SLC45A4 promotes glycolysis and prevents AMPK/ULK1‐induced autophagy in TP53 mutant pancreatic ductal adenocarcinoma
Wen‐Ying ChenFengting HuangJing HuangYuanhua LiJuanfei PengYanyan ZhuangXianxian HuangLiting LuZhe ZhuShineng Zhang
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Abstract Background Somatic mutations of the TP53 gene occur frequently in pancreatic ductal adenocarcinoma (PDA). Solute carrier family 45 member A4 (SLC45A4) is a H + ‐dependent sugar cotransporter. The role of SLC45A4 in PDA, especially in TP53 mutant PDA, remains poorly understood. Methods We explored the TCGA datasets to identify oncogenes in TP53 mutant PDA. MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium], colony formation and 5‐ethynyl‐2′‐deoxyuridine (Edu) assays were performed to investigate the function of SLC45A4 in vitro . Glucose consumption, lactate production and ATP production were detected to evaluate glucose utilization. Extracellular acidification rate and oxygen consumption rate assays were used to evaluate glycolysis and oxidative phosphorylation. The subcutaneous xenotransplantation models were conducted to explore the function of SLC45A4 in vivo . RNA‐sequencing and gene set enrichment analysis were employed to explore the biological alteration caused by SLC45A4 knockdown. Western blotting was performed to evaluate the activation of glycolysis, as well as the AMPK pathway and autophagy. Results SLC45A4 was overexpressed in PDA for which the expression was significantly higher in TP53 mutant PDA than that in wild‐type PDA tissues. Moreover, high level of SLC45A4 expression was tightly associated with poor clinical outcomes in PDA patients. Silencing SLC45A4 inhibited proliferation in TP53 mutant PDA cells. Knockdown of SLC45A4 reduced glucose uptake and ATP production, which led to activation of autophagy via AMPK/ULK1 pathway. Deleting SLC45A4 in TP53 mutant HPAF‐II cells inhibited the growth of xenografts in nude mice. Conclusions The present study found that SLC45A4 prevents autophagy via AMPK/ULK1 axis in TP53 mutant PDA, which may be a promising biomarker and therapeutic target in TP53 mutant PDA.Inducer
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Considerable attention has been paid to the topic of autophagy induction. In part, this is because of the potential for modulating this process for therapeutic purposes. Of course we know that induced autophagy can also be problematic--for example, when trying to eliminate an established tumor that might be relying on autophagy for its own cytoprotective uses. Accordingly, inhibitory mechanisms have been considered; however, the corresponding studies have tended to focus on the pathways that block autophagy under non-inducing conditions, such as when nutrients are available. In contrast, relatively little is known about the mechanisms for inhibiting autophagy under inducing conditions. Yet, this type of regulation must be occurring on a routine basis. We know that dysregulation of autophagy, e.g., due to improper activation of Beclin 1 leading to excessive autophagy activity, can cause cell death. Accordingly, we assume that during starvation or other inducing conditions there must be a mechanism to modulate autophagy. That is, once you turn it on, you do not want to let it continue unchecked. But how is autophagy downregulated when the inducing conditions still exist?
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macroautophagy 的角色(此后 autophagy ) 在癌症，到临床的干预的生物学和反应是复杂的。autophagy 是在许多肿瘤背景的 dysregulated，是清楚的，在肿瘤开始和前进期间，并且响应治疗。然而，在控制房间行为的 autophagy 的多种的机械学的角色使在一个给定的肿瘤背景预言困难 autophagy 的角色，并且，由扩展，指向 autophagy 的治疗学的结果，力量。在这评论，我们在在癌症支持 pro-tumorigenic 和 anti-tumorigenic 和 autophagy 的治疗学的角色的文学总结证据。这概述在滋养的管理，房间死亡，房间老朽， proteotoxic 应力的规定和细胞的动态平衡包含 autophagy 的角色，在在新陈代谢的变化的肿瘤主人相互作用和参予的规定。在可能的地方，我们也试着理解，为 autophagy 的这些角色的机械学的底。我们明确地阐述在在 vivo.We 使这些问题清楚些的癌症的模型也考虑 autophagy 蛋白质的任何东西或上述所有函数怎么可能是可指向的由的遗传上设计的老鼠的新兴的角色现存或 pharmacologic 代理人的未来类。我们由简短在细胞的过程为关键 autophagy 蛋白质的子集探索不在经典中的角色得出结论，并且这些怎么可能在癌症之上影响。
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Excessive light exposure can potentially cause irreversible damage to the various photoreceptor cells, and this aspect has been considered as an important factor leading to the progression of the different retinal diseases. AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) are crucial intracellular signaling hubs involved in the regulation of cellular metabolism, energy homeostasis, cellular growth and autophagy. A number of previous studies have indicated that either AMPK activation or mTOR inhibition can promote autophagy in most cases. In the current study, we have established an in vitro as well as in vivo photooxidation-damaged photoreceptor model and investigated the possible influence of visible light exposure in the AMPK/mTOR/autophagy signaling pathway. We have also explored the potential regulatory effects of AMPK/mTOR on light-induced autophagy and protection achieved by suppressing autophagy in photooxidation-damaged photoreceptors. We observed that light exposure led to a significant activation of mTOR and autophagy in the photoreceptor cells. However, intriguingly, AMPK activation or mTOR inhibition significantly inhibited rather than promoting autophagy, which was termed as AMPK-dependent inhibition of autophagy. In addition, either indirectly suppressing autophagy by AMPK activation/ mTOR inhibition or directly blocking autophagy with an inhibitor exerted a significant protective effect on the photoreceptor cells against the photooxidative damage. Neuroprotective effects caused by the AMPK-dependent inhibition of autophagy were also verified with a retinal light injured mouse model in vivo. Overall, our findings demonstrated that AMPK / mTOR pathway could inhibit autophagy through AMPK-dependent inhibition of autophagy to significantly protect the photoreceptors from photooxidative injury, which may aid to further develop novel targeted retinal neuroprotective drugs.
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Small interfering RNA (siRNA) molecules achieve sequence- specific gene silencing through a process known as RNA interference (RNAi). Compared to other nucleic acid-based therapeutics aimed at post-transcriptional gene silencing, such as antisense oligodeoxynucleotides, siRNA molecules achieve greater magnitude and duration of gene silencing at significantly lower doses. While the duration of gene knockdown by siRNA typically lasts around 1 week in rapidly dividing cells, recent reports of knockdown lasting for several weeks in nondividing cells indicate that dilution due to cell division may be a limiting factor in rapidly dividing cells. To determine if cell division directly impacts the duration of gene knockdown by siRNA, we chose to investigate the kinetics of siRNA-mediated gene silencing in luciferase-expressing cell lines with different observed doubling times using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function. In vitro and in vivo, the duration of gene knockdown is inversely proportional to the rate of cell division. Consistent with previous reports, luciferase protein levels recover to pre-treatment values within less than 1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting around 1 week in subcutaneous tumors in A/J mice and 3-4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. Here, we will present our latest results describing the effects of cell doubling time, siRNA stability, and dosing schedule on siRNA- mediated gene silencing. Specifically, we will investigate whether the duration of knockdown using chemically modified siRNA molecules exhibits a similar dependence on cell doubling time. The implications of these findings will be highlighted using model calculations to determine the dosing schedule required to maintain persistent silencing of target proteins and to predict when maximum mRNA or protein knockdown will occur, an especially important factor when trying to observe a therapeutic effect resulting from protein knockdown. The approach of bioluminescent imaging combined with mathematical modeling provides insights into siRNA function that will hopefully be of practical use for both researchers and clinicians alike.
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Autophagy 是导致长寿蛋白质和不正常的细胞器的降级的高度调整的细胞的机制。这个过程处于与神经病学的疾病相关的许多生理、病理学的条件被含有。最近的研究证明在服的局部缺血的 autophagy 的存在，而是没有一致还处于这个条件关于 autophagy 的功能被到达了。这篇文章在服的局部缺血或灌注期间加亮 autophagy 的激活，特别在神经原和星形细胞，以及在 neuronal 或 astrocytic 房间死亡和幸存的 autophagy 的角色。我们建议那 autophagy 的生理的层次，大概引起了由对谦虚组织缺氧或局部缺血温和，看起来保护。然而，严重组织缺氧或局部缺血或灌注引起的 autophagy 的高水平可以引起自我消化和最终的 neuronal 和 astrocytic 房间死亡。我们也讨论那氧化并且 endoplasmic 蜂窝胃(嗯) 在服的组织缺氧或局部缺血或灌注的压力是在神经原和星形细胞的 autophagy 的有势力刺激。另外，我们考察一方面建议在 autophagy 之间的可观的重叠的证据，和 apoptosis，坏死和 necroptosis 在另一方面，在决定结果和损坏神经原和星形细胞的最后的形态学。
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Autophagy is a highly conserved cell degradation process which can decompose the organelles and recycling the macromolecules by lysosomes. Autophagy is induced by multiple factors, such as starvation, ischemia, and oxidative stress, and plays a key role in development and differentiation of cells. It has been shown that autophagy is closely related to cardiovascular diseases. Moderate autophagy protects myocardial cells from injury, whereas insufficient or excessive autophagy triggers or aggravates diseases. The present article reviews the progress of autophagy in cardiovascular diseases.
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最近的研究揭示了那 autophagy，基本细胞内部的进程，在淋巴细胞开发和功能起许多不同作用。Autophagy 调整天真的 T 淋巴细胞动态平衡，明确地由调整 mitochondrial 质量和周转，并且为成熟 T 房间的增长是必要的。Autophagy 也在淋巴细胞充当一条细胞的死亡小径，在延长 cytokine 退却之上并且在尖锐抗原受体刺激期间如果不正确地调整了。在 HIV 感染期间，而且， autophagy 的 hyperinduction 在 uninfected CD4+ T 房间导致巨大的 T 房间死亡，并且被禁止 autophagic 救开始。在 thymic 的 autophagy 组成地高级上皮的房间为最佳的处理和内长的抗原的表示是必要的，并且为开发 thymocytes 的合适的积极、否定的选择要求。Autophagy 也支持早 B 房间祖先的 B 淋巴细胞，以及发展的幸存。在 B 房间， autophagy 是一条其他的死亡小径，当当 costimulation 不在时的抗原受体刺激导致有势力 autophagic 死亡。因此， autophagy 在淋巴细胞起一个复杂作用并且在他们的 lifespan 期间被调整保证一个健康免疫系统。
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Objective To develope gene knockdown cell model with artificial microRNA in setting up gene knockdown cell model.Methods We constructed vectors,and prepared siRNA fragments targeting on DJ-1.Then we transciently transfected the artificial miRNA and siRNA into MN9D cells by lipofectamine2000 reagent,the mRNA and protein expression level of DJ-1 gene were detected by RT-PCR and Western blot.Results Compared with control group,DJ-1 expression level was significantly decreased in both artificial miRNA and siRNA groups.DJ-1 was knockdowned and DJ-1 was decreased 90%(P0.05)at mRNA expression level,and decreased 70%~85%(P0.05) at protein level in MN9D cells transfected with the artificial miRNA.While DJ-1 was decreased by 50%~70%(P0.05)at mRNA level,and decreased by 20%~50%(P0.05)at protein level in MN9D cells transfected with siRNA.Comparing with siRNA,miRNA was more effective in silencing DJ-1.Conclusion The artificial miRNA and siRNA are both effective in silencing gene.miRNA has more significant function in knockingdown DJ-1 than siRNA.
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