Hou Xunbo, Xu Qiannan, Liu Ruibao
Department of Interventional, Harbin Medical University Cancer Hospital, No. 150, Haping Rd, Nangang District, Harbin, Heilongjiang 150081, PR China.
Department of Anesthesiology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, PR China.
Transl Oncol. 2024 Jun;44:101684. doi: 10.1016/j.tranon.2023.101684. Epub 2023 Nov 29.
Drug resistance greatly limits the therapeutic effect of a drug. This study aimed to explore the role of long noncoding RNA ZFAS1 in Donafenib resistance of hepatocellular carcinoma (HCC) cells.
The expression of CREB3, ZFAS1, and p65 in HCC cell lines was measured by RT-qPCR and western blotting. After transfection with sh-ZFAS1, sh-CREB3, or sh-CREB3 + oe-p65 in Donafenib-resistent (DR) HCC cell lines, the transfection efficiency was evaluated by RT-qPCR and western blotting. The proliferation and IC to Donafenib of HCC cell lines was examined by MTT assay. Cell proliferation and apoptosis were examined by colony formation and flow cytometry assays. Then, the correlation amongst CREB3, ZFAS1, LSD1/CoREST, and p65 was analysed by ChIP, dual-luciferase reporter gene, and RIP assays.
ZFAS1, CREB3, and p65 were upregulated in HepG2-DR and Huh7-DR cells. Silencing of ZFAS1 or CREB3 enhanced the sensitivity of HCC cells to Donafenib, inhibited cell proliferation and IC, and increased cell apoptosis, which were reversed by p65 overexpression. Mechanistically, CREB3 bound to ZFAS1 promoter to augment ZFAS1 transcriptional expression, and ZFAS1 recruited LSD1/CoREST to the p65 promoter region to decrease H3K4 methylation and elevate p65 transcriptional expression.
CREB3 overexpression contributed to Donafenib resistance in HCC cells by activating the ZFAS1/p65 axis.
耐药性极大地限制了药物的治疗效果。本研究旨在探讨长链非编码RNA ZFAS1在肝癌（HCC）细胞对多纳非尼耐药中的作用。
通过RT-qPCR和蛋白质免疫印迹法检测HCC细胞系中CREB3、ZFAS1和p65的表达。在多纳非尼耐药（DR）的HCC细胞系中转染sh-ZFAS1、sh-CREB3或sh-CREB3+oe-p65后，通过RT-qPCR和蛋白质免疫印迹法评估转染效率。采用MTT法检测HCC细胞系对多纳非尼的增殖和IC。通过集落形成和流式细胞术检测细胞增殖和凋亡。然后，通过染色质免疫沉淀（ChIP）、双荧光素酶报告基因和RNA免疫沉淀（RIP）实验分析CREB3、ZFAS1、赖氨酸特异性去甲基化酶1（LSD1）/REST核心抑制因子（CoREST）和p65之间的相关性。
在HepG2-DR和Huh7-DR细胞中，ZFAS1、CREB3和p65表达上调。沉默ZFAS1或CREB3可增强HCC细胞对多纳非尼的敏感性，抑制细胞增殖和IC，并增加细胞凋亡，而p65过表达可逆转这些作用。机制上，CREB3与ZFAS1启动子结合以增强ZFAS1转录表达，ZFAS1招募LSD1/CoREST至p65启动子区域以降低组蛋白H3第4位赖氨酸（H3K4）甲基化并提高p65转录表达。
CREB3过表达通过激活ZFAS1/p65轴导致HCC细胞对多纳非尼耐药。
