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

Biological Significance of High Expression of Serum Exosome MiR-96-5p to Suppress FoxO1 in Patients with Head and Neck Squamous Carcinoma

目的:通过证实头颈部鳞状细胞癌(HNSCC)患者血清外泌体(Exos)中miR-96-5p对叉头框转录因子O亚族1(FoxO1)的靶向调节作用,探讨Exo-miR-96-5p对HNSCC细胞增殖、侵袭、迁移等恶性生物学行为的影响及分子机制。方法:取2020年9月至2022年1月我院收治的HNSCC患者(25例)及同期体检健康者(40例)血清标本,超速离心法提取Exos,RT-PCR检测Exos中miR-96-5p表达;PKH67染液标记Exos后,与HNSCC细胞系-AGZY-973共培养,观察HNSCC细胞对Exos摄取的影响。AGZY-973细胞分为:Control组(加入DMEM培养基正常培养)、HNSCC患者血清Exos组(加入DMEM培养基和50μL HNSCC患者血清Exos悬液共培养)、健康对照者血清Exos组(加入DMEM培养基和50μL健康对照者血清Exos悬液共培养),CCK-8法检测细胞增殖;流式细胞术检测凋亡率;Transwell检测迁移、侵袭;RT-PCR法检测miR-96-5p表达;Western blot法检测细胞FoxO1及促凋亡蛋白Bax、死亡调解子(Bim)、凋亡蛋白p21表达。双荧光素酶报告试验验证miR-96-5p与FoxO1靶向关系;36只裸鼠分为Control组、Exos组、Exos-miR-96-5p inhibitor组、Exos-inhibitor-NC组、Exos-pcDNA FoxO1组、Exos-pcDNA NC组,每组6只。Control组接种AGZY-973细胞培养液,其余各组分别接种含50μL HNSCC患者血清Exos悬液、Exos-miR-96-5p inhibitor悬液、Exos-inhibitor-NC悬液、Exos-pcDNA-FoxO1悬液、Exos-pcDNA-NC悬液的AGZY-973细胞培养液,28d后,检测瘤体体积及瘤体重量,以验证HNSCC患者血清Exos对miR-96-5p及FoxO1调控的影响。结果:HNSCC患者血清Exos及健康对照者血清Exos均有完整膜,形态呈杯口状,均可被AGZY-973细胞摄取。HNSCC患者血清Exos中miR-96-5p表达高于健康对照者血清Exos(P<0.05)。与Control组相比,HNSCC患者血清Exos组AGZY-973细胞miR-96-5p表达升高,FoxO1及Bax、Bim、p21等凋亡蛋白表达降低,细胞增殖、迁移及侵袭能力升高,凋亡率降低(P<0.05);健康对照者血清Exos对AGZY-973细胞miR-96-5p/FoxO1轴及生物学行为影响不大(P>0.05)。miR-96-5p与FoxO1之间有靶向调控关系。HNSCC患者血清Exos中转染miR-96-5p inhibitor或pcDNA-FoxO1,均可部分逆转Exos发挥的促瘤体生长作用(P<0.05)。结论:HNSCC患者血清Exos可通过传递miR-96-5p,靶向下调FoxO1,发挥促进HNSCC细胞增殖、侵袭、迁移、生长等恶性生物学行为的发展。

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;36nude mice were divided into Control group,Exos group,Exos-miR-96-5p inhibitor group,Exos-inhibitorNC 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 50μL 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.