A novel protective role for microRNA-3135b in Golgi apparatus fragmentation induced by chemotherapy via GOLPH3/AKT1/mTOR axis in colorectal cancer cells
A novel protective role for microRNA-3135b in Golgi apparatus fragmentation induced by chemotherapy via GOLPH3/AKT1/mTOR axis in colorectal cancer cells
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DOI：
10.1038/s41598-020-67550-0
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发表时间：
2020-06-29
影响因子：
4.6
通讯作者：
Lopez-Camarillo, Cesar
中科院分区：
文献类型：
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作者：
Nunez-Olvera, Stephanie, I;Chavez-Munguia, Bibiana;Lopez-Camarillo, Cesar
化疗激活了一种新的细胞质DNA损伤反应，导致高尔基体断裂和癌细胞存活。该机制受Golgi磷蛋白-3（GOLPH 3）/Myo 18 A/F-actin轴调节。通过分析miR-3135 b（一种功能未知的小分子非编码RNA）的功能，我们发现其强制过表达可减弱化疗药物诱导的结直肠癌（CRC）细胞高尔基体断裂。首先，我们发现miR-3135 b在CRC细胞系和临床肿瘤中下调。生物信息学预测表明，miR-3135 b可能调控参与细胞存活、化疗抗性和高尔基体动力学的蛋白质编码基因。与此一致，在HCT-15癌细胞中异位转染miR-3135 b显著抑制细胞增殖，使细胞对5-氟尿嘧啶（5-FU）敏感，并促进晚期凋亡和坏死。此外，miR-3135 b过表达损害了HCT-15和SW-480癌细胞的细胞周期进程。因为GOLPH 3，一个参与维持高尔基体结构的基因，被预测为miR-3135 b的潜在靶点，我们研究了它们在响应化疗诱导的DNA损伤中的功能关系。免疫荧光和细胞超微结构实验，使用抗体对TGN 38蛋白，一个trans-Golgi网络标记，表明5-FU和阿霉素治疗的结果在HCT-15和SW-480细胞中的高尔基带结构的非依赖性堆叠分散。值得注意的是，这些细胞效应被miR-3135 b模拟物的转染显著阻碍。此外，我们的功能研究证实，miR-3135 b与GOLPH 3原癌基因的3 ' -UTR结合，并且还降低了p-AKT 1（Ser 473）和p-mTOR（Ser 2448）信号转导物的水平，这是细胞存活和自噬激活的关键。此外，我们发现用5-FU治疗后，TGN 38因子与beclin-1自噬蛋白在与片段化的高尔基体相关的离散结构中共免疫定位，这表明促生存自噬的激活与高尔基体完整性的丧失有关。自噬和高尔基体分散中的这些细胞效应被miR-3135 b逆转。总之，我们提供的实验证据首次表明miR-3135 b在结肠直肠癌细胞中通过GOLPH 3/AKT 1/mTOR轴保护化疗诱导的高尔基体片段化和保护性自噬中的新作用。
Chemotherapy activates a novel cytoplasmic DNA damage response resulting in Golgi apparatus fragmentation and cancer cell survival. This mechanism is regulated by Golgi phosphoprotein-3 (GOLPH3)/Myo18A/F-actin axis. Analyzing the functions of miR-3135b, a small non-coding RNA with unknown functions, we found that its forced overexpression attenuates the Golgi apparatus fragmentation induced by chemotherapeutic drugs in colorectal cancer (CRC) cells. First, we found that miR-3135b is downregulated in CRC cell lines and clinical tumors. Bioinformatic predictions showed that miR-3135b could be regulating protein-encoding genes involved in cell survival, resistance to chemotherapy, and Golgi dynamics. In agreement, ectopic transfection of miR-3135b in HCT-15 cancer cells significantly inhibited cell proliferation, sensitized cells to 5-fluoruracil (5-FU), and promoted late apoptosis and necrosis. Also, miR-3135b overexpression impaired the cell cycle progression in HCT-15 and SW-480 cancer cells. Because GOLPH3, a gene involved in maintenance of Golgi structure, was predicted as a potential target of miR-3135b, we studied their functional relationships in response to DNA damage induced by chemotherapy. Immunofluorescence and cellular ultrastructure experiments using antibodies against TGN38 protein, a trans-Golgi network marker, showed that 5-FU and doxorubicin treatments result in an apoptosis-independent stacks dispersal of the Golgi ribbon structure in both HCT-15 and SW-480 cells. Remarkably, these cellular effects were dramatically hindered by transfection of miR-3135b mimics. In addition, our functional studies confirmed that miR-3135b binds to the 3 ' -UTR of GOLPH3 proto-oncogene, and also reduces the levels of p-AKT1 (Ser473) and p-mTOR (Ser2448) signaling transducers, which are key in cell survival and autophagy activation. Moreover, we found that after treatment with 5-FU, TGN38 factor coimmunolocalizes with beclin-1 autophagic protein in discrete structures associated with the fragmented Golgi, suggesting that the activation of pro-survival autophagy is linked to loss of Golgi integrity. These cellular effects in autophagy and Golgi dispersal were reversed by miR-3135b. In summary, we provided experimental evidence suggesting for the first time a novel role for miR-3135b in the protection of chemotherapy-induced Golgi fragmentation via GOLPH3/AKT1/mTOR axis and protective autophagy in colorectal cancer cells.