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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.


Synthetic miR166b entry into silkworm hemolymph and fat body after silkworm ingestion as determined by droplet digital PCR.The copy number counts of miR166b in 80 ng small RNA of silkworm hemolymph, 240 ng total RNA of fat body, and 240 ng total RNA of silk gland were investigated by droplet digital PCR before (0 h) and following ingestion of 300 pmol synthetic miR166b (0.5 h, 3 h, 6 h, and 12 h).
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f1: Synthetic miR166b entry into silkworm hemolymph and fat body after silkworm ingestion as determined by droplet digital PCR.The copy number counts of miR166b in 80 ng small RNA of silkworm hemolymph, 240 ng total RNA of fat body, and 240 ng total RNA of silk gland were investigated by droplet digital PCR before (0 h) and following ingestion of 300 pmol synthetic miR166b (0.5 h, 3 h, 6 h, and 12 h).

Mentions: To further investigate the potential role of miR166b, we synthesized this miRNA with modified 2’-O-methyl on its terminal nucleotide, which is characteristic of plant miRNAs19, and smeared 300 pmol on a piece of mulberry leaf to feed silkworm larvae. Droplet digital PCR was applied to detect the copy numbers of miR166b in silkworm hemolymph, fat body, and silk gland before and after ingestion. The copy number counts differed over time according to the tissue type (Fig. 1, supplementary Fig. S1 and Table S2). In hemolymph, the miR166b counts peaked at 0.5 h and 3 h, with 30 and 18 times the count at 0 h; the counts at 6 h and 12 h were similar to those at 0 h. The trend in fat body was similar to that in hemolymph. However, the trend in silk gland differed, with miR166b counts at 0.5 h, 3 h, 6 h, and 12 h slightly less than the count at 0 h. These results suggest that synthetic miR166b could enter to silkworm hemolymph and fat body, but not to silk gland. These results also suggest that it takes 0.5 h or less for synthetic miR166b to enter hemolymph and fat body and that miR166b lasts 3 hours before degradation.


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)

Synthetic miR166b entry into silkworm hemolymph and fat body after silkworm ingestion as determined by droplet digital PCR.The copy number counts of miR166b in 80 ng small RNA of silkworm hemolymph, 240 ng total RNA of fat body, and 240 ng total RNA of silk gland were investigated by droplet digital PCR before (0 h) and following ingestion of 300 pmol synthetic miR166b (0.5 h, 3 h, 6 h, and 12 h).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Synthetic miR166b entry into silkworm hemolymph and fat body after silkworm ingestion as determined by droplet digital PCR.The copy number counts of miR166b in 80 ng small RNA of silkworm hemolymph, 240 ng total RNA of fat body, and 240 ng total RNA of silk gland were investigated by droplet digital PCR before (0 h) and following ingestion of 300 pmol synthetic miR166b (0.5 h, 3 h, 6 h, and 12 h).
Mentions: To further investigate the potential role of miR166b, we synthesized this miRNA with modified 2’-O-methyl on its terminal nucleotide, which is characteristic of plant miRNAs19, and smeared 300 pmol on a piece of mulberry leaf to feed silkworm larvae. Droplet digital PCR was applied to detect the copy numbers of miR166b in silkworm hemolymph, fat body, and silk gland before and after ingestion. The copy number counts differed over time according to the tissue type (Fig. 1, supplementary Fig. S1 and Table S2). In hemolymph, the miR166b counts peaked at 0.5 h and 3 h, with 30 and 18 times the count at 0 h; the counts at 6 h and 12 h were similar to those at 0 h. The trend in fat body was similar to that in hemolymph. However, the trend in silk gland differed, with miR166b counts at 0.5 h, 3 h, 6 h, and 12 h slightly less than the count at 0 h. These results suggest that synthetic miR166b could enter to silkworm hemolymph and fat body, but not to silk gland. These results also suggest that it takes 0.5 h or less for synthetic miR166b to enter hemolymph and fat body and that miR166b lasts 3 hours before degradation.

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.