盐酸小檗碱对宫颈癌HeLa细胞外泌体的影响及其机制

doi:10.13481/j.1671-587x.20200418

摘要/Abstract

摘要： 目的：观察盐酸小檗碱对宫颈癌HeLa细胞释放外泌体的干预作用，探讨其对癌细胞增殖和转移能力的影响。方法：将HeLa细胞分为对照组和低、中、高剂量盐酸小檗碱组，分别采用0、10、20和40 mg·L^-1盐酸小檗碱干预HeLa细胞24 h，高速离心提取外泌体，Western blotting法检测外泌体标志物CD81和CD63及磷脂酰肌醇3-激酶（PI3K）/蛋白质丝氨酸苏氨酸激酶（Akt）信号通路相关蛋白表达水平，MTT比色法检测细胞体外生长抑制率，划痕实验检测细胞迁移率，Transwell小室实验检测穿膜细胞数。结果：与对照组比较，低、中和高剂量盐酸小檗碱组细胞外泌体中外泌体标志物CD81和CD63蛋白表达水平明显降低（P<0.05），细胞生长抑制率明显升高（P<0.05），细胞迁移率和穿膜细胞数明显降低（P<0.05），细胞中PI3K、Akt和磷酸化Akt（p-Akt）蛋白表达水平明显降低（P<0.05）。与低剂量盐酸小檗碱组比较，中和高剂量盐酸小檗碱组细胞外泌体中CD81和CD63蛋白表达水平明显降低（P<0.05），细胞生长抑制率明显升高（P<0.05），细胞迁移率和穿膜细胞数明显降低（P<0.05），细胞中PI3K、Akt和p-Akt蛋白表达水平明显降低（P<0.05）。与中剂量盐酸小檗碱组比较，高剂量盐酸小檗碱组细胞外泌体中CD81和CD63蛋白表达水平明显降低（P<0.05），细胞生长抑制率明显升高（P<0.05），细胞迁移率和穿膜细胞数明显降低（P<0.05），细胞中PI3K、Akt和p-Akt蛋白表达水平明显降低（P<0.05），具有浓度依赖性。结论：盐酸小檗碱能够抑制宫颈癌细胞外泌体分泌和癌细胞增殖，降低癌细胞的侵袭和转移能力，其作用机制可能与下调PI3K/Akt信号通路相关蛋白表达有关。

关键词: 盐酸小檗碱, 外泌体, 宫颈肿瘤, 磷脂酰肌醇3-激酶, 蛋白质丝氨酸苏氨酸激酶, 信号通路

Abstract: Objective: To observe the intervention effect of berberine hydrochloride on the exosomes released by cervical cancer HeLa cells, and to explore its effects on the abilities of proliferation and metastasis of cancer cells. Methods: The cervical cancer HeLa cells were divided into control group and low, medium and high doses of berberine hydrochloride groups, and they were intervented with 0, 10, 20 and 40 mg·L^-1berberine hydrochloride for 24 h. High-speed centrifugation was used to extract the exosomes and Western blotting method was used to detect the expression levels of exosome markers CD81, CD63 and phosphatidylinositol 3-kinase(PI3K)/protein-serine-threonine kinase(Akt) signaling pathway related proteins. MTT assay was used to determine the inhibitory rates of growth of the HeLa cells.Scratch assay was used to measure the migration rate of cells. Transwell assay was used to detect the number of transmembrane cells. Results: Compared with control group, the expression levels of cervical cancer cell exosome markers CD81 and CD63 proteins in the exosomes in low, medium and high doses of berberine hydrochloride groups were significantly decreased (P<0.05), while the inhibitory rates of growth of cancer cells were increased significantly (P<0.05); the migration rates of cells and the number of transmembrane cells were decreased significantly (P<0.05),and the expression levels of PI3K, Akt and phosphorylated Akt (p-Akt) proteins in the cancer cells were significantly decreased (P<0.05).Compared with low dose of berberine hydrochloride group, the expression levels of CD81 and CD63 proteins in the exosomes in medium and high doses of berberine hydrochloride groups were significantly decreased (P<0.05), while the inhibitory rates of growth of cancer cells were increased significantly (P<0.05); the migration rates of cells and the number of transmembrane cells were decreased significantly (P<0.05), and the expression levels of PI3K, Akt and p-Akt proteins in the cancer cells were significantly decreased (P<0.05).Compared with medium dose of berberine hydrochloride group, the expression levels of CD81 and CD63 proteins in the exosomes in high dose of berberine hydrochloride group were significantly decreased (P<0.05), while the inhibitory rate of growth of cancer cells were increased significantly (P<0.05); the migration rates of cells and the number of transmembrane cells were decreased significantly (P<0.05), and the expression levels of PI3K, Akt and p-Akt proteins in the cancer cells were significantly decreased (P<0.05) in a concentration-dependent manner. Conclusion: Berberine hydrochloride can inhibit the exosome secretion of cervical cancer cells and the proliferation of cancer cells, and reduce the the abilities of invasion and metastasis of cancer cells. Its mechanism may be related to the down-regulation of the expressions of the PI3K/Akt signaling pathway related proteins.

Key words: berberine hydrochloride, exosome, cervical neoplasms, phosphatidylinositol-3-kinases, protein-serine-threonine kinase, signaling pathway

中图分类号:

R737.33

王景, 葛静, 王旭, 房桂英, 代丽丽, 刘晶. 盐酸小檗碱对宫颈癌HeLa细胞外泌体的影响及其机制[J]. 吉林大学学报(医学版), 2020, 46(04): 779-785.

WANG Jing, GE Jing, WANG Xu, FANG Guiying, DAI Lili, LIU Jing. Effect of berberine hydrochloride on exosomes of cervical cancer HeLa cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 779-785.

参考文献

[1] LUCIDO C T, WYNJA E, MADEO M, et al. Innervation of cervical carcinoma is mediated by cancer-derived exosomes[J].Gynecol Oncol, 2019, 154(1):228-235.
[2] BHAT A, SHARMA A, BHARTI A C. Upstream Hedgehog signaling components are exported in exosomes of cervical cancer cell lines[J]. Nanomedicine (Lond), 2018, 13(17):2127-2138.
[3] 王留军, 曾韬, 王越. 肿瘤外泌体在微环境细胞间通讯中的作用及其作为肿瘤标志物的研究进展[J]. 临床检验杂志, 2019, 37(5):338-341.
[4] LI J, LIU F, JIANG S L, et al. Berberine hydrochloride inhibits cell proliferation and promotes apoptosis of non-small cell lung cancer via the suppression of the MMP2 and Bcl-2/Bax signaling pathways[J]. Oncol Lett, 2018, 15(5):7409-7414.
[5] LI H L, WU H, ZHANG B B, et al. MAPK pathways are involved in the inhibitory effect of berberine hydrochloride on gastric cancer MGC803 cell proliferation and IL-8 secretion in vitro and in vivo[J]. Mol Med Rep, 2016, 14(2):1430-1438.
[6] WANG Y H, WEN B F, YU H L, et al. Berberine hydrochloride-loaded chitosan nanoparticles effectively targets and suppresses human nasopharyngeal carcinoma[J]. J Biomed Nanotechnol, 2018, 14(8):1486-1495.
[7] 朱泽民, 刘飞, 郭子依. 肝癌细胞分泌的外泌体microRNA-103通过靶向连接蛋白增加血管通透性并促进癌转移[J]. 临床肝胆病杂志, 2018, 34(5):1127.
[8] EPPLE L M, GRIFFITHS S G, DECHKOVSKAIA A M, et al. Medulloblastoma exosome proteomics yield functional roles for extracellular vesicles[J]. PLoS One, 2012, 7(7):e42064.
[9] 周建军, 傅忠星, 王烨菁, 等. 2004~2011年上海市卢湾区户籍人口常见妇科恶性肿瘤发病和死亡趋势分析[J]. 中国肿瘤, 2016, 25(11):854-859.
[10] 曾智豪, 陈小伍, 朱达坚, 等. 盐酸小檗碱对MCF-7乳腺癌小鼠体内抑制肿瘤生长的实验研究[J]. 河北医学, 2016, 22(6):988-990.
[11] LIU D, MENG X, WU D L, et al. A natural isoquinoline alkaloid with antitumor activity:studies of the biological activities of berberine[J]. Front Pharmacol, 2019, 10:9.
[12] KOMAL K. Abstract C38:Berberine/cisplatin-based radiotherapy for cervical cancer:The new and effective method to treat cervical cancer[J]. Cancer Res, 2011, 69(1):171-174.
[13] WANG H Y, YU H Z, HUANG S M, et al. P53, Bcl-2 and cox-2 are involved in berberine hydrochloride-induced apoptosis of HeLa229 cells[J]. Mol Med Rep, 2016, 14(4):3855-3861.
[14] 李娜, 尹艳英, 刘锦辉. 盐酸小檗碱对人宫颈癌细胞株转移侵袭研究[J]. 中医药导报, 2014, 20(3):76-78.
[15] 王超群, 孙豪庭, 钦伦秀. 外泌体与肿瘤侵袭转移相关研究进展[J]. 复旦学报(医学版), 2016, 43(1):99-103.
[16] 侯智为,李玉莹,宋海旭,等.糖尿病小鼠血清外泌体在H9C2心肌细胞损伤调控中的作用及机制[J]. 解放军医学杂志,2019,44(10):837-842.
[17] 易红艳, 周琛斐, 梁莉, 等. 宫颈癌细胞分泌外泌体介导上皮-间质转化提高癌前细胞侵袭能力的体外研究[J]. 现代妇产科进展, 2017, 26(3):165-168.
[18] HUANG C H, MANDELKER D, SCHMIDT-KITTER O, et al. The structure of a human p110α/p85α complex elucidates the effects of oncogenic PI3Kα mutations[J]. Science, 2007, 318(5857):1744-1748.
[19] 孙龙和, 夏建国, 钱春华, 等. 白藜芦醇通过Pi3K/AKT通路抑制胃癌SGC-7901细胞增殖和迁移[J]. 现代生物医学进展, 2016, 16(17):3237-3240, 3267.
[20] LI Y, XU Q H, SHI M L, et al. Low-level laser therapy induces human umbilical vascular endothelial cell proliferation, migration and tube formation through activating the PI3K/Akt signaling pathway[J]. Microvasc Res, 2020, 129:103959.
[21] 陈凤霞. 宫颈癌病情进展过程中PI3K/AKT/mTOR信号通路的变化及靶基因的探究[J]. 海南医学院学报, 2018, 24(19):1757-1761.
[22] HUO X, SUN H Z, LIU Q, et al. Clinical and expression significance of AKT1 by co-expression network analysis in endometrial cancer[J]. Front Oncol, 2019, 9:1147.