Effects of exosomes derived from hepatocellular carcinoma cells on their biological behaviors of proliferation, apoptosis and autophagy
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摘要:
目的 外泌体在肝癌的发生发展中发挥重要作用,本研究旨在探讨HepG2肝癌细胞增殖、凋亡及自噬与外泌体释放的影响及相关机制。 方法 通过超速离心法提取HepG2细胞外泌体,采用透射电镜、纳米颗粒跟踪、Western blotting分析HepG2细胞中外泌体形态、径粒分布及标志蛋白表达;将外泌体与HepG2细胞共同培养以后,采用CCK-8检测HepG2细胞增殖变化、流式细胞术检测HepG2细胞凋亡率,Western blotting检测凋亡相关蛋白(Bax、Bcl-2)和自噬相关蛋白(LC3-Ⅱ/LC3-Ⅰ、p62、Beclin-1)表达。 结果 提取的HepG2细胞外泌体形态符合外泌体特征,直径在30~150 nm之间,标志蛋白Alix和CD63表达明显。CCK-8结果显示,24 h外泌体组与对照组吸光度相比差异无统计学意义(P>0.05),48 h及72 h外泌体组吸光度均高于对照组(P<0.05)。流式细胞术检测结果显示,对照组细胞凋亡率为(14.96±0.28)%,外泌体组细胞凋亡率为(11.16±0.50)%,细胞凋亡率下降(t=11.485,P<0.001)。Western blotting结果显示,与对照组比较,外泌体组凋亡相关蛋白Bcl-2表达上调(P<0.01)、Bax表达下调(P<0.01),自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ表达上调(P<0.01)、Beclin-1表达上调(P<0.05)、p62表达下调(P<0.01)。 结论 HepG2细胞来源的外泌体可以促进细胞增殖,通过诱导自噬上调而抑制细胞凋亡。 Abstract:Objective Exosomes play an important role in the occurrence and development of hepatocellular carcinoma. This study aims to investigate the effects of exosomes on the proliferation, apoptosis, autophagy, and exosome release of HepG2 hepatocellular carcinoma cells and the related mechanisms. Methods Exosomes of HepG2 cells were extracted by ultrafast centrifugation. Exosome morphology, diameter distribution and marker protein expression of HepG2 cells were analyzed by transmission electron microscopy, nanoparticle tracking and Western blotting analysis. After the co-culture of exosomes and HepG2 cells, a CCK-8 kit was used to detect the proliferation of HepG2 cells, the apoptosis of HepG2 cells was detected by flow cytometry, and the expressions of apoptosis-related proteins (Bax, Bcl-2) and autophagy-related proteins (LC3-Ⅱ/LC3-Ⅰ, p62, Beclin-1) were detected by Western blotting. Results The morphology of exosomes extracted from HepG2 cells was consistent with the characteristics of exosomes, and the diameter between 30 and 150 nm. The marker proteins Alix and CD63 were significantly expressed. The results of CCK-8 showed that the absorbance of HepG2 cells in the 24 h exosome group was not significantly different from that in the control group(P > 0.05), but the absorbance of the exosome group at 48 hours and 72 hours were higher than that of the control group (P < 0.05). Flow cytometry showed that the apoptosis rate was (14.96±0.28) % in the control group and (11.16±0.50) % in the exosome group, and the apoptosis rate decreased (t=11.485, P < 0.001). Western blotting results showed that compared with the control group, the expression of apoptosis-related protein Bcl-2 in exosome group was up-regulated (P < 0.01), and the expression of Bax was down-regulated (P < 0.01). Autophagy associated protein LC3-Ⅱ/LC3-Ⅰ expression was up-regulated (P < 0.01), Beclin-1 expression was up-regulated (P < 0.05), and p62 expression was down-regulated (P < 0.01). Conclusion Exosomes derived from HepG2 cells can promote cell proliferation and inhibit apoptosis by inducing up-regulation of autophagy. -
Key words:
- Hepatocellular carcinoma cells /
- Exosome /
- Proliferation /
- Apoptosis /
- Autophagy
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表 1 CCK-8法检测不同时间点细胞吸光度值(x±s)
Table 1. Absorbance of cells at different time measured by CCK-8 method(x±s)
组别 孔数 24 h 48 h 72 h 对照组 3 1.72±0.06 1.94±0.06 2.30±0.02 外泌体组 3 1.75±0.05 2.12±0.06 2.69±0.12 F值 0.601 14.394 33.483 P值 0.481 0.019 0.004 注:F时间=199.236,P时间<0.001;F组间=40.178,P组间=0.003;F交互=11.407,P交互=0.005。 -
[1] SUNG H, FERLAY J, SIEGEL R L, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249. doi: 10.3322/caac.21660 [2] CHEN R, XU X, TAO Y Q, et al. Exosomes in hepatocellular carcinoma: a new horizon[J]. Cell Commun Signal, 2019, 17(1): 1. doi: 10.1186/s12964-018-0315-1 [3] ZELLI V, COMPAGNONI C, CAPELLI R, et al. Role of exosomal microRNAs in cancer therapy and drug resistance mechanisms: focus on hepatocellular carcinoma[J]. Front Oncol, 2022, 12: 940056. DOI: 10.3389/fonc.2022.940056. [4] SU Y Y, CHAO C H, HSU H Y, et al. DDX3 suppresses hepatocellular carcinoma progression through modulating the secretion and composition of exosome[J]. Am J Cancer Res, 2023, 13(5): 1744-1765. [5] XI L, PENG M X, LIU S Q, et al. Hypoxia-stimulated ATM activation regulates autophagy-associated exosome release from cancer-associated fibroblasts to promote cancer cell invasion[J]. J Extracell Vesicles, 2021, 10(11): e12146. DOI: 10.1002/jev2.12146. [6] XIE Q H, ZHENG J Q, DING J Y, et al. Exosome-mediated immunosuppression in tumor microenvironments[J]. Cells, 2022, 11(12): 1946. doi: 10.3390/cells11121946 [7] BAI J F, HUANG M, SONG B H, et al. The current status and future prospects for conversion therapy in the treatment of hepatocellular carcinoma[J]. Technol Cancer Res Treat, 2023. DOI: 10.1177/15330338231159718. [8] QU Z, FENG J W, PAN H, et al. Exosomes derived from HCC cells with different invasion characteristics mediated EMT through TGF-β/Smad signaling pathway[J]. Onco Targets Ther, 2019, 12: 6897-6905. doi: 10.2147/OTT.S209413 [9] LIU C J, REN C N, GUO L, et al. Exosome-mediated circTTLL5 transfer promotes hepatocellular carcinoma malignant progression through miR-136-5p/KIAA1522 axis[J]. Pathol Res Pract, 2023, 241: 154276. DOI: 10.1016/j.prp.2022.154276. [10] LI X, LI C Y, ZHANG L Q, et al. The significance of exosomes in the development and treatment of hepatocellular carcinoma[J]. Mol Cancer, 2020, 19(1): 1. doi: 10.1186/s12943-019-1085-0 [11] HAN Q J, ZHAO H J, JIANG Y, et al. HCC-Derived Exosomes: critical player and target for cancer immune escape[J]. Cells, 2019, 8(6): 558. doi: 10.3390/cells8060558 [12] YANG K K, ZHOU Q B, QIAO B B, et al. Exosome-derived noncoding RNAs: function, mechanism, and application in tumor angiogenesis[J]. Mol Ther Nucleic Acids, 2022, 27: 983-997. doi: 10.1016/j.omtn.2022.01.009 [13] GE Y, MU W, BA Q, et al. Hepatocellular carcinoma-derived exosomes in organotropic metastasis, recurrence and early diagnosis application[J]. Cancer Lett, 2020, 477: 41-48. doi: 10.1016/j.canlet.2020.02.003 [14] 邹敏, 游逾, 何松, 等. 缺氧外泌体对肝癌Huh7细胞增殖、迁移及侵袭的影响[J]. 中华肝脏病杂志, 2019, 27(5): 363-368. doi: 10.3760/cma.j.issn.1007-3418.2019.05.008ZOU M, YOU Y, HE S, et al. Effects of hypoxic exosomes on the proliferation, migration and invasion of hepatocellular carcinoma Huh7 cells[J]. Chin J Hepatol, 2019, 27(5): 363-368. doi: 10.3760/cma.j.issn.1007-3418.2019.05.008 [15] WEI X N, YANG Z H, CHEN G M, et al. VMP1 promotes exosome secretion and enhances 5-FU resistance in colon cancer cells[J]. Tissue Cell, 2022, 77: 101851. DOI: 10.1016/j.tice.2022.101851. [16] 符山花, 包利利, 赵达, 等. ABT-737对M2型TAM来源外泌体处理的卵巢癌细胞SKOV3自噬性凋亡与干性特征的影响[J]. 西部医学, 2023, 35(5): 654-661, 669. doi: 10.3969/j.issn.1672-3511.2023.05.005FU S H, BAO L L, ZHAO D, et al. Effects of ABT-737 on autophagy apoptosis and stemness characteristics of SKOV3 ovarian cancer cells treated with M2 tumor-associated macrophage-derived exosomes[J]. Med J West China, 2023, 35(5): 654-661, 669. doi: 10.3969/j.issn.1672-3511.2023.05.005 [17] YAZDANI H O, HUANG H, TSUNG A. Autophagy: dual response in the development of hepatocellular carcinoma[J]. Cells, 2019, 8(2): 91. doi: 10.3390/cells8020091 [18] 童里, 郑小飞, 顾旺, 等. 细胞自噬在肝细胞肝癌中的研究现状[J]. 中华全科医学, 2023, 21(4): 672-676. doi: 10.16766/j.cnki.issn.1674-4152.002957TONG L, ZHENG X F, GU W, et al. Research status of autophagy in hepatocellular carcinoma[J]. Chinese Journal of General Practice, 2023, 21(4): 672-676. doi: 10.16766/j.cnki.issn.1674-4152.002957 [19] KOUROUMALIS E, TSOMIDIS I, VOUMVOURAKI A. Pathogenesis of hepatocellular carcinoma: the interplay of apoptosis and autophagy[J]. Biomedicines, 2023, 11(4): 1166. doi: 10.3390/biomedicines11041166 [20] MA W J, ZHOU Y, LIU M, et al. Long non-cod-ing RNA LINC00470 in serum derived exosome: a critical regulator for proliferation and autophagy in glioma cells[J]. Cancer Cell Int, 2021, 21(1): 149. doi: 10.1186/s12935-021-01825-y [21] YAO W J, GUO P, MU Q M, et al. Exosome-derived circ-PVT1 contributes to cisplatin resistance by regulating autophagy, invasion, and apoptosis via miR-30a-5p/YAP1 axis in gastric cancer cells[J]. Cancer Biother Radiopharm, 2021, 36(4): 347-359. [22] LIAO M M, QIN M R, LIU L H, et al. Exosomal microRNA profiling revealed enhanced autophagy suppression and anti-tumor effects of a combination of compound Phyllanthus urinaria and lenvatinib in hepatocellular carcinoma[J]. Phytomedicine, 2024, 122: 155091. DOI: 10.1016/j.phymed.2023.155091. -