miR-200c靶向UBQLN1介导的自噬调节乳腺癌细胞辐射抵抗和EMT转化

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. The miR-200 family was demonstrated to be an important regulator of EMT as well as radiation resistance. Notably, recent studies suggest that autophagy may be also important in the regulation of cancer development and progression and in determining the response of tumor cells to radiotherapy. In our preliminary studies, we found that autophagy could be regulated by over- or repressed expression of miR-200c in mesenchymal-like MDA-MB-231 cells and epithelial-like MCF7 cells, respectively. However, the exact relationship between miR-200c, autophagy, and EMT and radiation resistance is unknown. In this project, we will explore the exact relationship between miR-200c, autophagy, and EMT and radiation resistance. The mechanism of the regulation of autophagy by miR-200c was analyzed using mesenchymal-like MDA-MB-231 cells and epithelial-like MCF7 cells. Moreover, we further determine whether JAK/STAT3 signal, which is an important EMT-inducing signal, are regulated by miR-200c and autophagy. We also evaluate the effect of miR-200c expression and autophagy states on EMT and radiation resistance in breast cancer cells with mesenchymal-like or epithelial-like properties. Finally, in order to further determine whether the relationship between miR-200c expression with autophagy consistently exists in tumors from clinical breast cancer patients, we quantify miR-200c expression, ubiquilin1, autophagy-associated proteins and active STAT3 protein by situ hybridization, western blot and immunohistochemistry. Altogether, our project tries to validate our hypothesis that autophagy regulated by miR-200c targeting to UBQLN1 control radioresistance and EMT in breast cancer cells. These findings provide new insights into the molecular functions of miR-200c as a breast cancer suppressor as well as how activation of the EMT program and why induction of radiation resistance in breast cancer cells.

近期发现miR-200c和自噬均参与调节乳腺癌细胞的辐射抵抗形成和EMT转化，但两者的联系及机制未见报道。我们前期在乳腺癌细胞中也证实了miR-200c可抑制其辐射抵抗形成和EMT，而自噬、Ubiquilin1、JAK/ STAT3信号活性在乳腺癌细胞辐射抵抗及EMT过程中均发生改变，同时经生物信息预测发现UBQLN1（翻译Ubiquilin1）是miR-200c的靶基因。基于以上，本项目拟在乳腺癌细胞上进行miR-200c表达的人工干预，验证miR-200c靶向UBQLN1抑制自噬，阻截JAK/STAT3信号，从而抑制细胞辐射抵抗和EMT，并在临床病理组织中分析验证。以证明miR-200c-UBQLN1-autophagy-JAK/STAT3通路是miR-200c调节乳腺癌细胞辐射抵抗及EMT的新机制。为miR-200c的表达异常在促进乳腺癌多种恶性生物学行为的产生提供了新的科学依据。

辐射抵抗是乳腺癌治疗的重要挑战，深入探讨其中的机制可以为乳腺癌治疗提供新的策略。在我们的研究中，我们比较了4种乳腺癌细胞株的miR-200c的表达，发现基底型乳腺癌细胞miR-200c的表达低而官腔型乳腺癌细胞则高，我们在基底型乳腺癌细胞MDA-MB-231中过表达miR-200c后出现了对辐射诱导的自噬的抑制，并且使细胞的放射敏感性提高；我们还鉴定出miR-200c 调控辐射诱导的自噬的靶基因为UBQLN1，并与乳腺癌细胞辐射敏感性密切相关。我们还在35例人乳腺癌组织中检测到了miR-200c和UBQLN1、自噬活性表达呈负相关。这些结果提示miR-200c/UBQLN1介导的自噬通路降低乳腺癌的辐射抵抗，可能为乳腺癌提供一个治疗策略。p53功能障碍和乏氧也是乳腺癌发生辐射抵抗的特点，线粒体自噬具有维持乏氧细胞稳态的保护作用，且在正常细胞中受p53抑制。为此我们在体内外探讨了融合蛋白TAT-ODD-p53对乏氧乳腺癌的放疗增敏作用及其分子机制。我们发现TAT-ODD-p53选择性聚集在乏氧乳腺癌细胞及组织区域，并且明显提高癌细胞的辐射敏感性；线粒体自噬具有维持乳腺癌细胞乏氧诱导辐射抵抗的重要作用，这种作用可以被TAT-ODD-p53抑制，而TAT-ODD-p53的抑制作用可经过表达Parkin蛋白减退。线粒体自噬可以经敲除p53诱发，这个过程在乏氧情况下可以经Parkin敲除减弱。我们还发现p53可以结合Parkin从而抑制其在线粒体上的定位，由此抑制线粒体自噬过程。这些结果提示TAT-ODD-p53经Parkin通路抑制线粒体自噬以发挥靶向乏氧乳腺癌细胞放射增敏作用。精确放疗已是肿瘤治疗的主要手段，但由于过程复杂，可能需要更长的剂量输出时间，我们还不清楚这对正常组织会产生怎样的影响。为此我们发现延长的照射会提高正常组织细胞的生存，且晚反应组织较早反应组织会获得更高的生存和更少的辐射所至DNA损伤，并发现其中经ATM-AMPK调解的辐射诱发自噬升高；经自噬抑制剂3-MA及ATM抑制剂KU55933处理后产生ROS升高，且延长照射的辐射保护作用减弱。这些结果提晚反应组织可经增强的自噬途径从精确放疗的延长照射剂量输出时间中获益示。