Difference between revisions of "Part:BBa K2976015"

Latest revision as of 01:24, 22 October 2019

targeting exosome-NF-κB induced hsa-let-7f

targeting exosome-NF-κB induced hsa-let-7f

Usage

LAMP2, highly expressed on exosome membrane and linked with PD-L1-targeting peptide by (Gly4Ser)3, could endow exosomes with macrophage-targeting ability. What´s more, the glycosylation protection help avoid excision of signal peptides and increase LAMP2 expression on exosomes. With the utilization of such targeting exosomes, miRNA hsa-let-7f delivered could be increased in infected macrophages and the treatment of intracellular Mtb could be realized. HA tag is inserted into the fusion proteins on the membrane of exosomes for the immunoblotting assay.

Biology

MicroRNA hsa-let-7f is reported to regulate human immune responses to Mtb via control of A20, an inhibitor of the NF-κB pathway, which plays an essential role in regulating the expression of pro-inflammatory cytokine and chemokine genes. However, when Mtb infects macrophages, hsa-let-7f inside macrophages decreases. So exogenous supplementation of let-7f will increase the level of endogenous let-7f and further upregulate NF-κB, thus can boost the secretion of cytokines, chemokines and NO to kill intracellular Mtb. To improve the targeting effectiveness of hsa-let-7f contained exosome , anti-PD-L1 peptide is displayed on the surface of the exosome to specifically target to the Mtb-infected macrophages. At the same time, Lamp 2, a protein which is highly expressed on the surface of exosomes assists anti-PD-L1 peptide to find Mtb-infected macrophages.

Characterization

According to the design of our plasmid, the sequence of lamp2 and HA tag was combined for expressing fusion protein, since the HA tag was characterized by western blot(Figure.1), the protein lamp2 was also proved to be expressed together with HA tag.

Figure 1: Western blot result of exosome protein.

The exosomal RNA was extracted for reverse transcription and then detection of let-7f amount by Real-time Quantitative PCR Detecting System(QPCR)(Figure.2). The results demonstrate that the amount of let7 after transfection is 174 times greater than that of control group, indicating that a considerable amount of hsa-let7f miRNA was successfully encapsulated in the exosomes.

Figure 2: qPCR result of microRNA.

We verified the effect of miRNA-carrying exosomes on the enhancement of the microbicidal ability of infected macrophages. The RNA extraction from the macrophages was conducted to be quantified by qPCR to monitor changes in the level of IL1β, IL6, TNFα, MCP1 and MIP2 genes (Figure.3). Elevated expression levels of these gene improve the inflammation and enhance microbicidal efficacy. The results showed that exosomes significantly increased IL1β, IL6 and MIP2 expression, while the level of MCP1 and TFNα was only lifted for a little bit. This demonstrated that our miRNA-carrying exosomes did and enhance microbicidal efficacy of macrophages and activate immunological function.

Figure 3: qPCR result of cytokines and chemokines.

Sequence and Features

Sequence and Features