Consumption of Coconut Oil Affects adipose miRNA Prole in Pigs.

Circulating miRNA molecules are intensively studied for their usefulness as biomarkers of civilization diseases. At the same time, it is known that diet can inuence the level of miRNA expression in tissues. Our research aimed to determine how a diet containing various sources of fat (rapeseed oil, beef tallow, coconut oil) and different amounts of cDDGS (corn Dried Distilled Grains with Solubles) affects the miRNA prole in pig fat – the main source of circulating miRNAs. For this purpose, we used Next Generation Sequencing of miRNA libraries. We observed the highest number of differentially expressed miRNAs in the samples from animals that were fed with coconut oil in the diet compared to all other treatments. In contrary, cDDGS appeared to have little effect on miRNA expression. We propose a subset of di-et-related, adipose-specic, conservative miRNAs among mammals, namely: ssc-miR-99b, ssc-miR-4334-3p, ssc-miR-146b, ssc-miR-23a. Moreover, we observed that several miRNAs regulated by dietary fats are considered as biomarkers in human and animal diseases. Conceptualization, M.O., M.Ś.; methodology, M.O., M.Ś., K.P; software, T.S, K.P.; validation, M.O., K.P.; formal analysis, M.O..; investigation, K.P..; resources, M.Ś..; data curation, K.P. T.S.; writing—original draft preparation, M.O, K.P..; writing—review and editing, K.P., MŚ., T.S.; visualization, M.O., K.P..; supervision, M.O..; project administration, M.O..; fund-ing acquisition, M.O. All


Introduction
A properly balanced diet, in addition to physical activity, is of colossal importance in maintaining health in humans and animals. One of the most critical components of the diet is the appropriate content of fatty acids. At the same time, it is believed that today we consume an excessive amount of saturated fatty acids and omega -6-fatty acids. In general, vegetable fats are considered to be healthier than animal fats. The cardioprotective effect of some vegetable fats has already been very well documented, e.g. olive oil, but other vegetable fats in the diet, such as coconut oil, are still quite controversial.
Understanding the molecular mechanisms by which dietary fat affects health may help clarify these controversies. It is known that fatty acids affect many processes because they are the main component of cell membranes, substrates of important biological molecules, and are involved in transmitting signals between cells. We have recently found that diet enriched with cDDGS (corn Dried Distilled Grains with Solubles -additive often used in pig breeding) changes mRNA expression in porcine adipose tissues [1].
Moreover, we observed that the source of dietary fats changes the expression of mRNA connected to neurodegenerative, cardiovascular diseases, or cancer [2] in this tissue. In this paper, we focus on miRNA pro les of porcine adipose tissue from these two previous nutrigenomic experiments [1,2]. Nutrigenomics of miRNA is especially interesting given its crucial role in cancer and other non-communicable diseases. However, the effect of nutrients on miRNA expression is relatively poorly studied, especially for the whole miRNAome scale.
Nevertheless, using the NGS technique, a number of miRNAs with different expressions in the adipose tissue of pigs with divergent backfat deposition were identi ed [3]. Moreover, in mice, it was shown that the overall miRNA expression pro le in adipose tissue is in uenced by a high-fat diet and other factors like sex hormones [4]. In pigs, the addition of CLA (Conjugated Linoleic Acid) to the diet changed the expression of 14 miRNAs in adipose tissue [5], while in rats, consumption of distinct dietary lipids during early pregnancy modulated the expression of microRNAs in mothers and offspring [6]. Recently, it has been established that adipose tissue is the primary source of circulating miRNAs in mice. Moreover, it has been shown that these miRNAs can alter gene expression in other organs [7]. Such observations make the adipose tissue a vital secretory organ and miRNA as endocrine molecules.
Our study aimed to establish the effect of two dietary factors: cDDGS and source of fat (rapeseed oil, beef tallow and coconut oil) on the miRNA pro le in the backfat of pigs.

Animals
Animals for the study were 24 crossbred fatteners originating from sows (Polish Landrace × White Large Polish) mated with a boar (Duroc × Pietrain) divided into four dietary groups: -cDDGS+rapeseed oil (group I, n=7), +cDDGS+rapeseed oil (group II, n=6), +cDDGS+beeftallow (group III, n=6), +cDDGS+coconut oil (group IV, n=5). All procedures relating to the use of live animals were in agreement with the local Ethics Committee for Experiments with Animals in Cracow (Resolution No. 912 dated 26 April 2012). All animals were healthy and as similar regarding weight as possible and kept in individual straw-bedded pens in uniform conditions. The ingredients, the nutritive value of the diets and the fatty acid compositions of the feed mixtures are presented elsewhere [8]. Brie y, all diets were isoenergetic and isoprotein, but differed in fatty acids composition: the group II feed mixture contained 80% of UFA content (44% MUFA and 36% PUFA), group III contained 67% of UFA (32% MUFA and 35% PUFA), and group IV encompassed 45% of UFA (16% MUFA and 29% PUFA). At the end of the experiment -when the animals reached the weight of 118 kg -all the pigs were slaughtered by stunning with high-voltage electric tongs (voltage 240-400 V), and samples of subcutaneous adipose tissue from the area between the last thoracic and the rst lumbar vertebrae were collected for transcriptome analysis. All samples were stored in a freezer (−85 °C) until further analysis.

miRNA isolation, library construction and NGS.
Total RNA, including miRNA, was isolated from the samples of backfat using a Di-rect-zol RNA isolation Kit (Zymo Research). RNA of appropriate quality and quantity was used for library preparation. Next, miRNA-seq libraries were created using NEBNext Mul-tiplex Small RNA Library Prep Set for Illumina (New  The obtained raw reads were conversed to FastQ les, demultiplexed with the bcl2fastq software (Illumina), and quality controlled using the FastQC software [9]. The resultant sequences were analyzed with UEA sRNA Workbench V4.6 [10] to identify potentially novel and known miRNA sequences using the Sus scrofa reference genome (Sscrofa 10.2) and miRBase v22.1 [11,12]. The default animal parameters except for minimum abundance (6 reads), minimum length (17 bp), and maximum length (25 bp) were set [13]. Predicted novel microRNA precursors were analyzed to exclude those belonging to other noncoding RNA species using the RNA central database v14 [14]. Detected microRNAs were subjected to differential expression analysis with the DESeq2 software [15]  3.2 Coconut oil induces changes in the miRNA pro le of backfat.
Principal component analysis revealed a relatively small variance between analyzed samples (12 and 15%). However, samples obtained from coconut oil-fed animals tended to separate from other samples ( Figure 1).

miRNA-mRNA interaction analysis
Next, we compared the obtained results with our previous RNAseq based identi cation of differentially expressed genes in the animals from the same nutritional experiment [1,2]. In these two previous experiments, we observed a strong effect of the addition of cDDGS on the gene expression in the backfat and gene expression differences between animals obtaining rapeseed oil and beef tallow in the diet  Table 2). We expected a correlation between the number of identi ed differentially expressed genes and differentially ex-pressed miRNAs in each comparison; however, contrary to the gene expression study, we observed no differentially expressed miRNAs after comparison between the group which obtained cDDGS in the feedstuff and without it. On the other hand, the number of DEGs was the highest in the comparison between group I and IV and the same was noted for miRNAs (Table2).
We also attempted to identify the miRNA-mRNA interactions in our datasets from the present and previous experiments. For this purpose, we chose the comparison between group I (-cDDGS + rapeseed oil) and group IV (+ cDDGS + coconut oil) because this comparison showed the largest number of both altered genes and miRNAs (Table 2). Interestingly, we observed the predominance of upregulated genes in the (+cDDGS+coconut oil) group [1] and the predominance of downregulated miRNA in the same group. This agrees with the assumption that although some exceptions exist, miRNAs downregulate the expression of target genes.
Using the miRNet 2.0 database [16], we identi ed 23 interactions between the identi ed differentially expressed miRNAs and mRNAs (Table 3). Four identi ed genes were targeted by more than one miRNA, while ve miRNAs targeted more than one gene. Two genes: RCSD1 and CCDC93, were targeted by three different miRNAs, but only in the case of CCDC93, an increase of mRNA expression was accompanied by a decrease in expression of targeting miRNA (Table 3). Table 3 miRNA-gene interactions identi ed by analysis of differentially expressed genes and differentially expressed miRNAs in (-cDDGS+rapessed oil) vs (+cDDGS+coconut oil) comparison. Shaded interactions in which increase in miRNA expression was accompanied by increase in mRNA expression.

Discussion
In recent years, adipose tissue has ceased to be treated only as a covering that insulates internal organs and protects against heat loss and has begun to be considered as a vital metabolic and endocrine organ due to the multitude of substances it secretes. It also turned out that adipose tissue is the main source of circulating miRNAs and low-grade adipose in ammation is one of the main factors responsible for atherosclerosis, metabolic diseases and cancer. Therefore, the identi cation of miRNAs formed in the fat is of great importance in humans and model animals such as pigs. In our NGS-based experiment, we identi ed eleven miRNAs with more than 10 000 read counts in porcine backfat, among which the most abundant were: ssc-miR-148a-3p, ssc-miR-143-3p, ssc-miR-126-3p (Fig. 1). This result is in good agreement with the report of Wang et al., [21], who observed that the same miRNAs are highly expressed in porcine adipose tissue. Similarly, Solexa sequencing revealed abundant expression of ssc-miR-143, ssc-let-7a and ssc-miR-148 in porcine adipose [22]. Some concordance is also maintained compared to the results obtained by Gaffo et al. [23], although the authors observed a signi cant enrichment of ssc-miR-10b, while in our study, miRNAs with the highest expression in adipose tissue was miR-148a-3p.
Remarkably, seven highly abundant miRNA were differentially expressed depend-ing of the source of fat in the diet. Ssc-miR-148a-3p was downregulated in pigs obtaining coconut oil compared to all other groups. Interestingly, it was reported recently that oleanolic acid induces upregulation of miR-148a-3p in chondrocytes [24] and consequently protects against IL1beta induced chondrocyte in ammation and dysfunction. Moreover, miR-143 (downregulated in coconut oil group) was downregulated in adipo-cytes from obese mice and upregulated during adipogenesis [18]. In our experiment, ani-mals of all groups received isocaloric diets, which differed only slightly in the amount of fat but substantially in a fatty acid composition [1,2]. Dietary regimes did not affect ani-mal's body weight or backfat thickness. However, the fatty acid composition of adipose tissue collected from the animals was strongly correlated with fatty acid composition in the feedstuff. Backfat from animals receiving coconut oil contained the lowest iodine value and the highest ratio of saturated to unsaturated fatty acids [8], which are known to in-duce a proin ammatory phenotype. Moreover, morphologically, it had the most solid consistency re ecting the high content of saturated fatty acids.
Generally, a high proportion of miRNAs identi ed by us as differentially expressed, has been previously shown as affected by CLA feeding, neonatal diet, or mother diet in pigs [5,20] (Table 1). Moreover, a huge part of them was over-or underexpressed in obesity in mice and humans [18,19]. This indicates on a subset of diet-related, adipose-speci c, conservative miRNAs among mammals, namely: ssc-miR-99b, ssc-miR-4334-3p, ssc-miR-146b, ssc-miR-23a (Table 1). On the other hand, however, we found poor agreement with the results of the analysis of the effect of the diet on the miRNA expression in the cattle and sheep [25,26].
The adipose samples taken from the animals in the current analysis were subjected to detailed transcriptome studies beforehand [1,2]. Therefore, we decided to try to integrate the results obtained now regarding miRNAs with those from earlier analyzes. We identi ed 23 potential interactions between miRNAs and mRNAs, of which, in 16 cases, an in-crease in the level of miRNA expression was accompanied by a decrease in the level of expression of target genes ( Table 2). Among the genes with the highest number of miRNA interactions, we identi ed CCDC93, RCSD1, MPHOSPH8, OCLN and MAP4K4. These are important regulators of in ammation, proliferation, epithelial barriers and metabolism. Thus, further investigation on diet regulation of these genes through miRNA may be of great clinical signi cance.
Among the KEGG pathways overrepresented by miRNAs with a different expression, we observed pathways associated with pathways in cancer, fatty acids biosynthesis, and stem cell pluripotency ( Table   4). The latter pathway is interesting because pluripotency, and thus the ability to proliferate adipose cells, may in uence the course of metabolic dis-eases associated with adipose tissue, such as diabetes. It is assumed that the proliferation of adipose cells is a favourable phenomenon in insulin resistance as opposed to adipocyte hypertrophy [27]. It would be of clinical importance if a diet high in certain fatty acids could modulate adipose stem cell pluripotency. Another overrepresented pathway -ECM (Extracellular matrix) receptor interaction -is strictly connected with adiposity hyperpha-gia/hyperplasia balance. Excess extracellular matrix deposition in the form of brosis can limit adiposity hyperphagia and, in some cases, may be adaptive to counteract diabetes [28]. Thus, knowing the effect of different dietary fats on adipose ECM through differential miRNA expression is crucial.
In recent times, circulating miRNAs are increasingly considered biomarkers of various diseases and physiological conditions in humans and animals [29][30][31]). Most studies of this type concern the search for biomarkers for various types of cancer, but there are also many reports on biomarkers in Alzheimer's disease [32], atherosclerosis [33] and diabetes [34]. Recently, such markers have also been analyzed in veterinary medicine [35][36][37]. It is di cult not to notice the bene ts of using circulating miRNAs in the diagnosis of civilization diseases, as it is a non-invasive method that allows for the detection of very early lesions. However, the clinical value of miRNA biomarkers will ultimately only be estimated when we consider the effects of extracorporeal factors affecting the expression of circulating miRNA: sex, age or physical exercise. Our research and others show that diet is also of great importance when it comes to in uencing the level of miRNA expression in adipose tissue -the main source of circulating miRNA [7].
Among the miRNAs we identi ed as potentially sensitive to dietary fatty acid composition, several are considered as miRNA biomarkers of civilization diseases. Recently, miR-21 and miR-210 were established as novel non-invasive biomarkers for (CRC) Colorectal cancer diagnosis and prognosis [38]. Furthermore, miR-133b and miR-21 were proposed as possible candidates of novel biomarkers in the early prediction of (CAD) Coronary Artery Disease [39].
Similarly, plasma miR-126 and miR-143 are potential novel biomarkers for cerebral atherosclerosis 40]. The progression of civilization diseases is closely related to the type of fats consumed. Therefore, at times it can be di cult to distinguish whether the changes in the miRNA expression are due to the diet or the development of a pathologic state.

Conclusions
In this paper, we describe a subset of miRNA molecules whose expression changes after consuming a diet rich in coconut oil. Many of them have been identi ed previously as over-or under-expressed after a different diet. What is more, some of them are proposed as biomarkers of civilization diseases. Our results suggest caution and the need for further studies before miRNAs are used routinely as biomarkers in medicine.

Declarations
Author Contributions:     Graphical representation of differentially expressed miRNAs-mRNAs interac-tion identi ed in the adipose of pigs fed different diets (-cDDGS+rapeseed oil) vs (+cDDGS+coconut oil). Purple circles indicate DEGs identi ed in [2] while the green ones indicate those targeted by DE miRNAs identi ed in this study. Green squares in-dicate DE miRNAs identi ed in the present study, while blue squares indicate all miRNAs which may target identi ed DEGs [2].