Abstract:

Objective To discuss the effect of long non-coding RNA （lncRNA） DUXAP8 in exosomes （Exo） derived from the lung cancer A549 cells on the growth and immune escape of the lung cancer cells， and to clarify the mechanism. Methods The human lung cancer cell line A549 was cultured， and its exosomes were extracted and identified. The A549 cells were treated with PKH67-labeled Exo to observe the uptake of Exo by A549 cells. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of lncRNA DUXAP8 in A549 cells before and after Exo treatment. The A549 cells were divided into control group （no treatment）， Exo group （A549 cells treated with Exo）， Exo+sh-NC group （A549 cells treated with Exo and then transfected with sh-NC）， and Exo+ sh-DUXAP8 group （A549 cells treated with Exo and then transfected with sh-DUXAP8）. RT-qPCR method was used to detect the expression level of lncRNA DUXAP8 in A549 cells in various groups； colony formation assay was used to detect the colony formation abilities of the A549 cells in various groups； 5-ethynyl-2'-deoxyuridine （EdU） staining method was used to detect the proliferation abilities of the A549 cells in various groups. After co-culturing A549 cells in various groups with human peripheral blood lymphocytes， flow cytometry was used to detect the percentages of activated CD8+ T lymphocytes in the human peripheral blood lymphocytes in various groups； 3-（4，5-dimethylthiazol-2-yl）-2， 5-diphenyltetrazolium bromide （MTT） method was used to detect the killing rates of human peripheral blood lymphocytes on the A549 cells in various groups. Results The diameter of Exo vesicles was 50-150 nm， and the exosome-specific marker proteins cluster of differentiation 63（CD63）， cluster of differentiation 9（CD9）， tumor susceptibility gene 101（TSG101）， and heat shock protein 70（HSP70） were positively expressed， indicating successful exosome extraction. A549 cells efficiently took up PKH67-labeled Exo. The RT-PCR results showed that compared with A549 cells cultured alone， the expression level of lncRNA DUXAP8 in the A549 cells was increased after treatment with Exo derived from A549 cells （P<0.05）. compared with control group， the expression level of lncRNA DUXAP8 in the A549 cells in Exo group was increased （P<0.05）； compared with Exo group， the expression level of lncRNA DUXAP8 in the A549 cells in Exo+sh-DUXAP8 group was decreased （P<0.05）， while there were no significant difference in the expression level of IncRNA DUXAP8 in the cells in Exo+sh-NC group （P>0.05）. The colony formation assay results showed that compared with control group， the number of colony formation of the A549 cells in Exo group was increased （P<0.05）； compared with Exo group， the number of colony formation of the A549 cells in Exo+sh-DUXAP8 group was decreased （P<0.05）， while there was no significant difference in the number of colony formation of the A549 cells in Exo+sh-NC group （P>0.05）. The EdU staining results showed that compared with control group， the EdU-positive rate of the A549 cells in Exo group was increased （P<0.05）； compared with Exo group， the EdU-positive rate in A549 cells in Exo+sh-DUXAP8 group was decreased （P<0.05）， while there was no significant difference in the EDU-positive rate in the cells in Exo+sh-NC group （P>0.05）. The flow cytometry results showed that compared with control group， the percentage of activated CD8+T lymphocytes in the human peripheral blood lymphocytes in Exo group was decreased （P<0.05）； compared with Exo group， the percentage of activated CD8+T lymphocytes in the human peripheral blood lymphocytes in Exo+ sh-DUXAP8 group was increased （P<0.05）， while there was no significant difference in the percentage of activated CD8+T lymphaytes in Exo+sh-NC group （P>0.05）. The MTT assay results showed that compared with control group， the killing rate of human peripheral blood lymphocytes on the A549 cells in Exo group was decreased （P<0.05）； compared with Exo group， the killing rate of human peripheral blood lymphocytes on A549 cells in Exo+sh-DUXAP8 group was increased （P<0.05）， while no significant difference was observed in Exo+sh-NC group （P>0.05）. Conclusion The lncRNA DUXAP8 in exosomes derived from the lung cancer A549 cells promotes the proliferation of lung cancer cells and tumor immune escape.

Key words: Lung neoplasms, Exosomes, Long non-coding RNA, DUXAP8, Cell proliferation, Immune escape