Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing

<div><p>miRNAs are a class of non-coding endogenous small RNAs. They play vital roles in plant growth, development, and response to biotic and abiotic stress by negatively regulating genes. Mulberry trees are economically important species with multiple uses. However, to date, little is known about mulberry miRNAs and their target genes. In the present study, three small mulberry RNA libraries were constructed and sequenced using high-throughput sequencing technology. Results showed 85 conserved miRNAs belonging to 31 miRNA families and 262 novel miRNAs at 371 loci. Quantitative real-time PCR (qRT-PCR) analysis confirmed the expression pattern of 9 conserved and 5 novel miRNAs in leaves, bark, and male flowers. A total of 332 potential target genes were predicted to be associated with these 113 novel miRNAs. These results provide a basis for further understanding of mulberry miRNAs and the biological processes in which they are involved.</p></div

The expression profile of 10 target genes belonging to 6 miRNAs in three mulberry tissues (leaf, bark, and male flower) using RT-PCR.

<p>Morus015493 and Morus018032 are the predicted target genes of miR156, so as Morus012124, Morus012122, and Morus012121 for miRn51, Morus008520 for miRn247, Morus019289 for miRn67, Morus011908 for miRn62, Morus002508 and Morus014466 for miRn157. The X axis represents different tissues. L indicates leaf tissue. B indicates bark tissue. MF indicates male flower tissue. The Y axis represents the relative expression level of target genes. MorusXXXXXX represented the target gene ID.</p

Relative expression of 14 miRNAs in three mulberry tissues (leaf, bark, and male flower) using stem-loop RT-PCR.

<p>The X axis represents different tissues. L indicates leaf tissue. B indicates bark tissue. MF indicates male flower tissue. The Y axis represents the relative expression level of miRNAs.</p

Classification of small RNAs in the mulberry leaves, bark, and male flowers.

<p>Classification of small RNAs in the mulberry leaves, bark, and male flowers.</p

Reads per million of conserved miRNAs in mulberry leaves, bark, and male flowers.

<p>A) miRNAs with >1000 reads; B) miRNAs with 30–1000 reads; C) miRNAs with <30 reads. The X axis represents different conserved miRNAs. The Y axis represents the reads per million for different miRNAs. RPM means reads per million.</p

Additional file 1 of A humanized 4-1BB-targeting agonistic antibody exerts potent antitumor activity in colorectal cancer without systemic toxicity

Additional file 1: Fig. S1 The interaction model between 4-1BB and HuB6 Fab. One copy of the complex is composed of chain A and chain E and F. Chain D interacts with chain B’ and C’ in another asymmetric unit form another copy with the same mode as chain B and C. 4-1BB is in blue and orange, HuB6 Fab H chain is in red, green, cyan and magenta. 4-1BB and HuB6 Fab in another asymmetric unit are in grey. Fig. S2 The dependence on FcγR of the 4-1BB agonist activity. CD8 + T cells were cocultured with CHO-K1 cells expressing different FcγRs and treated with HuB6, utomilumab, urelumab or IgG control for 3 days in a CO2 incubator at 37 °C. After incubation, the secreted IFN-γ and IL-2 levels in the cell supernatants were determined by ELISA. Fig. S3 Tumor photos of the MC38 and CT26 model mice. a the tumors of MC38 and CT26 model mice treated with the three doses of HuB6. b the tumors of MC38 and CT26 model mice treated with the three 4-1BB agonistic mAbs. Fig. S4 Hematoxylin and eosin staining for major organs of the mice treated with HuB6. Major organs included heart, liver, lung, kidney and spleen