LncRNA H19/miR-141调控细胞自噬在光老化中的作用与机制研究

Recent studies find that abnormally reduced autophagy is one of the important factors that cause aging of the skin and the whole body. More than 40 proteins are required to participate in the procedure of autophagy. Our preliminary studies found that 1) the autophagy activity was significantly reduced in photoaged skin cells; 2) autophagy activator can reverse many performance of skin photoaging; 3) lncRNA H19 expression was significantly decreased and miR-141 expression was increased in skin photoaging models which was confirmed by miRNAs chip and RT-PCR, and miR -141 predicted downstream target sites have some relationship with autophagy. In this condition, we propose the hypothesis: lncRNA H19/miR-141 may participate in the development of skin photoaging through regulation of autophagy. The present project intends to observed the impact of lncRNA H19/miR-141 and autophagy on established skin photoaging cells. On the basis of verify the roles of lncRNA H19/miR-141 in autophagy, clarify the exact mechnisms of lncRNA H19/miR-141 regulation of photoaging. Our expeirments will provide an experimental basis of specific mechanisms of new targets for the prevention and treatment of skin photodamage.

新近发现细胞自噬异常减少是导致机体老化的重要因素之一；发生细胞自噬需要40余种蛋白参与。前期研究发现：皮肤光老化细胞的自噬活性显著降低，提高自噬水平可逆转皮肤光老化诸多表现；lncRNA芯片及RT-PCR检测证实光老化模型中lncRNA H19表达明显减低，miR-141显著升高，且miR-141预测的下游靶位点为自噬调控相关基因。为此提出假说：lncRNA H19可能通过ceRNA机制调控miR-141的表达调控靶基因进而影响细胞自噬而参与皮肤光老化的发生发展。本项目拟通过已建立的皮肤老化细胞，观察lncRNA H19/miR-141与细胞自噬对皮肤光老化的影响，验证其通过调控自噬改善老化的具体机制。在确证“紫外线- lncRNA H19/miR-141-细胞自噬-皮肤光老化”调控环节的基础上，明确调控光老化的具体机制，为防治皮肤光老化明确新靶点提供理论基础和实验参考。

新近发现细胞自噬异常减少是导致机体老化的重要因素之一；发生细胞自噬需要40余种蛋白参与。前期研究发现：皮肤光老化细胞的自噬活性显著降低，提高自噬水平可逆转皮肤光老化诸多表现；lncRNA芯片及RT-PCR检测证实皮肤光老化模型中lncRNA H19表达明显减低，miR-141表达显著升高，且miR-141预测的下游靶位点为自噬调控相关基因。为此提出假说：lncRNA H19极有可能通过ceRNA机制调控miR-141的表达，并进一步调控靶基因进而参与皮肤光老化的发生发展。在本研究中，我们发现紫外线辐射可致LncRNA H19表达量下降，miR-141表达增高，而PTEN表达量明显增加。进一步的，我们在UVB诱导的光老化模型中将LncRNA H19过表达后，可进一步降低PTEN的表达，而这种作用可被miR-141 mimics所逆转。而LncRNA H19可通过PI3K/AKT通路进一步调控细胞周期相关蛋白。我们发现UVB辐照足以使成纤维细胞发生凋亡。黄芩苷可提高细胞活力并抑制凋亡。黄芩苷的加入可下调Bax并增加Bcl-2，表明黄芩苷可能通过下调p53信号通路抑制UVB诱导的细胞凋亡。Western blot显示与单独的UVB照射相比，黄芩苷处理可抑制凋亡期间被激活caspase-3和PARP。加入Bafilomycin A1，黄芩苷处理仍然可以增加含GFP-LC3的细胞数量，并上调beclin-1的表达和LC3-II / LC3-I的比例。表明黄芩苷通过诱导自噬体产生而不是通过抑制自噬体-溶酶体融合来促进UVB诱导的自噬活性。自噬体形成抑制剂3-MA显着减少了含有GFP-LC3的细胞数目，抑制了beclin-1的表达和UVB +黄芩苷诱导的LC3-II / LC3-I的比例，上调了UVB +黄芩苷诱导的凋亡水平和caspase-3水平。这表明黄芩苷可通过诱导自噬流对UVB诱导的自噬和凋亡起作用。此外，黄芩苷可以增加UVB诱导的AMPK并降低mTOR的磷酸化水平，通过应用compound C和siRNA抑制AMPK，黄芩苷对UVB诱导的AMPK和mTOR的影响被消除。本研究证实了以下假说：黄芩苷→AMPK高表达→mTOR低表达→自噬能力增强→老化和受损的细胞器被清除→皮肤光损伤表现减轻，明确黄芩苷调控光损伤的具体机制，为防治皮肤光老化明确新靶点提供理论基础和实验参考。