外泌体为载体的微小RNA-1基因运送抑制人口腔鳞状细胞癌CAL-27细胞增殖的研究

doi:10.7518/hxkq.2021.02.003

华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (2): 136-142.doi: 10.7518/hxkq.2021.02.003

外泌体为载体的微小RNA-1基因运送抑制人口腔鳞状细胞癌CAL-27细胞增殖的研究

伍宝琴¹^,²(), 黎春晖¹^,², 张梦莲¹, 聂敏海¹^,²()

^1.西南医科大学口腔医学院口颌面修复重建和再生实验室，泸州 646000
^2.西南医科大学附属口腔医院牙周黏膜病科，泸州 646000

收稿日期:2020-06-26 修回日期:2020-09-09 出版日期:2021-04-01 发布日期:2021-04-09
通讯作者: 聂敏海 E-mail:wubaoqinasd@163.com;nieminhai@126.com
作者简介:伍宝琴，医师，硕士，E-mail：wubaoqinasd@163.com
基金资助:
四川省教育厅重点项目(17ZA0443);四川省卫生和计划生育委员会科研课题(16PJ535);西南医科大学附属口腔医院项目(201707)

microRNA-1 gene delivery mediated by exosomes suppresses CAL-27 cell proliferation

Wu Baoqin¹^,²(), Li Chunhui¹^,², Zhang Menglian¹, Nie Minhai¹^,²()

^1.Orofacial Reconstruction and Regeneration Laboratory, School of Stomatology of Southwest Medical University, Luzhou 646000, China
^2.Dept. of Periodontal and Oral Medicine, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, China

Received:2020-06-26 Revised:2020-09-09 Online:2021-04-01 Published:2021-04-09
Contact: Nie Minhai E-mail:wubaoqinasd@163.com;nieminhai@126.com
Supported by:
Key Project of Education Department of Sichuan Province(17ZA0443);Project of Health and Family Planning Commission of Sichuan Province(16PJ535);Project of The Affiliated Stomatology Hospital of Southwest Medical University(201707)

摘要/Abstract

摘要： 目的

用供体细胞基因过表达的方法构建微小RNA-1（miR-1）高表达的细胞内源性外泌体，探讨以外泌体为载体，运送miR-1对口腔鳞状细胞癌CAL-27细胞增殖的作用。

方法

采用超速离心法提取过表达miR-1的HEK293细胞的外泌体，并用透射电子显微镜、纳米颗粒分析仪、Western blot及定量聚合酶链反应（qPCR）进行外泌体鉴定。过表达miR-1的HEK293细胞外泌体（miR1-EXO）、HEK293细胞的外泌体（CON-EXO）及等体积磷酸盐缓冲液（PBS）分别与CAL-27细胞共培养，用免疫荧光检测外泌体向细胞的转运，用qPCR检测CAL-27细胞miR-1及下游靶基因MET的表达变化，用噻唑蓝比色法（MTT）检测CAL-27细胞增殖情况，用流式细胞术进行细胞周期检测。同时，miR1-EXO、CON-EXO及等体积PBS分别与人正常口腔黏膜上皮角化细胞（NOK）共培养，用MTT法检测NOK细胞增殖情况。

结果

miR1-EXO、CON-EXO均呈典型的球形或杯状结构，直径约110 nm，并高表达外泌体标志性蛋白CD9、Alix及Tsg101。miR1-EXO中miR-1的表达量为285.80±14.33，CON-EXO的表达量为1.00±0.06，二者间有统计学差异（P<0.000 1）。免疫荧光及qPCR结果显示，与CAL-27细胞共培养后，miR1-EXO被CAL-27细胞摄取，miR1-EXO CAL-27细胞的miR-1表达水平高于CON-EXO及PBS，miR-1下游靶基因MET表达水平下调。MTT及细胞周期结果显示，与CAL-27细胞共培养后，miR1-EXO G0/G1期细胞比例高于CON-EXO和PBS，抑制CAL-27细胞增殖，而miR1-EXO对NOK细胞增殖的影响很小。

结论

供体细胞基因过表达的方法可使过表达miR-1的HEK293细胞分泌高表达miR-1的外泌体，并且高表达miR-1的外泌体可以将miR-1运送到CAL-27细胞，下调MET基因表达，抑制CAL-27细胞增殖。

关键词: 外泌体, 微小RNA-1, 口腔鳞状细胞癌, 细胞增殖

Abstract: Objective

This study aims to construct endogenous exosomes abundantly loaded with miR-1 and investigate the role of exosome-mediated microRNA-1 (miR-1) delivery on CAL-27 cell proliferation.

Methods

Exosomes secreted by miR-1-overexpressing HEK293 cells (miR1-EXO) were purified via ultracentrifugation and subjected to transmission electron microscopy, nanoparticle analysis, Western blot analysis, and quantitative polymerase chain reaction (qPCR). CAL-27 cells were cocultured with exosomes secreted by HEK293 cells (CON-EXO) and miR1-EXO and equivalent phosphate buffer saline. The intracellular transport of exosomes was measured by using immunofluorescence, the expression of miR-1 and its target gene MET were investigated via qPCR, CAL-27 cell proliferation was measured through MTT assay, and cell cycle state was determined by applying flow cytometry.

Results

Electron microscopy revealed that miR1-EXO and CON-EXO were spherical or cup-shaped with an average diameter of approximately 110 nm. The well-known exosome markers CD9, Tsg101, and Alix were enriched. The expression of miR-1 in miR1-EXO was higher than that in CON-EXO (285.80±14.33 vs 1.00±0.06, P<0.000 1). After coculture with CAL-27 cells, miR1-EXO was internalized and unloaded miR-1 into CAL-27 cells. After coculture with miR1-EXO, the expression of miR-1 in CAL-27 cells was upregulated, whereas that of MET, the target gene of miR-1, was suppressed and the proliferation of CAL-27 cells was inhibited significantly. Normal oral keratinocyte cell proliferation was negligibly affected after coculture with miR1-EXO.

Conclusion

Exosomes secreted from miR1-EXO cells could load abundant miR-1. Exosomal miR-1 delivered into CAL-27 cells by using miR1-EXO suppressed the expression of MET mRNA and inhibited cell proliferation.

Key words: exosomes, microRNA-1, oral squamous cell carcinoma, cell proliferation

中图分类号:

R 739.8

伍宝琴, 黎春晖, 张梦莲, 聂敏海. 外泌体为载体的微小RNA-1基因运送抑制人口腔鳞状细胞癌CAL-27细胞增殖的研究[J]. 华西口腔医学杂志, 2021, 39(2): 136-142.

Wu Baoqin, Li Chunhui, Zhang Menglian, Nie Minhai. microRNA-1 gene delivery mediated by exosomes suppresses CAL-27 cell proliferation[J]. West China Journal of Stomatology, 2021, 39(2): 136-142.

图/表 4

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外泌体为载体的微小RNA-1基因运送抑制人口腔鳞状细胞癌CAL-27细胞增殖的研究

microRNA-1 gene delivery mediated by exosomes suppresses CAL-27 cell proliferation

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