毛茛总苷通过circ_0079593/miR-324-5p通路抑制肝癌细胞增殖、迁移及侵袭的机制研究

摘要/Abstract

摘要： 目的: 探讨毛茛总苷对肝癌细胞增殖、迁移、侵袭的影响及其对circ_0079593/miR-324-5p通路的调控作用。
方法: 体外培养人肝癌细胞MHCC97H,并分为对照组,毛茛总苷低、中、高剂量组,si-NC组,si-circ_0079593组,毛茛总苷+pcDNA组,毛茛总苷+pcDNA-circ_0079593组;采用CCK-8法、Transwell实验分别检测细胞活力、迁移及侵袭;采用定量反转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction, qRT-PCR）检测circ_0079593和miR-324-5p的表达水平;采用双荧光素酶报告基因实验检测circ_0079593和miR-324-5p的靶向关系;采用蛋白质印迹法检测基质金属蛋白酶-2（matrix metalloproteinase-2, MMP-2）、基质金属蛋白酶-9（matrix metalloproteinase-9, MMP-9）的表达水平。
结果: 与对照组比较,毛茛总苷不同剂量组细胞增殖抑制率和miR-324-5p表达水平均升高（P<0.05）,迁移及侵袭细胞数均减少（P<0.05）,circ_0079593表达水平及MMP-2、MMP-9蛋白水平均降低（P<0.05）,且呈剂量依赖性;circ_0079593能靶向调控miR-324-5p;与si-NC组比较,si-circ_0079593组细胞增殖抑制率升高（P<0.05）,迁移及侵袭细胞数均减少（P<0.05）,MMP-2、MMP-9蛋白水平均降低（P<0.05）;与毛茛总苷+pcDNA组比较,毛茛总苷+pcDNA-circ_0079593组细胞增殖抑制率降低（P<0.05）,迁移及侵袭细胞数均增多（P<0.05）,MMP-2、MMP-9蛋白水平均升高（P<0.05）。
结论: 毛茛总苷可通过调控circ_0079593/miR-324-5p通路降低肝癌细胞增殖、迁移及侵袭能力。

关键词: 毛茛总苷, 肝癌, 细胞增殖, 细胞迁移, 细胞侵袭

Abstract: Objective:To explore the effect of total glycosides of ranunculus japonicus on the proliferation, migration and invasion of liver cancer MHCC97H cells and its regulation of circ_0079593/miR-324-5p pathway.
Method:Human hepatocarcinoma cells MHCC97H were cultured in vitro, experimental groups: control group, total glycosides of ranunculus japonicus low-dose group, total glycosides of ranunculus japonicus medium-dose group, total glycosides of ranunculus japonicus high-dose group, si-NC group, si-circ_0079593 group, total glycosides of ranunculus japonicus+pcDNA group, total glycosides of ranunculus japonicus+pcDNA-circ_0079593 group. CCK-8 and Transwell chamber experiments were used to detect cell viability, migration and invasion. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect the expression of circ_0079593 and miR-324-5p. The dual luciferase reporter gene experiment was used to detect the targeting relationship between circ_0079593 and miR-324-5p. Western blotting was used to detect the protein expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9).
Result:Compared with the control group, the cell proliferation inhibition rate and the expression level of miR-324-5p in different dose groups of total glycosides of ranunculus japonicus were increased (P<0.05), while the number of migrating and invading cells was decreased (P<0.05), and the expression level of circ_0079593 and the protein levels of MMP-2, MMP-9 were decreased (P<0.05), and were dose-dependent. circ_0079593 could target and regulate miR-324-5p. Compared with the si-NC group, the cell proliferation inhibition rate in the si-circ_0079593 group was increased (P<0.05), while the number of migrating and invading cells was decreased (P<0.05), and the protein levels of MMP-2 and MMP-9 were decreased (P<0.05). Compared with the total ranunculus glycosides+pcDNA group, the inhibition rate of cell proliferation in the total ranunculus glycosides+pcDNA-circ_0079593 group was reduced (P<0.05), while the number of migration and invasion cells was increased (P<0.05), and the protein levels of MMP-2, MMP-9 were increased (P<0.05).
Conclusion:Total glycosides of ranunculus japonicus could reduce the proliferation, migration and invasion of liver cancer cells by regulating the circ_0079593/miR-324-5p pathway.

Key words: Total glycosides of ranunculus japonicus, Liver cancer, Cell proliferation, Cell migration, Cell invasion

杜红蕾, 张锋, 张咏华, 班志超, 王旭. 毛茛总苷通过circ_0079593/miR-324-5p通路抑制肝癌细胞增殖、迁移及侵袭的机制研究[J]. 肝癌电子杂志, 2022, 9(1): 48-54.

Du Honglei, Zhang Feng, Zhang Yonghua, Ban Zhichao, Wang Xu. Study on the mechanism of total glycosides of ranunculus japonicus inhibiting the proliferation, migration and invasion of liver cancer cells through the circ_0079593/miR-324-5p pathway[J]. Electronic Journal of Liver Tumor, 2022, 9(1): 48-54.

参考文献

[1] 伍振辉, 孟娴, 丁永芳, 等. 白首乌C-21甾体总苷对人肝癌HepG2细胞增殖、侵袭与凋亡的影响[J]. 中国中药杂志, 2018, 43(9): 1915-1921.
[2] 卢文显, 周志华, 陈素珠. 无患子总皂苷对人肝癌细胞Huh7增殖与凋亡的影响[J]. 福建师范大学学报(自然科学版), 2019, 35(2): 85-88.
[3] 雷佳蕾, 张志宇, 杨天歌, 等. 文冠果种仁总皂苷诱导肝癌HepG2细胞凋亡的实验研究[J]. 陕西师范大学学报(自然科学版), 2020, 48(1): 80-86.
[4] 杨星. 毛茛总苷诱导卵巢癌SKOV-3细胞凋亡的体外研究[J]. 中国药物与临床, 2015, 15(6): 775-777.
[5] LU J, LI Y.Circ_0079593 facilitates proliferation, metastasis, glucose metabolism and inhibits apoptosis in melanoma by regulating the miR-516b/GRM3 axis[J]. Mol Cell Biochem, 2020, 475(1-2): 227-237.
[6] CAO L, XIE B, YANG X, et al.MiR-324-5p Suppresses Hepatocellular Carcinoma Cell Invasion by Counteracting ECM Degradation through Post-Transcriptionally Downregulating ETS1 and SP1[J]. PLoS One, 2015, 10(7): e0133074-e0133084.
[7] 傅士龙, 朱峰妍, 曹志飞, 等. 毛茛总苷对人胃癌MGC803细胞血管生成拟态的影响及其机制[J]. 中草药, 2013, 44(16): 2267-2274.
[8] 刘涛, 栾昕, 赵菊梅, 等. 延龄草总皂苷通过促进人β防御素2(HBD-2)的表达抑制HuH-7细胞侵袭和迁移[J]. 细胞与分子免疫学杂志, 2019, 35(6): 526-532.
[9] 郑雪莹, 曹宁宁, 裴帅, 等. 人参皂苷Rh2 抗肝癌作用机制的研究进展[J]. 中国实验方剂学杂志, 2019, 25(20): 208-213.
[10] YU J, YANG M, ZHOU B, et al.CircRNA-104718 acts as competing endogenous RNA and promotes hepatocellular carcinoma progression through microRNA-218-5p/TXNDC5 signaling pathway[J]. Clin Sci (Lond), 2019, 133(13): 1487-1503.
[11] 虞迪, 朱峰妍, 曹志飞, 等. 毛茛有效部位D1对人胃癌细胞MGC803血管生成拟态的作用及其机制[J]. 中国药理学通报, 2013, 29(2): 261-265.
[12] ZHAO W T, LIN X L, LIU Y, et al.miR-26a promotes hepatocellular carcinoma invasion and metastasis by inhibiting PTEN and inhibits cell growth by repressing EZH2[J]. Lab Invest, 2019, 99(10): 1484-1500.
[13] GONG X, ZHU Z.Long Noncoding RNA HOTAIR Contributes to Progression in Hepatocellular Carcinoma by Sponging miR-217-5p[J]. Cancer Biother Radiopharm, 2020, 35(5): 387-396.
[14] CHANG L, YUAN Y, LI C, et al.Upregulation of SNHG6 regulates ZEB1 expression by competitively binding miR-101-3p and interacting with UPF1 in hepatocellular carcinoma[J]. Cancer Lett, 2016, 383(2): 183-194.
[15] YANG Z, LI C, FAN X Y, et al.Circular RNA circ_0079593 promotes glioma development through regulating KPNA2 expression by sponging miR-499a-5p[J]. Eur Rev Med Pharmacol Sci, 2020, 24(3): 1288-1301.
[16] QU Y, ZHU J, LIU J, et al.Circular RNA circ_0079593 indicates a poor prognosis and facilitates cell growth and invasion by sponging miR-182 and miR-433 in glioma[J]. J Cell Biochem, 2019, 120(10): 18005-18013.
[17] GU C, ZHANG M, SUN W, et al.Upregulation of miR-324-5p Inhibits Proliferation and Invasion of Colorectal Cancer Cells by Targeting ELAVL1[J]. Oncol Res, 2019, 27(5): 515-524.
[18] ZHANG X, ZHANG L, CHEN M, et al.miR-324-5p inhibits gallbladder carcinoma cell metastatic behaviours by downregulation of transforming growth factor beta 2 expression[J]. Artif Cells Nanomed Biotechnol, 2020, 48(1): 315-324.