Department of Gynaecology and Obstetrics, People's Hospital of Bishan County, Chongqing, China.
Neoplasma. 2021 May;68(3):509-518. doi: 10.4149/neo_2021_201112N1212. Epub 2021 Jan 28.
This study aims to investigate the role of the long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) in the regulation of anoikis resistance of ovarian cancer cells, a prerequisite for metastasis and chemoresistance in ovarian cancer cells. Ovarian cancer SKOV3 cells were cultured in an ultra-low attachment system to establish an anoikis model. The relationship between cellular anoikis capability and HOTAIR expression level was studied by flow cytometry and RT-PCR. The ability of spheroid formation, migration, and invasion of the suspended cells was assessed following the knockdown of HOTAIR expression. The expression of EZH2, H3K27me3, representative targets of EZH2, and anoikis-related biomarkers was also detected. An increase in the duration of suspension culture time rendered the SKOV3 cells anoikis-resistant with a significantly lower apoptotic rate compared to the adherent cells. HOTAIR expression in the suspension cells increased significantly, while that in the adherent cells did not. Following small interfering RNA (siRNA)-mediated knockdown of HOTAIR expression, the abilities of anoikis resistance, migration, and invasion decreased in the suspension cells. Knockdown of HOTAIR levels also reduced the spheroid forming ability of the tumor cells in continuous suspension cultures. Moreover, EZH2 expression correlated with HOTAIR expression, thus regulating the expression of miR-193a and DOK2 via introducing H3K27me3. Western blot analysis of anoikis-related markers showed that N-cadherin, ZEB1, and TWIST1 were downregulated following inhibition of HOTAIR, while E-cadherin and ErbB3 were upregulated. In conclusion, HOTAIR enhances the anoikis resistance and spheroid forming ability of ovarian cancer cells by recruiting EZH2 and influencing H3K27 methylation that may contribute to migration, invasion, and chemoresistance of ovarian cancer cells.
本研究旨在探讨长链非编码 RNA（lncRNA）HOX 转录反义 RNA（HOTAIR）在调节卵巢癌细胞抗失巢凋亡中的作用，而抗失巢凋亡是卵巢癌细胞转移和化疗耐药的前提。我们在超低附着系统中培养卵巢癌细胞 SKOV3 以建立失巢凋亡模型。通过流式细胞术和 RT-PCR 研究细胞抗失巢凋亡能力与 HOTAIR 表达水平之间的关系。敲低 HOTAIR 表达后，评估悬浮细胞的球体形成、迁移和侵袭能力。还检测了 EZH2、H3K27me3（EZH2 的代表性靶标）和与失巢凋亡相关的生物标志物的表达。延长悬浮培养时间会使 SKOV3 细胞获得抗失巢凋亡能力，与贴壁细胞相比，其凋亡率显著降低。悬浮细胞中的 HOTAIR 表达显著增加，而贴壁细胞中的 HOTAIR 表达没有增加。在小干扰 RNA（siRNA）介导的 HOTAIR 表达敲低后，悬浮细胞中的抗失巢凋亡、迁移和侵袭能力下降。HOTAIR 水平的敲低也降低了肿瘤细胞在连续悬浮培养中的球体形成能力。此外，EZH2 表达与 HOTAIR 表达相关，通过引入 H3K27me3 调节 miR-193a 和 DOK2 的表达。Western blot 分析失巢凋亡相关标志物显示，抑制 HOTAIR 后 N-钙黏蛋白、ZEB1 和 TWIST1 下调，而 E-钙黏蛋白和 ErbB3 上调。总之，HOTAIR 通过招募 EZH2 并影响 H3K27 甲基化来增强卵巢癌细胞的抗失巢凋亡和球体形成能力，这可能有助于卵巢癌细胞的迁移、侵袭和化疗耐药性。
