Expression profiles of goat rumens during pre- and post-weaning periods
In the present study, we mainly focused on the expression characteristics of genome-wide microRNAs involved in rumen development and their potentially affects especially on the functional transition of the rumen epidermal layer in goats. Seven highly expressed miRNAs mainly involving in cell proliferation, growth and apoptosis were identified at the four tested periods. Among them, miR-143-3p has been verified to be related with the regeneration of skeletal muscle cells by targeting IGFBP5 [25], and it could target the CTGF and inhibit the Akt/mTOR signaling pathway to regulate the proliferation and apoptosis of fibroblasts [26]. miR-21-5p and miR-26a-5p have also been shown to be associated with the proliferation of melanoma and hepatocellular carcinoma cells, respectively [27, 28]. As for the members of let-7, expressions of let-7i-5p, let-7f-5p and let-7g-5p have been revealed to regulate the regeneration rate of liver in mice [29].
Compared with the rumens from post-weaning period (D150), a similarity number of DEMs (82 in E60 and 111 in E135) were detected the rumens at prenatal stages (Additional file 6: Figure S2). It was notable that the rumen function was absorption maternal glucose in embryonic stage, while solely obtain energy from SCFA in feed after weaning. From fetal to birth and adult stage, the morphology, structure and function of rumen have significantly changed, especially the surface area of the rumen papillae of goats under supplementary feeding significantly increased during the pre- and post-weaning periods [30]. 66 DEMs were observed between the rumens at D30 and D150 (Fig. 1a). We previously reported that miRNAs expression in embryonic rumens and less number of DEMs (22 up-regulated and 20 down-regulated) was identified between E60 and E135 [18].
Histomorphological analyses have found that rumen development in cattle [31] and sheep [32] were slightly faster than that in goats during embryonic period. During weaning, the length and surface area of the rumen papillae of goats and calves increased, and rumen development significantly changed [33, 34]. Accompanied by the development of rumen size, rumen papillae and muscular tissues, gene expression were also remarkably altered though transcriptional and post-transcriptional regulation. Plenty of differential expressed genes (DEGs) have been identified in cattle between pre- and post-weaning [2, 35, 36]. During weaning, the DEGs in calf rumen epithelium were mainly involved in the processes of lipid metabolism, cell morphology, growth and proliferation, molecular transport [2]. Later, more DEGs (4,104) were revealed between the pre- and the post-weaning periods in bovine rumen and several highly DEGs with immune functions (LY6D, MUC1, EMB and EYA2) were pointed out. Similarity, immunity and lipid metabolism were also significantly enriched for DE genes in the rumen [35]. Subsequently, 122 DEMs were identified from 260 known and 35 novel miRNAs in the same rumen tissues. Among them, six miRNAs (miR-143, miR-29b, miR-145, miR-493, miR-26a and miR-199) were identified as the key regulators of rumen development in cattle [36]. However, the studies about miRNAs in goat rumens still limited.
miRNAs related with the pathways involved in rumen development
With regard to biological functions of the target genes of DEMs, four relevant pathways were enriched significantly in goat rumens, including the MAPK signaling pathway, Jak-STAT signaling pathway, Rap1 signaling pathway, Ras signaling pathway and PI3K-Akt signaling pathway (Fig. 2b). Several factors could effect cell proliferation and differentiation mediated by the epidermal growth factor receptor (EGFR) with respond to EGF and transmembrane transforming growth factor (TGF) through the MAPK signaling pathway [37], it promoted the growth of gastric epithelium during development and the renewal of the gastric tissue [38]. It has been shown that inhibition of Jak-STAT3 signaling pathway could inhibit cell proliferation in gastric cancer cells [39]. Ras signaling was a downstream pathway of EGFR and played an important role in the cell fate and proliferation of intestinal epithelial cells [40]. In addition, phosphorylation of PI3K-Akt signaling pathway can promote ghrelin-mediated intestinal cell proliferation [41]. These findings indicated that epithelial cell proliferation during rumen development is closely related to MAPK signaling pathway, Ras signaling pathway and PI3K-Akt signaling pathway.
Compared with prenatal rumens (E60 and E135), the differently enriched pathways at the post-weaning stage (D150) were mainly clustered into carbohydrate digestion and absorption, vitamin digestion and absorption, adherens junction, gap junction, amino acid metabolism (Additional file 8: Table S6). Subsequently, the specific pathways including tryptophan and metabolism, ether lipid metabolism and glycosaminoglycan biosynthesis were revealed in comparison of pre- and post-weaning rumens (D30 and D150). During the processes of rumen development, the histomorphology changes and functional transitions of rumen should be followed by gene regulation [42]. These results indicated that some DEMs may be involved in epithelial cell junctions, digestion and metabolism of nutrients.
To date, many investigations have focused on the molecular mechanism of rumen epithelial cell proliferation and related transporter regulatory pathways, such as insulin-like growth factor (IGF) and epidermal growth factor (EGF) involved in the regulation of glucose transport, NHE, MCTs and GPR involved in the transport of SCFA in rumen epithelial cells [43, 44]. With respect to other external effects, diet composition and nutrient level can affect rumen development, and the expression of epithelial cell genes can also affect gastrointestinal development. Malmuthuge and colleagues demonstrated that the VFAs produced by microbiota could stimulate processes of rumen tissue metabolism and epithelium development via interacting with the host transcriptome and microRNAome. Approximately half of miRNAs and over 25% of mRNAs were correlated with the diversities of VFAs in neonatal calves [45]. In addition, ratio of non-fibrous carbohydrate/neutral detergent fiber (NFC/NDF) in diet could affect the the length and width of rumen papillae. One differently expressed bta-miR-128 induced by NFC/NDF was found to regulate the rumen development via regulating PPARG and SLC16A1 genes [46]. Moreover, in grass- and grain-fed cattle could also resulted in alternations of miRNA expression potentially influencing rumen function, as well as mRNA expression and DNA methylation [47].
miR-148a-3p functions in proliferation, differentiation and apoptosis
In the current study, we have found that miRNA-148a-3p was highly expressed in rumen tissues, especially in D30 (2-fold), while its underlying regulatory mechanism in rumen development remains unclear. FISH results (Fig. 6) demonstrated that miR-148a-3p and QKI were co-expressed in rumen, reticulum, omasum and bomasum, compared with in other gastrointestinal tract samples, miR-148a-3p and QKI are expressed in different locations (Additional file 10: Figure S3). Furthermore, a high correlation of miR-148a-3p and QKI expression was found at all the four periods (Additional file 9: Table S7). As revealed in GES-1 cells, miR-148a-3p suppressed the cell proliferation by regulating QKI, suggesting that a similarity effects on the rumen epithelial cells.
A previous study has revealed that miR-148a-3p was significantly up-regulated during fetal liver development, and its target gene also participated in the differentiation process of liver cells [48]. miR-148a-3p could induce hepatocytic differentiation by inhibiting the IKKα/NUMB/NOTCH signaling pathway [49]. In domestic animals, miR-148a-3p was highly expressed in fetal bovine skeletal muscle, but decreased in the growing myocytes. It was found that the overexpression of miR-148a-3p could inhibit the proliferation of myocytes and promote the proliferation of myocytes after interference [50]. In rabbits, miR-148a-3p is not only highly expressed in white adipose tissue at early growth stage, but also gradually increased in the differentiation process of preadipocytes cultured in vitro [51]. In pigs, miR-148a-3p was always highly expressed during the development of skeletal muscle, which was speculated to be related to the proliferation, generation and apoptosis of muscle cells [52].
Here, QKI was confirmed to be a target gene of miR-148a-3p in goat rumens. QKI is a kind of RNA binding protein, which is a member of RNA signal transduction and activation family and plays an important role in mammalian embryo development. QKI protein has a maxi-KH domain, which is transcriptionally controlled by QKI-RNA recognition element CUAAC sequence, which is involved in many RNA metabolic processes, and has a target regulatory role in the localization, translation, transport and stability of mRNA [53]. The main subtypes of QKI are QKI5, QKI6 and QKI7, in which QKI5 mainly exists in the nucleus, QKI6 is distributed in all parts of the cell, and QKI7 mainly exists in the cytoplasm. The expression of QKI was regulated by various miRNAs, which affected its biological functions. For example, miR-214 can target and regulate the expression of QKI and participate in the differentiation of vascular smooth muscle cells [54]. miR-148a-3p could affect the proliferation of cancer cells by targeting QKI5 [55]. In addition, QKI can also act on miRNAs precursor and feedback regulate miRNAs expression [16].
Previous studies reported that the expression of miR-148a-3p was affected by methylation. The expression of QKI was also affected by the methylation level of its promoter region, which contained CpG islands in the upstream region of pre-miR-148a-3p [56]. Meanwhile, miR-148a-3p also regulated the expression of DNMT1 and affected the physiological processes of different cells [57-59]. The binding site of miR-148a-3p was also confirmed to be existance in the 3′-UTR of DNMT1. Thus, It is speculated that the expression of miR-148a-3p should be regulated by epigenetics [60]. In this study, we found that the expression of miR-148a-3p was significantly different in the rumen tissues between embryonic stage and after weaning. miR-148a-3p may affect cell proliferation by binding to QKI5 3'-UTR and inhibiting its expression. Therefore, during rumen development, DNA methylation whether involve in and how to regulate the expression of miR-148a-3p and QKI remains to be further studied.