School of Pharmaceutical Sciences, Binzhou Medical University, Yantai 264003, China.
School of Pharmaceutical Sciences, Taishan Medical University, Taian 271016, China.
Cell Death Dis. 2017 Oct 26;8(10):e3137. doi: 10.1038/cddis.2017.500.
Emerging evidence suggests that microRNA (miRNA) and long noncoding RNA (lncRNA) play important roles in disease development. However, the mechanism underlying mRNA interaction with miRNA and lncRNA in idiopathic pulmonary fibrosis (IPF) remains unknown. This study presents a novel lnc-PCF that promotes the proliferation of TGF-β1-activated epithelial cells through the regulation of map3k11 by directly targeting miR-344a-5p during pulmonary fibrogenesis. Bioinformatics and in vitro translation assay were performed to confirm whether or not lnc-PCF is an actual lncRNA. RNA fluorescent in situ hybridization (FISH) and nucleocytoplasmic separation showed that lnc-PCF is mainly expressed in the cytoplasm. Knockdown and knockin of lnc-PCF indicated that lnc-PCF could promote fibrogenesis by regulating the proliferation of epithelial cells activated by TGF-β1 according to the results of xCELLigence real-time cell analysis system, flow cytometry, and western blot analysis. Computational analysis and a dual-luciferase reporter system were used to identify the target gene of miR-344a-5p, whereas RNA pull down, anti-AGO2 RNA immunoprecipitation, and rescue experiments were conducted to confirm the identity of this direct target. Further experiments verified that lnc-PCF promotes the proliferation of activated epithelial cells that were dependent on miR-344a-5p, which exerted its regulatory functions through its target gene map3k11. Finally, adenovirus packaging sh-lnc-PCF was sprayed into rat lung tissues to evaluate the therapeutic effect of lnc-PCF. These findings revealed that lnc-PCF can accelerate pulmonary fibrogenesis by directly targeting miR-344a-5p to regulate map3k11, which may be a potential therapeutic target in IPF.
新兴证据表明，微小 RNA（miRNA）和长非编码 RNA（lncRNA）在疾病发展中发挥重要作用。然而，特发性肺纤维化（IPF）中 mRNA 与 miRNA 和 lncRNA 相互作用的机制尚不清楚。本研究提出了一种新型 lnc-PCF，它通过直接靶向 miR-344a-5p 调控 map3k11，在肺纤维化过程中促进 TGF-β1 激活的上皮细胞增殖。通过生物信息学和体外翻译实验来确认 lnc-PCF 是否为实际的 lncRNA。RNA 荧光原位杂交（FISH）和核质分离显示 lnc-PCF 主要在细胞质中表达。通过 lnc-PCF 的敲低和敲入实验表明，lnc-PCF 可以通过调节 TGF-β1 激活的上皮细胞增殖来促进纤维化，这是根据 xCELLigence 实时细胞分析系统、流式细胞术和 Western blot 分析的结果得出的。计算分析和双荧光素酶报告系统用于鉴定 miR-344a-5p 的靶基因，而 RNA 下拉、抗 AGO2 RNA 免疫沉淀和挽救实验用于确认该直接靶基因的身份。进一步的实验验证了 lnc-PCF 促进激活的上皮细胞增殖依赖于 miR-344a-5p，通过其靶基因 map3k11 发挥其调节功能。最后，通过腺病毒包装 sh-lnc-PCF 喷入大鼠肺组织来评估 lnc-PCF 的治疗效果。这些发现表明，lnc-PCF 可以通过直接靶向 miR-344a-5p 调控 map3k11 来加速肺纤维化，这可能是 IPF 的一个潜在治疗靶点。
