腫瘤微環境和癌細胞之間會以許多不同的方式彼此相互作用，包括作為介質之一的外泌體 (exosomes)，其為一種細胞外囊泡 (extracellular vesicles，EVs)。許多研究已經報導，外來體攜帶的特定貨物 (cargo) 具有調節癌細胞特性的能力。 HuR 蛋白 (HuR protein) 是一種RNA結合蛋白 (RNA-binding protein)，可藉由結合不同的 RNA 片段加以控制基因的表達，進而改變細胞的生理活性。在本項研究中，我們主要想探討間質幹細胞 (mesenchymal stem cells，MSCs) 所分泌的外泌體是否可以攜帶 HuR 蛋白/HuR 相關 RNAs (HuR-associated RNAs) 並影響癌細胞的特性，使其更相似於癌症幹細胞 (cancer stem cells，CSCs)。

　　我們使用 CRISPR/Cas9 技術在肺癌細胞 LM-V252 (上皮型) 和 HM20-V252 (間質型) 中剔除 ELAVL1 基因 (HuR)，再以含有 HuR 蛋白的 MSCs 衍生外泌體進行作用，觀察是否影響其功能特性，包含測試使癌細胞獲得更多 CSC 特性的潛力。首先，我們通過西方墨點法 (western blot) 和免疫螢光染色 (immunofluorescence staining) 發現 HuR / HuR 相關 RNAs 可以轉移至 MSC 衍生的外泌體之中，並且於作用 24 小時後大多數的癌細胞皆能吞入 MSC 衍生的外泌體。此外，使用 RT-PCR測定上皮-間質轉化 (epithelial-mesenchymal transition，EMT) 相關和CSC相關的基因表達。測定和比較加入MSC衍生的外來體作用後CRISPR-control V257和ELAVL1 (HuR)-剔除 V252 細胞之間的差異。有趣的是，我們發現經過 MSC 衍生的外來體作用可以增加 HM20-ELAVL1 (HuR) 剔除細胞中 EMT 相關基因的表達，而在 LM-ELAVL1 (HuR) 剔除細胞中卻有相反的效應。除此之外，加入外泌體作用後，HM20-V252 的球體形成能力增加，表示癌細胞加強了 CSC 的性質，而LM-V252 則是降低了此能力。

　　整體而言，根據上述研究結果我們認為 MSC 衍生的外泌體所攜帶的 HuR 蛋白結合了特定的短/長非編碼 RNA (short/long non-coding RNAs)，並進入癌細胞內調節其惡化的程度以及 CSC 的特性。

　　The tumor microenvironment and cancer cells interact with each other in many different ways. Which is including exosomes, a type of extracellular vesicles (EVs), as one of the mediums. Many studies have reported that the specific cargo carried by exosomes has the ability to regulate the characteristics of cancer cells. HuR protein, an RNA-binding protein, can bind to different RNA segments to control the expression of genes, and then change physiological activities of cells. In this study, we mainly would like to study whether the exosomes secreted by mesenchymal stem cells (MSCs) can carry HuR protein/HuR-associated RNAs and affect the properties of cancer cells to make them more similar to cancer stem cells (CSCs).

　　We used the CRISPR/Cas9 technology to knockout ELAVL1 gene (HuR) in lung cancer cells LM-V252 (epithelial-type) and HM20-V252 (mesenchymal-type), and then treated with MSC derived-exosome which containing HuR to observe whether that would affect its functional properties, including testing for the potential to make cancer cells gaining more the CSC properties. First, we found that HuR/HuR-associated RNAs can translocate into MSC-derived exosomes by western blot and immunofluorescence staining, and then most of cancer cells can uptake MSC-derived exosomes after treatment for 24 hours. Furthermore, RT-PCR was used to respectively determine the expression of epithelial-mesenchymal transition (EMT)-related and CSC-related genes. The differences between CRISPR-control V257and ELAVL1 (HuR)-knockout V252 after treatment with MSC derived-exosomes were determined and compared. Interestingly, we found that treatment of MSC-derived exosomes could increase the expression of EMT-related genes in HM20-ELAVL1 (HuR)-knockout cells, whereas the effects were opposite in LM-ELAVL1 (HuR)-knockout cells. In addition, the ability of sphere-forming was increased in HM20-V252 cells after treatment with exosomes but reduced the ability in LM-V252, indicating that the CSC properties of cancer cells could be altered by tumor microenvironment via exosome.

　　Overall, according to these studies, we expect that HuR protein carried by MSC derived-exosomes binds to specific short/long non-coding RNAs and enters into cancer cells to regulate their deterioration degree and promote the CSC properties.

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