探討陽離子性高分子聚乙烯亞胺誘導細胞產生自噬之分子機制

Exploring autophagy related molecular mechanisms in cells induced by cationic polymer polyethylenimine

林嘉緯(Chia-Wei Lin)

指導教授 ： 詹明修

中山醫學大學/醫學院/生化微生物免疫研究所/博士(2017年)

摘要

陽離子性高分子聚乙烯亞胺 (plyethylenimine, PEI) 和聚賴胺酸 (poly(L-lysine), PLL) 主要應用於核酸傳遞之載體，其帶正電之特性會造成細胞毒性，但所導致的細胞死亡確切之分子機轉仍不完全清楚。在本論文研究中，我們證實聚乙烯亞胺在人類癌細胞 (human cervical cancer cell line, HeLa) 及老鼠胚胎纖維母細胞 (mouse embryonic fibroblast, MEF) 中會誘導細胞自噬產生，從細胞型態或是具有綠色螢光蛋白表達的細胞中皆有明顯細胞自噬特徵，在西方墨點法分析中細胞自噬主要指標 LC3、Beclin-1 表現量增加及 p62 之降解。同時由流式細胞儀分析 annexin V-FITC/PI 之比例及 caspase-3 蛋白表達量發現聚乙烯亞胺也會同時造成細胞凋亡與壞死，接著使用凋亡抑制劑 (Z-VAD) 發現細胞自噬與細胞凋亡可能是獨立發生或者在細胞凋亡上游發生。此外，聚乙烯亞胺在剔除掉 atg5 基因之老鼠胚胎纖維母細胞與野生型細胞相比會造成更高程度之細胞死亡，表示細胞自噬扮演著保護細胞免於死亡之角色。這些結果表明，聚乙烯亞胺能夠同時觸發死亡與存活路徑，當細胞自噬啟動時會保護細胞免於走向死亡。我們亦應用生物晶片進行細胞自噬相關基因之分析，以了解聚乙烯亞胺在野生型和剔除掉 atg5 基因之老鼠胚胎纖維母細胞中細胞自噬的分子機轉差異，結果顯示在野生型和剔除掉 atg5 基因之老鼠胚胎纖維母細胞中大多數細胞自噬相關基因表現量下降 (down-regulated)。此外聚乙烯亞胺也會誘導細胞自噬與凋亡並影響彼此間相關基因表現。因細胞自噬與 mTOR 訊息路徑有關，因此我們更進一步分析在聚乙烯亞胺處理後老鼠胚胎纖維母細胞之 miRNA 表達，結果顯示聚乙烯亞胺在野生型老鼠胚胎纖維母細胞中有三個 miRNA (miR-3090-5p、miR-346-3p 及 miR-494-3p) 表現量增加並參與 mTOR 特定訊息傳遞路徑誘導細胞自噬。並利用聚合酶連鎖反應及西方墨點法檢測這些 miRNAs 共同目標基因生長因子 Igf1，並觀察到 Igf1 比例減少，我們進一步將這三個表現量增加之人工合成 miRNA 轉染至野生型老鼠胚胎纖維母細胞中觀察到 Igf1 之基因和蛋白質比例有明顯減少，也證實會抑制 mTOR 訊息傳遞路徑相關蛋白，經 PEI/miR-494-3p 複合物處理後在老鼠胚胎纖維母細胞中，觀察到細胞自噬啟動後 miR-494-3p 入核比例明顯增加，本研究使我們更完整了解陽離子性高分子聚乙烯亞胺導致的細胞死亡分子機轉。

關鍵字

聚乙烯亞胺 ； 細胞自噬 ； 微型核醣核酸

並列摘要

Polyethylenimine (PEI) and poly(L-lysine) (PLL), which are cationic polymers used for gene therapy, are known to be cytotoxic, but their molecular mechanisms of cell death are not fully understood. In this study, we provide evidence that PEI and PLL induced autophagy in HeLa cervical cancer cells. In cells overexpressed with green fluorescent protein (GFP) microtubule associated protein 1 light chain 3 (LC3) fusion protein, PEI and PLL induced fluorescent puncta formations that represent LC3 recruitment to autophagosomes. In Western blot analysis, conversions of the LC3-I to LC3-II were significant, Beclin-1 and p62 degradation was observed in cells treated with PEI and PLL. From the analysis of annexin V flourescein isothiocyanate (FITC) and propidium iodide (PI) staining by flow cytometry, both apoptosis and necrosis occurred in PEI and PLL treated cells. Significant activated caspase 3 expression was detected in PLL and PEI treated cells. By applying ZVAD apoptotic inhibition, apoptosis and autophagy may occur independently or autophagy may be in the upstream of apoptosis on PEI and PLL treated cells. In addition, PEI and PLL induced higher degree of cell death in atg5-/- mouse embryonic fibroblast (MEF) cells than in wild-type cells. These results indicate that PEI and PLL can trigger both death and survival pathways simultaneously, and autophagy played a role in cell survival in PEI and PLL treated cells. The molecular mechanisms of autophagy in PEI treated cells are not well understood because of the use of nonspecific autophagy inhibitors. Here, we applied autophagy related gene expression analysis to pinpoint the molecular mechanisms of autophagy in PEI treated wild-type and atg5-/- MEF cells. It was demonstrated that the majority of induced genes are downregulated in wild-type and atg5-/- MEF cells, indicating that autophagy exhibits a trend toward downregulation after treatment with PEI. In addition to regulating genes encoding autophagy machinery components, genes related to coregulation of autophagy and apoptosis were induced in wild-type and atg5-/- MEF cells treated with PEI. These data indicate that autophagy and apoptosis are closely related in the PEI induced mechanism of cell death. In the absence of autophagy, the regulation of apoptosis was enhanced in atg5-/- MEF cells treated with PEI, indicating that inhibition of autophagy may lead to higher levels of apoptosis. The toxicology of PEI in gene expression levels has been previously investigated, little is known about the effects of PEI on the expression of microRNAs (miRNAs) that regulate gene expression at the post-transcriptional level. In this study, we explored miRNA expression profiles related to cell death mechanisms in MEF cells treated with PEI by applying microarray analysis, in this study, we focused on the mTOR signaling pathway, which is also a key regulator of autophagy. Based on the analysis of the mTOR signaling pathway, three upregulated miRNAs (miR-3090-5p, miR-346-3p, and miR-494-3p) were verified in PEI treated MEF cells using real-time quantitative reverse transcriptase-polymerase chain reaction. We further demonstrated that these three upregulated miRNAs resulted in the decrease of gene and protein expressions of the target gene growth factor Igf1 in MEF cells treated with PEI or transfected with three upregulated miRNA mimics. Finally, we demonstrated that the proteins involved of mTOR signaling pathway is inhibited. The cellular uptake, nuclear localization, and quantitative miR-494 levels of the complexes of miR-494 with PEI (miR-494 polyplexes) were higher in MEF cells. The indicator of autophagic activity in cells treated with miR-494 polyplexes was higher. Our study therefore provides deeper insight into the molecular mechanisms of cell death caused by cationic polymers.

並列關鍵字

polyethylenimine ； autophagy ； miRNA

參考文獻

Amberg N. Adenovirus receptors: implications for targeting of viral vectors. Trends Pharmacol Sci. 2012;33:442-448.

Akhtar S. Cationic nanosystems for the delivery of small interfering ribonucleic acid therapeutics: a focus on toxicogenomics. Expert Opin Drug Metab Toxicol. 2010;6:1347-1362.

Alsaggar M, Liu D. Physical methods for gene transfer. Adv Genet. 2015;89:1-24.

Alonso-Curbelo D, Soengas MS. Self-killing of melanoma cells by cytosolic delivery of dsRNA: wiring innate immunity for a coordinated mobilization of endosomes, autophagosomes and the apoptotic machinery in tumor cells. Autophagy. 2010;1:148-150.

Behr JP. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA. 1995;92:7297-7301.