Down-regulation of Notch1 gene promotes autophagy of human liver cancer cell line HepG2

Abstract

Abstract: Objective To investigate the effect of Notch1 gene on the proliferation, apoptosis, autophagy and Akt/mTOR signaling pathway of HepG2 liver cancer cells. Methods Thirty-two cases of liver cancer tissues from patients undergoing liver cancer surgery in the Third Affiliated Hospital of Xinxiang Medical College were selected, and the expression of Notch1 mRNA was detected by fluorescence quantitative PCR. Notch1 siRNA eukaryotic expression vector was constructed, transfected into HepG2 cells by liposome, RT-qPCR and Western blot were applied to check the interference effect of Notch1, MTT method was used to detect cell proliferation and flow cyto- metry was used to detect cell apoptosis, Western blot was also applied in the detection of apoptosis-related proteins BCL-2, BAX, cleared-caspase-3, autophagy-related proteins LC3B, Beelinl, ATG7, ATG5 and Akt/mTOR signaling pathway related proteins Akt, p-Akt, p-mTOR, p-P70S6K. Results The relative expression of Notch1 gene in primary hepatocellular carcinoma tissue was significantly higher than that in adjacent tissues (1.865±0.791 vs 0.709±0.414) (P＜0.001). After Notch1 siRNA was transfected into HepG2 cells, the expression of Notch1 mRNA and protein in Notch1 siRNA group was significantly lower than that in NC siRNA group and in control group (P＜0.001). The cell proliferation rate of Notch1 siRNA group at 24, 48, 72 and 96 h was significantly lower than that of NC siRNA group and control group (P＜0.001). Forty-eight hours after transfection, the apoptosis rate and Bax level of Notch1 shRNA group were significantly higher than that of NC siRNA and control group, and the expression of Bcl-2 and cleaved caspase-3 were significantly lower than that of NC siRNA group and control group (P＜0.001). The expression of autophagy-related proteins LC3B, Beelinl, ATG7 and ATG5 in Notch1 siRNA group was significantly higher than that of NC siRNA group and control group (P＜0.001). The expression level of p-Akt, p-mTOR, and p-P70S6K in Notch1 siRNA group was significantly lower than those in NC siRNA group and control group (P＜0.001). Conclusions Down-regulation of expression of Notch1 gene can significantly inhibit the proliferation of liver cancer HepG2 cells, induce cell apoptosis, and increase cell autophagy. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway.

Key words: liver cancer, Notch1 gene, Akt/mTOR signal pathway, autophagy

CLC Number:

LI Chang-an, LIU Chun-xiu, XIE Xiao-juan, SUN Ai-ping. Down-regulation of Notch1 gene promotes autophagy of human liver cancer cell line HepG2[J]. Basic & Clinical Medicine, 2021, 41(4): 508-513.

References

[1] 杨文,李修红,张奇能.射频消融与手术切除治疗直径3～5 cm肝癌的倾向性评分匹配研究[J]. 现代肿瘤医学,2020,28:1689-1692.
[2] 陈耿,孔一帆,别平.剩余肝体积不足的肝癌手术切除策略[J]. 中华肝脏外科手术学电子杂志,2020,9:104-107.
[3] Xu L, Gu L, Tao X, et al. Effect of dioscin on promoting liver regeneration via activating Notch1/Jagged1 signal pathway[J]. Phytomedicine, 2018, 38:107-117.
[4] Wang Y, Li M, Dong C, et al. Linc00152 knockdown inactivates the Akt/mTOR and Notch1 pathways to exert its anti-hemangioma effect[J]. Life Sci, 2019,223:22-28.
[5] 庄志明,林天旗,林建贵, 等.靶向沉默Notch1基因对肾透明细胞癌增殖、凋亡及Akt/mTOR信号通路的影响[J]. 中华泌尿外科杂志,2017,38:136-140.
[6] 黄壮程,王宏浩,沈沛麟, 等.Notch信号通路相关蛋白在腺性膀胱炎和膀胱癌中的表达与意义[J]. 癌变·畸变·突变,2018,30:374-377,383.
[7] 王卫东,陈小伍,吴志强, 等.肝细胞癌组织中 Notch1和 Jagged1的表达及意义[J]. 广东医学,2015,36:763-766.
[8] 毛静芳,周建中,周家华.Notch1基因在调控人肝癌细胞HepG2侵袭转移中的作用[J]. 山东医药,2014,54:24-26.
[9] 宋园远,姜晓峰,梁红艳.细胞自噬在肿瘤治疗中的研究进展[J]. 医学综述,2019,25:4887-4891.
[10] 单钰莹,陆才德.自噬在肝癌中的双面性作用及自噬调节剂用于肝癌治疗的研究进展[J]. 浙江医学,2018,40:1020-1023.
[11] Rupert L, Christina N, Manuel K, et al. Expression analysis of autophagy related markers LC3B, p62 and HMGB1 indicate an autophagy-independent negative prognostic impact of high p62 expression in pulmonary squamous cell carcinomas[J]. Cancers, 2018, 10:281-293.
[12] You Z, XU Y, Wan W,et al. TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction.[J]. Autophagy, 2019, 15: 1309-1321.
[13] Kumar V, Zhang MX, Swank MW, et al. Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways[J]. J Neurosci, 2005, 25:11288-99.
[14] Milosevic Z, Pesic M, Stankovic T, et al. Targeting RAS-MAPK-ERK and PI3K-AKT-mTOR signal transduction pathways to chemosensitize anaplastic thyroid carcinoma[J]. Transl Res, 2014, 164:411-423.
[15] 周福安, 张志明, 张巍. PI3K/AKT/mTOR信号转导通路相关蛋白在脑胶质瘤中的表达及临床意义[J]. 临床神经病学杂志, 2012, 25:278-281.