Department of Surgical Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, No.3 Eastern Qingchun Road, Hangzhou, 310016, Zhejiang, China.
Biomedical Research Center and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, 310016, Zhejiang, China.
J Hematol Oncol. 2019 Jul 24;12(1):81. doi: 10.1186/s13045-019-0747-0.
Tamoxifen resistance remains a clinical challenge for hormone receptor-positive breast cancer. Recently, dysregulations in autophagy have been suggested as a potential mechanism for tamoxifen resistance. Although the long noncoding RNA H19 is involved in various stages of tumorigenesis, its role in tamoxifen resistance remains unknown. Here, we assessed the role of H19 in the development of tamoxifen-resistant breast cancer.
Quantitative real-time PCR analyzed expression of H19 in tamoxifen-resistant breast cancer tissues. Knockdown of H19 was used to assess the sensitivity to tamoxifen in vitro and in vivo. Both knockdown and overexpression of H19 were used to analyze the status of autophagy. Real-time quantitative methylation-specific polymerase chain reaction, chromatin immunoprecipitation, immunofluorescence, and Western blot were used to explore the tamoxifen resistance mechanism of H19.
In this study, we observed that the expression of H19 was substantially upregulated in tamoxifen-resistant breast cancer cell line and tumor tissues, and knockdown of H19 enhanced the sensitivity to tamoxifen both in vitro and in vivo. Furthermore, knockdown of H19 significantly inhibited autophagy in MCF7 tamoxifen-resistant (MCF7/TAMR) cells. Conversely, overexpression of H19 promoted autophagy. Interestingly, overexpression of H19 in MCF7 tamoxifen-sensitive cells could recapitulate tamoxifen resistance. Moreover, an increase in methylation in the promoter region of Beclin1 was observed in MCF7/TAMR-shH19 cells. In the double knockdown groups, both shH19+shSAHH and shH19+shDNMT3B rescued the Beclin1 promoter region methylation levels and reactivated autophagy functions. A chromatin immunoprecipitation assay further validated that DNMT3B binds to the Beclin1 promoter region and the knockdown of H19 increases this binding.
Our findings demonstrate that H19 induces autophagy activation via the H19/SAHH/DNMT3B axis, which could contribute to tamoxifen resistance in breast cancer.
他莫昔芬耐药仍是激素受体阳性乳腺癌的临床挑战。最近，自噬失调被认为是他莫昔芬耐药的潜在机制。虽然长链非编码 RNA H19 参与肿瘤发生的各个阶段，但它在他莫昔芬耐药中的作用尚不清楚。在这里，我们评估了 H19 在他莫昔芬耐药性乳腺癌发展中的作用。
定量实时 PCR 分析 H19 在他莫昔芬耐药性乳腺癌组织中的表达。敲低 H19 用于体外和体内评估对他莫昔芬的敏感性。同时敲低和过表达 H19 用于分析自噬状态。实时定量甲基化特异性聚合酶链反应、染色质免疫沉淀、免疫荧光和 Western blot 用于探索 H19 的他莫昔芬耐药机制。
在这项研究中，我们观察到 H19 在他莫昔芬耐药性乳腺癌细胞系和肿瘤组织中的表达显著上调，敲低 H19 可增强体外和体内对他莫昔芬的敏感性。此外，敲低 H19 可显著抑制 MCF7 他莫昔芬耐药（MCF7/TAMR）细胞中的自噬。相反，H19 的过表达促进自噬。有趣的是，在 MCF7 他莫昔芬敏感细胞中过表达 H19 可再现他莫昔芬耐药性。此外，在 MCF7/TAMR-shH19 细胞中观察到 Beclin1 启动子区域的甲基化增加。在双敲低组中，shH19+shSAHH 和 shH19+shDNMT3B 均挽救了 Beclin1 启动子区域的甲基化水平并重新激活了自噬功能。染色质免疫沉淀试验进一步证实，DNMT3B 结合到 Beclin1 启动子区域，并且敲低 H19 增加了这种结合。
我们的研究结果表明，H19 通过 H19/SAHH/DNMT3B 轴诱导自噬激活，这可能导致乳腺癌中他莫昔芬耐药。
