肺癌是全球癌症死亡的主要原因。半乳糖凝集素 1(Gal-1) 是一種多效同型二聚體 β-半乳糖苷結合蛋白，具有單個碳水化合物識別域，Gal-1在腫瘤過量表達並促進腫瘤細胞的功能，也能結合T細胞導致T細胞凋亡而使腫瘤細胞能夠逃避免疫攻擊，導致肺癌患者預後不良。適體是經由化學合成的單鏈DNA或RNA，對目標具有高親和力和專一性，對化學修飾具有良好的耐受性，可是適體容易透過腎臟代謝出體外。為了彌補單價適體的缺陷，多價適體被提出來，它們由相同或不同的適體經過或未經修飾組裝而成，與單體適體相比具有更好的功能。實驗室先前研究開發了一種新型適體名為AP-74 M-545，同時表現出對Gal-1的高親和力和抑制肺腫瘤的生長。本研究的目標是設計出可以針對 Gal-1 的四聚體適體 (AP-74 M-545@4)，並闡明它在阻斷 galectin-1 功能方面比單體 AP-74 M-545 更有效。 AP-74 M-545@4 由通過鹼基配對連接四個AP-74M545-linker到Scaffold所組成。首先，我們通過預先重新折疊 Ap-74M545-linker ，隨後添加Scaffold來合成 AP-74 M-545@4，然後將兩種材料在冷房中在搖床上共孵育過夜。透過10%丙烯酰胺電泳對產物進行驗證。我們發現凝膠上看不到Scaffold DNA，並且在約300 bp處形成清晰的條帶，表明AP-74 M-545@4已成功合成。 此外，AP-74 M-545@4 在 37℃ 孵育 24 小時後仍保持穩定。接下來，我們通過血液凝集試驗比較了 AP-74 M-545、AP-74 M-545-linker 和 AP-74 M-545@4 對 CS-Gal-1 的抑制能力。 結果表明，這兩種材料均不會抑制血液凝集。為了研究上述材料對癌細胞活性的影響，我們對兩種肺癌細胞系CL1-0和CL1-5進行了傷口癒合的細胞爬行實驗。數據表明有時AP-74 M-545@4能抑制CL1-0和CL1-5的遷移。此外，加入AP-74 M-545@4後能夠讓CL1-0和CL1-5傾向上皮特性。綜上所述，我們的研究表明 AP-74 M545@4 可能是治療肺癌的潛在治療劑。

Lung cancer is the leading cause of cancer deaths globally. Galectin 1 (Gal-1), a pleotropic homodimeric beta-galactoside binding protein with a single carbohydrate recognition domain. Gal-1 is overexpressed in lung cancer and promotes the function of cancer cells. Gal-1 can also bind T cells to cause T cell apoptosis, so that tumor cells can escape immune attack, resulting in poor prognosis of lung cancer patients. Aptamers are chemically synthesized single stranded DNA or RNA with high affinity and specificity to the targets and good tolerant to chemical modification but are easily lost through renal excretion. Multivalent aptamer is assembled by identical or different aptamers with or without modification and possess better function in comparison to monomeric aptamer. Our previous study developed a novel aptamer, AP-74 M-545, demonstrating high affinity to Gal-1 and inhibition of lung tumor growth. Our aim in this study is to design a Gal-1 specific tetrameric aptamer (AP-74 M-545@4) and to clarify that it is more potent than monomeric AP-74 M-545 in blocking gal-1 function. The AP-74 M-545@4 is composed of a scaffold connected to four AP-74M545-linker through base pairing. First, we synthesized the AP-74 M-545@4 by refolding the Ap-74M545-linker in advance subsequently added scaffold and then co-incubated the two materials overnight in the cold room on a shaker. The verification of the products was carried out through 10% acrylamide electrophoresis. We found that no scaffold DNA could be seen on the gel, and a clear band was formed at about 300 bp, indicating that AP-74 M-545@4 had been successfully synthesized. Furthermore, the AP-74 M-545@4 remained stable after incubating in 37°C for 24 hours. Next, we compared the inhibitory ability of AP-74 M-545, AP-74 M-545-linker, and AP-74 M-545@4 on CS-Gal-1 through hemagglutination assay. The results shown that neither of the materials could inhibit hemagglutination. To investigate the therapeutic effect of the above-mentioned materials, we performed wound healing assay on two lung cancer cell lines CL1-0 and CL1-5. The data suggested that the AP-74 M-545@4 could possibly inhibit the migration of CL1-0 and CL1-5. Since we discovered AP-74 M-545@4 affected CL1-0 and CL1-5 in some level. Thus, we explored how AP-74 M-545@4 would affect two lung cancer cell lines. We performed western blot assay. The result suggested CL1-0 and CL1-5 were prone to epithelial changes. Taken together, our research indicated that AP-74 M545@4 might be a potential therapeutic agent for treating lung cancer.

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