Limits...
Nonfunctional ingestion of plant miRNAs in silkworm revealed by digital droplet PCR and transcriptome analysis.

Jia L, Zhang D, Xiang Z, He N - Sci Rep (2015)

Bottom Line: The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues.Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable.The results provided a new aspect of cross-kingdom miRNA transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, P. R. China.

ABSTRACT
Since a plant miRNA (miR168) cross-regulating a mammalian transcript was reported, miRNA-mediated cross-kingdom communication has become one of the most compelling but controversial topics. In the present study, we used silkworm and mulberry, which is a model for studies on the interactions between the insect and its host plant, to address whether miRNA-mediated cross-kingdom communication is a common phenomenon. The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues. Synthetic miR166b was also detected in hemolymph and fat body. However, the ingestion of synthetic miR166b did not play roles in silkworm physiological progress, which was revealed by RNA-seq analyses, RT-PCR, and phenotypic investigations. Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable. The results provided a new aspect of cross-kingdom miRNA transfer.

No MeSH data available.


The expression level of 6 differentially expressed genes in silkworm ingested synthetic miR166c and miR167e.Differentially expressed genes were identified by transcriptome analysis. FED-control represents the silkworm larvae were fed on only mulberry leaves. FED-miR166c and FED-miR167e means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166c and miR167e, respectively. Statistical significance was determined by Student’s t test (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4508662&req=5

f3: The expression level of 6 differentially expressed genes in silkworm ingested synthetic miR166c and miR167e.Differentially expressed genes were identified by transcriptome analysis. FED-control represents the silkworm larvae were fed on only mulberry leaves. FED-miR166c and FED-miR167e means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166c and miR167e, respectively. Statistical significance was determined by Student’s t test (*p < 0.05).

Mentions: For a better understanding of the potential overall biological roles of ingested mno-miR166b in silkworms, we used RNA-seq to reveal the global expression patterns in whole body of silkworm larvae before and after feeding with synthetic miR166b for 3 hours. Compared with silkworm larvae fed on mulberry leaves, only 30 genes were differentially expressed, of which 17 were up-regulated and 13 were down-regulated in larvae feeding with synthetic miR166b. These genes can be classified into seven groups (supplementary Fig. S3, Table 4): immunity (11 genes), response to stress (1 gene), development (5 genes), cytoskeleton (1 gene), digestion (1 gene), hydrolyzing juvenile hormone (1 gene), and unknown function (10 genes). Genes involved in immunity and stress comprised about 40% of the differentially expressed genes. Thus, the ingestion of synthetic miR166b might activate the host response against non-self-molecules. We further fed silkworm larvae with two other synthetic miRNAs, miR166c and miR167e. Six genes randomly chosen from 30 DEGs (Differentially Expressed Genes) were subjected to RT-PCR analyses. The result showed that Bm_nscaf2556_13 was down-regulated by synthetic miR166c, Bm_nscaf2818_080 was up-regulated by miR167e, and Bm_nscaf1071_24 gene was up-regulated by both (Fig. 3). The results indicated that the different expression of these immunity-related genes was caused by the ingestion of nucleic acids, not specific to miR166b. Therefore, we speculate that the ingested synthetic miR166b basically did not play a significant physiological role to B. mori. Consistently, there was no significant overlap in the predicted miR166b target sites for the 30 differentially expressed genes (data not shown). In addition, the expression levels of 72 potential target genes for miR166b were not significantly changed in silkworm fed on syntetic miR166b (supplementary table S4). These results suggest that mulberry miR166b has no significant effect on the expression of silkworm genes.


Nonfunctional ingestion of plant miRNAs in silkworm revealed by digital droplet PCR and transcriptome analysis.

Jia L, Zhang D, Xiang Z, He N - Sci Rep (2015)

The expression level of 6 differentially expressed genes in silkworm ingested synthetic miR166c and miR167e.Differentially expressed genes were identified by transcriptome analysis. FED-control represents the silkworm larvae were fed on only mulberry leaves. FED-miR166c and FED-miR167e means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166c and miR167e, respectively. Statistical significance was determined by Student’s t test (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508662&req=5

f3: The expression level of 6 differentially expressed genes in silkworm ingested synthetic miR166c and miR167e.Differentially expressed genes were identified by transcriptome analysis. FED-control represents the silkworm larvae were fed on only mulberry leaves. FED-miR166c and FED-miR167e means the silkworm larvae were fed on a piece of mulberry leaf containing synthetic miR166c and miR167e, respectively. Statistical significance was determined by Student’s t test (*p < 0.05).
Mentions: For a better understanding of the potential overall biological roles of ingested mno-miR166b in silkworms, we used RNA-seq to reveal the global expression patterns in whole body of silkworm larvae before and after feeding with synthetic miR166b for 3 hours. Compared with silkworm larvae fed on mulberry leaves, only 30 genes were differentially expressed, of which 17 were up-regulated and 13 were down-regulated in larvae feeding with synthetic miR166b. These genes can be classified into seven groups (supplementary Fig. S3, Table 4): immunity (11 genes), response to stress (1 gene), development (5 genes), cytoskeleton (1 gene), digestion (1 gene), hydrolyzing juvenile hormone (1 gene), and unknown function (10 genes). Genes involved in immunity and stress comprised about 40% of the differentially expressed genes. Thus, the ingestion of synthetic miR166b might activate the host response against non-self-molecules. We further fed silkworm larvae with two other synthetic miRNAs, miR166c and miR167e. Six genes randomly chosen from 30 DEGs (Differentially Expressed Genes) were subjected to RT-PCR analyses. The result showed that Bm_nscaf2556_13 was down-regulated by synthetic miR166c, Bm_nscaf2818_080 was up-regulated by miR167e, and Bm_nscaf1071_24 gene was up-regulated by both (Fig. 3). The results indicated that the different expression of these immunity-related genes was caused by the ingestion of nucleic acids, not specific to miR166b. Therefore, we speculate that the ingested synthetic miR166b basically did not play a significant physiological role to B. mori. Consistently, there was no significant overlap in the predicted miR166b target sites for the 30 differentially expressed genes (data not shown). In addition, the expression levels of 72 potential target genes for miR166b were not significantly changed in silkworm fed on syntetic miR166b (supplementary table S4). These results suggest that mulberry miR166b has no significant effect on the expression of silkworm genes.

Bottom Line: The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues.Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable.The results provided a new aspect of cross-kingdom miRNA transfer.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, P. R. China.

ABSTRACT
Since a plant miRNA (miR168) cross-regulating a mammalian transcript was reported, miRNA-mediated cross-kingdom communication has become one of the most compelling but controversial topics. In the present study, we used silkworm and mulberry, which is a model for studies on the interactions between the insect and its host plant, to address whether miRNA-mediated cross-kingdom communication is a common phenomenon. The results of TA clone, Sanger sequencing and droplet digital PCR demonstrated that several mulberry-derived miRNAs could enter to silkworm hemolymph and multiple tested tissues. Synthetic miR166b was also detected in hemolymph and fat body. However, the ingestion of synthetic miR166b did not play roles in silkworm physiological progress, which was revealed by RNA-seq analyses, RT-PCR, and phenotypic investigations. Mulberry miRNAs are convincingly transferred to the silkworm orally and no physiological process associated with the miRNAs was demonstrable. The results provided a new aspect of cross-kingdom miRNA transfer.

No MeSH data available.