LncRNA-UCA1通过糖酵解途径调控膀胱癌细胞自噬的机制

Autophagy is a lysosomal degradation pathway that acts as a dynamic regulator in responsible for different stress such as hunger, hypoxia, during normal cellular homeostasis. Also in recent years, autophagy was found activated in cancer cells and contributed to the tumorigenesis and tumor progression by facilitating the survival of cancer cells. However, the underlying mechanisms by which autophagy promotes tumor progression is still unclear. Accumulating evidence showed that long noncoding RNAs (lncRNAs) were involved in the Warburg effect in tumor cells, which is crucial for tumor metabolism. In our previous studies, the data showed that Urothelial carcinoma associated 1(UCA1), which is a bladder cancer specific lncRNA, can promote Warburg effect by regulating the expression of HK2 via mTOR/STAT3/miR-143 pathway in bladder cancer cells. In addition, lncRNA-UCA1 can interact with microRNA-16 as ceRNA, thus increase the GLS2 expression, decrease the production of ROS and ATP, and change the autophagy associated protein LC3-II/I ratio. In further, it needs to be verified that whether UCA1 induces autophagy by activating Adenosine Monophosphate Activated Protein Kinase (AMPK) and altering ROS level via Warburg effect in bladder cancer cells. In this study, we will investigate the interaction of UCA1 and miR-16 in bladder cancer cells and in nude mice xenograft models, focus that whether the interaction between UCA1 and miR-16 could alter the ratio of mitochondria ATP/ADP, thus induce autophagy by activating AMPK. Moreover, the associated molecules such as mTOR, ULK1, Beclin1, VPS34 and LC3 will be detected to reveal the underlying mechanisms. In summary, in this project we aim to uncover the molecular mechanism by which lncRNA-UCA1 induces autophagy via glycosis in bladder cancer cells and provide the experimental basis for novel targets and strategies of tumor metabolic therapy.

细胞通过自噬应对各种应激以维持其正常生理状态的平衡，同时自噬也参与了肿瘤的发生发展过程，但其机制尚不清楚。研究表明lncRNAs可以调节肿瘤细胞的Warburg效应，我们发现UCA1通过mTOR/STAT3/miR-143通路调节HK2促进膀胱癌细胞Warburg效应；并与miR-16通过ceRNA作用增加GLS2表达，减少ROS和ATP生成；过表达UCA1改变LC3-II/I比值。UCA1是否通过Warburg效应激活AMPK激酶，改变ROS水平而引起膀胱癌细胞的自噬需要证实。本课题拟利用细胞、组织和动物模型，通过综合实验手段验证UCA1与miR-16的作用可以改变线粒体ATP/ADP比值，进而激活AMPK诱导自噬的发生，分析mTOR、ULK1、Beclin1、VPS34和LC3等分子改变，从而揭示UCA1通过糖酵解途径诱导膀胱癌细胞自噬发生的分子机制，为肿瘤代谢治疗提供新的策略和靶点。

本项目前期研究发现lncRNA-UCA1可促进膀胱癌细胞的葡萄糖消耗和乳酸生成，这一过程是通过mTOR/STAT3/miR-143通路调控 HK2实现的。而且，膀胱癌中高表达UCA1可改变线粒体膜电位、细胞 ATP浓度及ROS的水平，并调控自噬的发生，但其分子机制尚不清楚。本项目在前期研究基础上，深入研究UCA1对膀胱肿瘤细胞自噬发生的影响和调控的分子机制。研究结果显示，在膀胱肿瘤细胞中UCA1高表达可促进糖酵解的发生，并且UCA1可通过mTOR/STAT3/miR-143通路调节HK2促进膀胱癌细胞Warburg效应。本研究发现，在膀胱肿瘤细胞中高表达UCA1可通过抑制miR-16而促进谷氨酰胺代谢、细胞活力并抑制ROS水平。同时，UCA1可通过UCA1/miR-195-5p/ARL2信号轴促进膀胱肿瘤线粒体功能。此外，在膀胱癌细胞中高表达UCA1可导致自噬体及自噬溶酶体计数减少；敲低UCA1自噬体和自噬溶酶体计数增高。提示UCA1在膀胱癌细胞中抑制了自噬的发生。UCA1可升高ATP水平，上调p-mTOR、p-STAT3及HK2表达，并且使ROS水平及糖酵解途径相关分子p-AMPK表达下调。在使用AMPK激动剂及抑制剂处理后，验证了p-AMPK对糖酵解途径分子p-mTOR、HK2表达的抑制作用及对自噬标志物LC3、ATGs、Beclin1表达的促进作用。最终得出结论，UCA1通过AMPK/mTOR/HK2途径抑制了膀胱癌细胞自噬发生。

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