Fang Shun, Shen Yefeng, Chen Bin, Wu Yuanzhou, Jia Longfei, Li Yaling, Zhu Yaru, Yan Yusheng, Li Man, Chen Rui, Guo Linlang, Chen Xin, Chen Qunqing
Department of Pathology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Department of Cardiothoracic Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Ann Transl Med. 2018 Nov;6(22):440. doi: 10.21037/atm.2018.10.21.
The long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) serves as a powerful predictor of tumor progression and overall survival in patients. Our previous studies showed that HOTAIR modulated HOXA1 DNA methylation by reducing DNMT1 and DNMT3b expression in drug-resistant small cell lung cancer (SCLC). Moreover, H3 lysine 27 trimethylation (H3K27me3) is catalyzed by enhancer of zeste homolog 2 (EZH2) and plays a critical role in SCLC chemoresistance. However, it is not completely clear whether H3K27me3 affects HOXA1 DNA methylation or whether this effect is mediated by HOTAIR.
The levels of EZH2 and H3K27me3 were identified in SCLC tissues by immunohistochemical (IHC) staining and in SCLC multidrug-resistant cells by Western blotting. Cell counting kit-8 (CCK-8) and flow cytometry were used to detect and analyze the biological function of H3K27me3. Then, we assessed the role of HOTAIR in the regulation of EZH2 and H3K27me3 by using lentivirus and small interfering RNA. Further, bisulfite sequencing PCR was conducted to detect the methylation levels of HOXA1 DNA. Finally, Western blotting was performed to examine the regulatory role of H3K27me3 in controlling HOTAIR expression in SCLC.
In this study, we found that EZH2 and H3K27me3 levels were markedly higher in SCLC tissues and multidrug-resistant SCLC cells. The results indicated that H3K27me3 was related to multidrug resistance. HOTAIR overexpression and knockdown showed that EZH2 and H3K27me3 were regulated by HOTAIR. Moreover, H3K27me3 affected HOXA1 DNA methylation levels. Strikingly, we found that H3K27me3 acted as a negative feedback regulator of HOTAIR.
Our study showed that H3K27me3 affects HOXA1 DNA methylation via HOTAIR regulation, indicating that H3K27me3 may be a potential therapy target for SCLC chemoresistance.
长链非编码RNA（lncRNA）HOX转录反义RNA（HOTAIR）是患者肿瘤进展和总生存期的有力预测指标。我们之前的研究表明，HOTAIR通过降低耐药性小细胞肺癌（SCLC）中DNMT1和DNMT3b的表达来调节HOXA1的DNA甲基化。此外，组蛋白H3赖氨酸27三甲基化（H3K27me3）由zeste同源物2增强子（EZH2）催化，在SCLC化疗耐药中起关键作用。然而，H3K27me3是否影响HOXA1的DNA甲基化，以及这种影响是否由HOTAIR介导，目前尚不完全清楚。
通过免疫组织化学（IHC）染色在SCLC组织中以及通过蛋白质免疫印迹法在SCLC多药耐药细胞中鉴定EZH2和H3K27me3的水平。使用细胞计数试剂盒-8（CCK-8）和流式细胞术检测和分析H3K27me3的生物学功能。然后，我们使用慢病毒和小干扰RNA评估HOTAIR在EZH2和H3K27me3调控中的作用。此外，进行亚硫酸氢盐测序PCR以检测HOXA1 DNA的甲基化水平。最后，进行蛋白质免疫印迹法以检查H3K27me3在控制SCLC中HOTAIR表达的调控作用。
在本研究中，我们发现EZH2和H3K27me3水平在SCLC组织和多药耐药SCLC细胞中明显更高。结果表明H3K27me3与多药耐药有关。HOTAIR的过表达和敲低表明EZH2和H3K27me3受HOTAIR调控。此外，H3K27me3影响HOXA1 DNA甲基化水平。令人惊讶的是，我们发现H3K27me3作为HOTAIR的负反馈调节因子。
我们的研究表明，H3K27me3通过HOTAIR调控影响HOXA1 DNA甲基化，表明H3K27me3可能是SCLC化疗耐药的潜在治疗靶点。
