基于长链非编码RNA HOX转录反义RNA探讨次乌头原碱对人肝癌SK-Hep-1细胞自噬的影响实验研究OACSTPCD
Effect of protopine on autophagy in human liver cancer SK-Hep-1 cells based on lncRNA HOTAIR
目的:基于长链非编码RNA(lncRNA)HOX转录反义RNA(HOTAIR)探讨次乌头原碱对人肝癌SK-Hep-1细胞自噬的影响.方法:取对数期人肝癌SK-Hep-1细胞,将lncRNA HOTAIR抑制质粒lncRNA HOTAIR-shRNA转染至SK-Hep-1细胞,随机分为转染组和联合组,转染组常规培养,联合组加入次乌头原碱(终浓度200 μmol/L).另取SK-Hep-1细胞随机分为空白组和次乌头原碱组,空白组常规培养,次乌头原碱组加入次乌头原碱(终浓度200 μmol/L).48 h后收集细胞,实时荧光定量PCR(RT-qPCR)检测各组lncRNA HOTAIR表达量;流式细胞仪检测各组凋亡率;绿色荧光蛋白(GFP)-微管相关蛋白1轻链3(LC3)转染检测各组自噬情况;免疫印迹法检测细胞Beclin1、P62、微管相关蛋白1轻链3 Ⅰ(LC3 Ⅰ)蛋白相对表达量.结果:与空白组比较,次乌头原碱组lncRNA HOTAIR表达量升高,转染组lncRNA HOTAIR表达量降低(均P＜0.05).与次乌头原碱组比较,联合组lncRNA HOTAIR表达量降低(P＜0.05).与转染组比较,联合组lncRNA HOTAIR表达量升高(P＜0.05).与空白组比较,次乌头原碱组细胞凋亡率及Beclin1、P62、LC3 Ⅰ蛋白相对表达量升高,GFP-LC3斑点数增加(均P＜0.05);转染组细胞凋亡率及Beclin1、P62、LC3 Ⅰ蛋白表达量降低,GFP-LC3斑点数减少(均P＜0.05).与次乌头原碱组比较,联合组细胞凋亡率及Beclin1、P62、LC3 Ⅰ蛋白相对表达量降低,GFP-LC3斑点数减少(均P＜0.05).与转染组比较,联合组细胞凋亡率及Beclin1、P62、LC3 Ⅰ蛋白相对表达量升高,GFP-LC3斑点数增加(均P＜0.05).结论:次乌头原碱可能通过诱导人肝癌SK-Hep-1细胞过度自噬而促进细胞凋亡,其作用机制可能与上调LncRNA HOTAIR表达有关.
Objective:To investigate the effect of protopine on autophagy in human liver cancer SK-Hep-1 cells based on long non-coding RNA(lncRNA)HOX transcript antisense RNA(HOTAIR).Methods:Logarithmic phase human hepatoma SK-Hep-1 cells were taken,and lncRNA HOTAIR suppression plasmid lncRNA HOTAIR-shRNA was transfected into SK-Hep-1 cells,which were randomly divided into transfection group and combination group.The transfection group was cultured routinely,and the combination group was added with protopine(final concentra-tion 200 μmol/L).Another SK-Hep-1 cells were randomly divided into blank group and protopine group,and the blank group was cultured routinely,and the protopine group was added with protopine(final concentration 200 μmol/L).The cells were collected after 48 hours,and RT-qPCR was used to detect the expression of lncRNA HOTAIR in each group;flow cytometry was used to detect the apoptosis rate of each group;GFP-LC3 transfection was used to detect the autophagy of each group;immunoblotting was used to detect the expression of Beclin1,P62,LC3 Ⅰ protein in cells.Results:Compared with the blank group,the expression of lncRNA HOTAIR in the protopine group was in-creased,and the expression of lncRNA HOTAIR in the transfection group was decreased(all P＜0.05).Compared with the protopine group,the expression of lncRNA HOTAIR in the combination group was decreased(P＜0.05).Compared with the transfection group,the expression of lncRNA HOTAIR in the combination group was increased(P＜0.05).Compared with the blank group,the apoptosis rate and the expression of Beclin1,P62,LC3 Ⅰ protein in the protopine group was increased,and the number of GFP-LC3 dots was increased(all P＜0.05);the apoptosis rate and the expression of Beclin1,P62,LC3 Ⅰ protein in the transfection group were decreased,and the number of GFP-LC3 dots was decreased(all P＜0.05).Compared with the protopine group,the apoptosis rate and the expression of Bec-lin1,P62,LC3 Ⅰ protein in the combination group were decreased,and the number of GFP-LC3 dots was decreased(all P＜0.05).Compared with the transfection group,the apoptosis rate and the expression of Beclin1,P62,LC3 Ⅰprotein in the combination group were increased,and the number of GFP-LC3 dots was increased(all P＜0.05).Conclusion:Protopine may induce excessive autophagy and promote apoptosis of human hepatoma SK-Hep-1 cells,and its mechanism may be related to the up-regulation of lncRNA HOTAIR expression.
濮忠建;杨军;杨帆;王亚军
海安市中医院肿瘤科,江苏海安 226600
中医学
肝癌次乌头原碱长链非编码RNAHOX转录反义RNA自噬细胞凋亡
HepatomaProtopineLong non-coding RNAHOX antisense RNAAutophagyApoptosis
《陕西医学杂志》 2024 (006)
754-758,763 / 6
江苏省中医药科技发展计划项目(YB2020070);江苏省南通市中医医疗联盟科研课题(TZYK202218)
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