Changes in mRNA expression in lung tissue after long-term supplementation with an increased dose of cholecalciferol.
Maintaining an appropriate concentration of vitamin D is essential for the proper functioning of the body regardless of age. Nowadays, there are more and more indications that vitamin D supplementation at higher than standard doses may show protective and therapeutic effects. Our study identifies differences in the body's response to long-term supplementation of cholecalciferol at an increased dose. Two groups of pigs were used in the experiment. The first group received a standard dose of cholecalciferol (grower- 2000 and finisher- 1500 IU/kg feed), and the second group received an increased dose (grower- 3000 and finisher- 2500 IU/kg feed). The animals were slaughtered when they reached breeding maturity. Then the lung tissue was collected from the animals. The material was used for mRNA sequencing. The results of mRNA sequencing showed a significant change in the expression of 195 genes. Functional analysis using the KEGG database showed that increased cholecalciferol supplementation significantly activated 42 pathways. Most of the activated pathways is associated with metabolism. Overall design: The libraries for mRNA-seq were prepared using the QuantSeq 3'mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Austria). Quantitative evaluation of the prepared cDNA libraries was performed using Qubit (Thermo Scientific™, USA), while qualitative evaluation was assessed using Tapestation 2200 device (Agilent, USA). Sequencing of mRNA pooled libraries (75 bp single read) was performed using Nextseq 5500 device (Illumina, USA). The libraries were prepared for sequencing according to the Standard Normalization Method from NextSeq 500 and NextSeq 550 Sequencing Systems-Denature and Dilute Libraries Guide protocol. We used a 2nM starting library concentration and a 10% PhiX addition.