvian maca (Lepidium meyenii) Root Extract on In Vitro Cultured Porcine Fibroblasts and Adipocytes
Peruvian maca (Lepidium meyenii) is a plant known for its nutritional and medicinal properties whose use as a supplement in animal diets has attracted much interest. We studied the effects of powdered maca root extract on the growth potential of in vitro cultured porcine cells prior to its use as an additive in animal nutrition. Fibroblast cell viability (MTT), cell proliferation (BrdU), and apoptosis level (TUNEL) were measured for a range of extract doses (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, and 10 mg/mL). Transcript levels of CCND1, MCM2, and PCNA genes as molecular markers of cell proliferation were also determined. Next, the effects of maca extract at 2 and 5 mg/mL on in vitro induced adipogenesis were evaluated over eight days of differentiation. The transcript levels of three adipocyte marker genes (CEBPA, PPARG, and FABPB4) were measured at days 0, 4, and 8 of adipose differentiation, and lipid droplet accumulation (BODIPY staining) was also noted. No cytotoxic effect was detected on fibroblast cell viability, and the inhibitory concentration (IC50) value was determined to be IC50 > 10 mg/mL. Doses of maca extract above 3 mg/mL decreased cell proliferation. The transcript level decreased in concentrations above 5 for the MCM2 and PCNA genes. For the CCND1 gene, the transcript level decreased when the greatest maca dose was used. In the in vitro adipogenesis experiment, it was found that the rate of lipid droplet formation increased on day 4 of differentiation for both doses, while decreased lipid droplet formation was observed on day 8 for 5 mg/mL of maca extract. Significant changes were seen in the mRNA level for CEBPA and PPARG on days 4 and 8, while the transcript of FABP4 increased only on day 8 at 2 mg/mL dose. It can be concluded that the addition of Peruvian maca in small doses (<3 mg/mL) has no negative effect on porcine fibroblast growth or proliferation, while 2 mg/mL of maca extract enhances adipocyte differentiation.