Samples similarity and difference of samples based on lncRNA transcriptome under diverse postnatal development stages and condition
In order to explore similarity and difference of samples based on lncRNA transcriptome under diverse postnatal development stages and condition. Hierarchical clustering method based on the PCCs was performed. We found all the samples of week 1 were clustered together (Fig. 1A). Samples of week 4 and week 12/16 cluster together obviously and couldn’t distinguish. The result indicated that samples of week 1 showed stronger similarity on lncRNA level. CONV and GF samples also could be distinguished at some extent. The results of PCA for lncRNA expression showed first three PCs had most proportion of variance (Fig. 1B). Especially, the first PC accounts for 85%. Thus, the first three PCs were analyzed and showed, separately. Samples of week1 were separated from other two stages including week 4 and week 12/16 according to both the developmental stage and microbial status based on PC1 and PC2 (Fig. 1C). It indicated that lncRNA expression changed dramatically during maturation of IECs, especially in the early postnatal period. In addition, PC2 also could distinguish CONV and GF within single developmental week 1 stage. PC3 could separated week 4 and week 12/16 (Fig. 1D, E). Thus, these first three PCs showed outperformance on distinguishing developmental stage and microbial status. All the results indicated that lncRNA expression could serve as effective biomarkers for developmental stage and microbial status.
Some lncRNAs were differential expressed between CONV and GF mice in diverse postnatal development stages
In order to further explore roles of lncRNAs for microbiota in diverse postnatal development stages, differential expressed lncRNAs between CONV and GF mice were identified. In each postnatal development stage, a certain number of differential expressed lncRNAs were identified. For example, there was 20 differential expressed lncRNAs in week 1 (Fig. 2A). In week 4 and week 12/16 CONV and GF mice, 21 and 42 differential expressed lncRNAs were identified (Fig. 2B, 2C). We also describe the interactions of differential expressed lncRNAs among three diverse kinds of postnatal development stages. We found there is little intersection between these differential expressed lncRNAs in diverse postnatal development stages (Fig. 2D). It also indicated that microbiota-specific lncRNAs showed diverse expression pattern during postnatal development. Different microbiota-related lncRNAs serve as their roles at specific postnatal development stage.
Differential expressed lncRNAs between CONV and GF mice showed specific features in respective postnatal development stage
All above results revealed that microbiota-related lncRNAs showed significant differences in diverse postnatal development stage. Thus, we further depicted the specific features of microbiota-related lncRNAs in respective postnatal development stage. We divided all the microbiota-related lncRNAs to up- and down-regulated microbiota-related lncRNAs. In mice with week 1, there were 6 and 14 up- and down-regulated microbiota-related lncRNAs. The expression pattern of up- and down-regulated microbiota-related lncRNAs were almost consistent (Fig. 3A, B). For example, fold-change values of almost all down-regulated microbiota-related lncRNAs showed declining trend. Fold-change values of almost all down-regulated microbiota-related lncRNAs showed rising trend. Similar pattern were also present in week 4 and week 12/16 (Figure S1). We also discovered that some up- and down-regulated lncRNAs showed similar expression patterns and clustered together (Fig. 3C, D). Specially, up-regulated lncRNAs Gm13067, Gm37459, F630040K05Rik, 4930519L02Rik, Gm16137 and Gm16540 form an independent cluster, obviously. Similarly, up- and down-regulated lncRNAs in other postnatal development stages also clustered diverse groups (Figure S2). Collectively, differential expressed lncRNAs between CONV and GF mice showed specific expression in diverse postnatal development stage. However, these lncRNAs also showed similar expression pattern in respective postnatal development stage.
Gut microbiota-specific lncRNA and protein coding gene interaction networks were constructed at different postnatal development stages
In order to describe biological mechanisms of gut microbiota-specific lncRNAs, lncRNA and protein coding gene interaction networks at different postnatal development stages were constructed. All the interactions were filtered by co-expression of lncRNAs and genes. Gut microbiota-specific lncRNA and gene interaction network in week 1 contained 11 lncRNAs and 209 genes (Fig. 4A). Only a little number of lncRNAs and genes had high degree in the network (Fig. 4B, C). It maybe could serve as a degree pattern of scale-free network. In addition, Gut microbiota-specific lncRNA and gene interaction network in week 4 contained 15 lncRNAs and 562 genes (Fig. 4D). Gut microbiota-specific lncRNA and gene interaction network in week 12/16 contained 31 lncRNAs and 461 genes (Fig. 4E). The degree of genes and lncRNAs in week 4 and week 12/16 also showed similar patterns which are only a small part of genes and lncRNAs had higher degree (Figure S3). The results revealed that gut microbiota-specific lncRNA could serve their roles by interacting with some genes in diverse postnatal development stages. Moreover, these gut microbiota-specific lncRNA and protein coding gene interaction networks showed specific features of meaningful biological network.
Gut Microbiota-specific Lncrnas Were Associated With Eukaryotic-type Abc Transporters
In order to further explore the biological functions of gut microbiota-specific lncRNAs in diverse postnatal development stages, functional analyses were performed for their interacted genes. In week 1, gut microbiota-specific lncRNAs were associated with key pathways including Rap1 signaling pathway, cAMP signaling pathway, Axon guidance and ATP Binding Cassette (ABC) transporters (Fig. 5A). In week 4, gut microbiota-specific lncRNAs were associated with key pathways including RNA transport, RNA degradation, regulation of actin cytoskeleton, ABC transporters and so on (Fig. 5B). In week 12/16, gut microbiota-specific lncRNAs were associated with key pathways including tight junction, Rap1 signaling pathway, focal adhesion, ABC transporters and so on (Fig. 5C). The gut microbiota-specific lncRNAs showed different functions in diverse postnatal development stages. Notably, ABC transporters pathway was a key and common pathway which were associated with gut microbiota-specific lncRNAs in all postnatal development stages. Accumulating evidence reported that ABC transporters could regulate the absorption, distribution, metabolism, secretion and toxicity of xenobiotics [28]. Thus, we inferred that these gut microbiota-specific lncRNAs may play their role in postnatal development by participating in ABC transporters pathway. Previous study indicated that there were potential associations between ABC transporters of the intestinal epithelial cell barrier and gut microbes in health and disease [29]. Transporters belonging to the ABC superfamily couple the energy released from ATP hydrolysis to the translocation of a wide variety of substances into or out of cells and organelles [30]. ABC transporter is one of the largest known protein superfamily and there are 48 ABC transporters in humans. Yin et al. reported that there were close relationships among ABC transporters pathway, gut microbiota and obesity in chinese children and adolescents [31]. ABCA and ABCC were two major subfamilies in ABC transporters (Fig. 5D). Some key genes in these two subfamilies could interact with gut microbiota-specific lncRNAs (Fig. 5E). And, most of them showed high degree. These results indicated that gut microbiota-specific lncRNAs could influence ABC transporters pathway in postnatal development stages.