Exosomes serve as a crucial mode of communication between tumor-associated macrophages (TAMs) and cancer cells. This study attempted to explore the function of M1-derived exosomes and clarify their specific mechanism in head and neck squamous cell carcinoma (HNSCC). Moreover, the functional roles of M1-derived exosomes and their key molecule long noncoding RNA (lncRNA) HOXA transcript at the distal tip (HOTTIP) in HNSCC were investigated by conducting a series of in vitro and in vivo experiments. The dual-luciferase test was utilized to clarify the binding capacities between HOTTIP/mRNA and miRNAs. Accordingly, HOTTIP was found to be upregulated in M1-derived exosomes. Meanwhile, the in vitro experiments indicated that M1 exosomes suppressed proliferation, migration and invasion but induced apoptosis of cancer cells. This function was noted to be enhanced by HOTTIP-overexpressed M1 exosomes but was weakened by HOTTIP-knockdown ones, indicating that HOTTIP serves as a key molecule in M1 exosomes. Therefore, the function of HOTTIP in cancer cells was explored, for which overexpression of HOTTIP was found to inhibit proliferation, migration and invasion but induced apoptosis of cancer cells in vitro. A mechanism study further showed that M1 exosomes and HOTTIP activated the TLR5/NF-κB signaling pathway by competitively sponging miR-19a-3p and miR-19b-3p. Furthermore, cancer cells expressing HOTTIP were noted to induce the polarization of both local M1 and M2 macrophages; however, M1 exosomes were observed to reprogram local TAMs into M1 macrophages. More importantly, both cancer cells expressing HOTTIP and M1 exosomes reeducated circulating monocytes to express the M1 phenotype. The corresponding data demonstrated that the M1 exosomal lncRNA HOTTIP suppresses HNSCC progression by upregulating the TLR5/NF-κB signaling pathway through competitively sponging miR-19a-3p and miR-19b-3p. In particular, M1 exosomes and HOTTIP induce the polarization of M1 in circulating monocytes, thus providing novel insight into HNSCC immunotherapy.
Adeno-associated virus (AAV) is the preferred vector for gene therapy of hereditary deafness, and different viral serotypes, promoters and transduction pathways can influence the targeting of AAV to different types of cells and the expression levels of numerous exogenous genes. To determine the transduction and expression patterns of AAV with different serotypes or promoters in hair cells and supporting cells in the neonatal mouse cochlea, we examined the expression of enhanced green fluorescent protein (eGFP) for five different types of AAV vectors [serotypes 2, 9, and Anc80L65 with promoter cytomegalovirus (CMV)-beta-Globin and serotypes 2 and 9 with promoter chicken beta-actin (CBA)] in in vitro cochlear explant cultures and we tested the transduction of AAV2/2-CBA, AAV2/9-CBA, and AAV2/Anc80L65-CMV by in vivo microinjection into the scala media of the cochlea. We found that each AAV vector had its own transduction and expression characteristics in hair cells and supporting cells in different regions of the cochlea. There was a tonotopic gradient for the in vitro transduction of AAV2/2-CBA, AAV2/9-CBA, AAV2/2-CMV, and AAV2/9-CMV in outer hair cells (OHCs), with more OHCs expressing eGFP at the base of the cochlea than at the apex. AAV2/2-CBA in vitro and AAV2/Anc80L65-CMV in vivo induced more supporting cells expressing eGFP at the apex than in the base. We found that AAV vectors with different promoters had different expression efficacies in hair cells and supporting cells of the auditory epithelium. The CMV-beta-Globin promoter could drive the expression of the delivered construct more efficiently in hair cells, while the CBA promoter was more efficient in supporting cells. The in vitro and in vivo experiments both demonstrated that AAV2/Anc80L65-CMV was a very promising vector for gene therapy of deafness because of its high transduction rates in hair cells. These results might be useful for selecting the appropriate vectors for gene delivery into different types of inner ear cells and thus improving the effectiveness of gene therapy.
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