头颈鳞癌患者血清外泌体miR-96-5p高表达抑制FoxO1的生物学意义

doi:10.3969/j.issn.1006-6233.2022.08.01

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Abstract Objective: To explore the effect and molecular mechanism of Exos-miR-96-5p on the malignant biological behaviors of HNSCC cells such as proliferation, invasion and migration by confirming the targeted regulation of miR-96-5p in serum exosomes (Exos) of head and neck squamous cell carcinoma (HNSCC) patients on forkhead box transcription factor O subfamily 1 (FoxO1). Methods: Serum specimens of HNSCC patients (25 cases) admitted to our hospital from September 2020 to January 2022 and healthy individuals (40 cases) examined during the same period were extracted by ultracentrifugation, and miR-96-5p expression in Exos was detected by RT-PCR; after labeling Exos with PKH67 dye, they were co-cultured with HNSCC cell line-AGZY-973 to observe the effect of HNSCC cells on Exos uptake. AGZY-973 cells were divided into: Control group (normal culture with DMEM medium), HNSCC patient serum Exos group (co-culture with DMEM medium and 50μL HNSCC patient serum Exos suspension), healthy control serum Exos group (co-culture with DMEM medium and 50μL healthy control serum Exos suspension). The cell proliferation was detected by CCK-8 method; apoptosis rate was detected by flow cytometry; migration and invasion were detected by Transwell; miR-96-5p expression was detected by RT-PCR; FoxO1 and pro-apoptotic protein Bax, death mediator (Bim) and apoptotic protein p21 expression were detected by Western blot. Dual luciferase reporter assay was performed to verify the relationship between miR-96-5p and FoxO1 targeting; 36 nude mice were divided into Control group, Exos group, Exos-miR-96-5p inhibitor group, Exos-inhibitor-NC group, Exos-pcDNA FoxO1 group, Exos-pcDNA NC group, 6 in each group. The control group was inoculated with AGZY-973 cell culture, and the remaining groups were inoculated with 50 μL of Exos suspension of HNSCC patient serum, Exos-miR-96-5p inhibitor suspension, Exos-inhibitor-NC suspension, Exos-pcDNA-FoxO1 suspension, and Exos-pcDNA-NC suspension in AGZY-973 cell culture, respectively, and after 28 days, tumor volume and tumor weight were examined to verify the effect of serum Exos on miR-96-5p and FoxO1 regulation in HNSCC patients. Results: Serum Exos of HNSCC patients and healthy controls had intact membranes with a cup-shaped morphology, and both could be taken up by AGZY-973 cells. The expression of miR-96-5p in serum Exos of HNSCC patients was higher than that in serum Exos of healthy controls (P<0.05). Compared with the Control group, the serum Exos group of HNSCC patients had elevated miR-96-5p expression, decreased expression of FoxO1 and apoptotic proteins such as Bax, Bim and p21, elevated cell proliferation, migration and invasion ability, and decreased apoptosis rate (P<0.05); serum Exos of healthy controls had higher miR-96-5p expression on AGZY-973 cells miR-96-5p/FoxO1 axis and biological behavior were not significantly affected (P>0.05). MiR-96-5p had a target-regulatory relationship with FoxO1. Transfection of miR-96-5p inhibitor or pcDNA-FoxO1 in serum Exos of HNSCC patients partially reversed the pro-tumor growth effect exerted by Exos (P<0.05). Conclusion: Serum Exos of HNSCC patients can target down FoxO1 by transmitting miR-96-5p, and plays a role in promoting the development of malignant biological behaviors such as proliferation, invasion, migration and growth of HNSCC cells.

Key words： Head and neck squamous cell carcinoma Exosomes MiR-96-5p Forkhead box transcription factor O subfamily 1

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http://www.hbyxzzs.cn/EN/10.3969/j.issn.1006-6233.2022.08.01 OR http://www.hbyxzzs.cn/EN/Y2022/V28/I8/1233

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