肺癌為發生率以及致死率最高的癌症，究其主要的原因，最大的關鍵在於治療期間病人會產生抗藥性，進而造成癌症復發及惡化。如何在治療過程中阻止抗藥性的發生是增加癌症病患存活率最重要的因素。實驗室已發現USP24會促進化療時所產生的抗藥性，並已發展新穎的藥物USP24-i抑制抗藥性的產生，進一步釐清其分子機制是重要的議題。首先，使用USP24-i處理肺癌抗藥性細胞A549-T24，發現自噬作用的重要蛋白LC3B大量表現。免疫螢光染色和電子顯微鏡觀察USP24-i處理的抗藥性細胞，顯示大量的自噬作用產生於細胞內。也發現實驗室建構USP24基因剔除鼠體重下降以及LC3B的表現量明顯增加。另外，也使用USP24-i處理正常的小鼠，發現LC3B表現量明顯增加。綜合體外和體內結果皆顯示抑制USP24會促進自噬作用的產生。本研究使用了對抑制抗藥性有不同效果的藥物處理抗藥性細胞，發現LC3B表現量與藥物的效果成正比。接著將自噬作用阻斷，發現抑制自噬作用也抑制了USP24-i對於抗藥性的抑制作用。接下來本研究嘗試探討USP24-i誘發自噬作用的分子機制。發現自噬作用上游的ULK, LC3及p62的mRNA和蛋白質表現量明顯的上升，代表抑制USP24會透過增加這些蛋白的表現量來誘發自噬作用，這些研究對於USP24在抗藥的角色及其機制提供了清楚的證據。

According to statistics from the World Health Organization, one in six deaths is due to cancer. Abnormal cell growth occurs in almost all organs and tissues in the body, and it can also metastasize to other parts of the body. Among all cancers, lung cancer has the highest incidence and mortality rate. The main reason is patient will develop drug resistance during the treatment, which will cause cancer to recur and worsen. How to prevent the occurrence of drug resistance during treatment is the most critical factor in increasing the survival rate of cancer patients. Our previous studies have shown that Ubiquitin-specific Peptidase 24 (USP24) can promote drug resistance during chemotherapy. We have developed a novel drug, USP24-i, to inhibit the development of drug resistance. Further clarification of its molecular mechanism is an important issue. First, we used USP24-i to treat lung cancer drug-resistant cells A549-T24. We found that the essential proteins of autophagy, LC3B, were expressed in large quantities. We also use immunofluorescence and electron microscope to observe drug-resistant cells treated with USP24-i. The results showed that USP24-i caused a large amount of autolysosome. In addition, we construct USP24 knockout mice (USP24C1695A) by using CRISPR/Cas9. We also found that the weight of USP24C1695A mice decreased significantly, and the expression of LC3B also increased significantly. In addition, we also used USP24-i to treat normal mice and found significantly increased expression of LC3B in mice. So far, our in vitro and in vivo results show that inhibiting USP24 can induce autophagy. Finally, we are wondering whether there is a causal relationship between autophagy and drug resistance. Therefore, we used Bafilomycin A1 to block autophagy. We found that inhibiting autophagy also inhibited the inhibitory effect of USP24-i on drug resistance. All the data showed that the autophagy induced by USP24-i is necessary for USP24-i to inhibit drug resistance. Next, we try to explore the molecular mechanism that inhibits the autophagy induced by USP24. We found that the inhibition of USP24 can induce autophagy by increasing the expression of ULK, LC3, and p62. Our research provides clear evidence for the role of USP24 in drug resistance and its mechanism.

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