KIF12在大腸癌細胞中調控自噬作用與細胞外囊泡形成之角色
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2022
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驅動蛋白家族成員 12（Kinesin Family Member 12；KIF12）是一種微管相關的運動蛋白，在細胞分裂及囊泡運輸中扮演重要角色。我們之前發現 KIF12 的表現量在大腸癌的組織比起正常組織有升高的現象，並且會促進大腸癌幹細胞的增殖。為更了解 KIF12 對大腸癌細胞的調控，我們進一步探討其對細胞自噬作用(autophagy)與外泌體(exosome)生成之影響。細胞中自噬作用泛指將細胞質內容物吞噬，形成自噬泡並送至溶酶體內降解的路徑，它的功能主要在於分解細胞內異常摺疊或堆積的蛋白質以及清除老化的胞器。細胞中自噬作用異常與癌症、神經及心血管退化性疾病形成皆有相關。外泌體是由內體(endosome)衍生的多囊泡體產生，包裹部分細胞內容物後分泌到細胞外。有研究指出外泌體的形成與自噬作用在維持細胞穩定及腫瘤細胞轉移中有協同作用。為深入探討 KIF12 是否調節大腸癌細胞以及其癌幹細胞自噬與外泌的能力，我們利用Lipofectamine 2000 將削弱 KIF12 的 shRNA 轉染至SW480(shKIF12)，並利用 CRISPR 系統建立剔除 KIF12 的穩定細胞株(CR-KIF12)，來探討 KIF12 對此二功能之影響。我們以 GFP-LC3融合蛋白質表現載體及酸性胞器染劑 Lysotracker red 來偵測細胞自噬作用。同時，我們利用外泌體的報告質體 PalmGRET reporter 來偵測細胞外泌體的生成與 KIF12 蛋白質之相關性。結果發現降低KIF12 的表現會抑制細胞因饑餓引發的自噬作用與外泌體生成。
Kinesin family member 12 (KIF12) is a microtubule-associated motorprotein that plays an important role in cytokinesis and vesicular transport.We have found that the expression level of KIF12 is higher in colorectalcancer tissues than in normal tissues. It also promotes the proliferation ofcolorectal cancer stem cells (CSC). To understand the regulation ofKIF12 on colorectal cancer cells, we further explored its effects onautophagy and exosome formation. Autophagy is a cellular process thatincludes autophagosome forming and fusing with lysosome to degradethe cellular components. Its main function is to degrade cellular proteinswith abnormal folding and aged organelles, and to recycle usefulmaterials to maintain the normal physiological function of the cell. It hasbeen known that aberrant autophagy correlates with cancer, neuronal andcardiovascular diseases. Exosomes are generated by multiple intraluminalvesicles derived from multivesicular bodies, which carry cell-specificcargos of proteins, lipids, and genetic materials. Studies have pointed outthat autophagy and the formation of exosomes have a synergistic effect inmaintaining cell stability and tumor cell metastasis. To further explorewhether KIF12 regulates the autophagy and exocytosis of colorectalcancer cells and their CSCs, we used Lipofectamine 2000 to transienttransfect KIF12 shRNA into SW480, and also establish the stable celllines that knocked out KIF12 by CRISPR system. Moreover, wequantified autophagosome formation-level by counting GFP-LC3colocalized with lysotracker red staining. At the same time, we took theadvantage of exosome reporter, palmGRET, to study the correlationbetween the production of cellular exosomes and KIF12. Our resultsshowed that downregulated KIF12 expression inhibits starvation-inducedautophagy and exosome formation.
Kinesin family member 12 (KIF12) is a microtubule-associated motorprotein that plays an important role in cytokinesis and vesicular transport.We have found that the expression level of KIF12 is higher in colorectalcancer tissues than in normal tissues. It also promotes the proliferation ofcolorectal cancer stem cells (CSC). To understand the regulation ofKIF12 on colorectal cancer cells, we further explored its effects onautophagy and exosome formation. Autophagy is a cellular process thatincludes autophagosome forming and fusing with lysosome to degradethe cellular components. Its main function is to degrade cellular proteinswith abnormal folding and aged organelles, and to recycle usefulmaterials to maintain the normal physiological function of the cell. It hasbeen known that aberrant autophagy correlates with cancer, neuronal andcardiovascular diseases. Exosomes are generated by multiple intraluminalvesicles derived from multivesicular bodies, which carry cell-specificcargos of proteins, lipids, and genetic materials. Studies have pointed outthat autophagy and the formation of exosomes have a synergistic effect inmaintaining cell stability and tumor cell metastasis. To further explorewhether KIF12 regulates the autophagy and exocytosis of colorectalcancer cells and their CSCs, we used Lipofectamine 2000 to transienttransfect KIF12 shRNA into SW480, and also establish the stable celllines that knocked out KIF12 by CRISPR system. Moreover, wequantified autophagosome formation-level by counting GFP-LC3colocalized with lysotracker red staining. At the same time, we took theadvantage of exosome reporter, palmGRET, to study the correlationbetween the production of cellular exosomes and KIF12. Our resultsshowed that downregulated KIF12 expression inhibits starvation-inducedautophagy and exosome formation.
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驅動蛋白家族成員12, 大腸癌, 自噬作用, 細胞外囊泡, KIF12, Colorectal Cancer, Autophagy, Extracellular vesicles