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標題: 利用IP-SELEX篩選HLJ1特異性適體及其功能探討
Functional characterization of specific HLJ1 aptamer by IP-SELEX
作者: 陳之容
Chih-Jung Chen
關鍵字: 非小細胞肺癌;HLJ1;介白素-8;適體;免疫沉澱法結合配體指數增強系統進化技術;NSCLC;HLJ1;Interleukin 8;aptamer;IP-SELEX
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在實驗室先前研究中發現屬於熱休克蛋白40家族成員的Human Liver DnaJ-like (HLJ1) 是腫瘤生長及轉移的抑制基因,在細胞株中HLJ1大量表現能抑制癌細胞增生 (proliferation)、聚落形成 (colony formation) 、侵襲 (invasion) 與移動 (migration) 能力。此外,在過去研究中也發現,非小細胞肺癌 (non-small cell lung cancer,NSCLC) 中HLJ1蛋白大量表現會促使下游途徑的介白素-8 (interleukin 8,IL-8) 表現增加,因此推測HLJ1蛋白可能參與免疫發炎反應。本研究建立指數式擴增配體的系統進化技術與免疫沉澱法做結合 (immunoprecipitation-coupled systematic evolution of ligands by exponential enrichment, IP-SELEX),篩選能夠辨認HLJ1的適體,探討若將HLJ1阻斷後是否會抑制IL-8的表現及對於癌細胞侵襲與移動能力之影響。首先,利用IP-SELEX技術篩選後,透過次世代定序並結合生物資訊分析的方式分析篩選結果獲得HLJ1候選適體序列,進一步藉由免疫沉澱法確認HLJ1適體能夠專一性結合目標蛋白HLJ1。此外,將HLJ1適體轉染至大量表現HLJ1蛋白的肺癌細胞株後,結果顯示HLJ1蛋白受到抑制時會使癌細胞的侵襲及移動能力增加。由即時定量聚合酶連鎖反應分析在IL-8 mRNA層次上的改變,也發現HLJ1蛋白會增加IL-8 mRNA的表現,且同時加入HLJ1適體後,IL-8 mRNA的表現會受到抑制。另外,共轉染 (co-transfection) HLJ1及HLJ1適體至癌細胞中,經由冷光報導基因分析結果證實HLJ1適體會抑制IL-8表現。綜合上述結果證實本研究所篩選出之HLJ1適體具有與HLJ1蛋白的專一性結合能力及拮抗能力。

Our previous studies have shown that Human Liver DnaJ-like protein (HLJ1), which belongs to the Hsp40 family, plays the role of a tumor suppressor gene in non-small cell lung cancer (NSCLC). Overexpression of HLJ1 could inhibit cancer cell proliferation, colony formation, invasion and migration. In addition, HLJ1 could induce Interleukin 8 (IL-8) expression in lung cancer, which implied it may be involved in the immune inflammatory response. This study is aimed to use the immunoprecipitation-coupled systematic evolution of ligands by exponential enrichment (IP-SELEX) to screen HLJ1-specific aptamer, and further to investigate whether cell motility and IL-8 induction would be altered by the HLJ1 aptamer. After IP-SELEX screening, the HLJ1 candidate aptamers were identified through next-generation sequencing and bioinformatics analysis. Immunoprecipitation and pull down assay showed that the aptamer can specifically bind to HLJ1. Furthermore, the invasion and migration ability of lung cancer cell transfected with HLJ1 aptamer were increased. Transfecting HLJ1 aptamer into cells could also down-regulate IL-8 mRNA expression as measured by real-time PCR. In addition, IL-8 promoter reporter construct, HLJ1 expression vector and HLJ1 aptamer were co-transfected into lung cancer cell line and indicated that HLJ1 aptamer decreased IL-8 promoter transcriptional activity. In conclusion, our results indicated that the identified HLJ1 aptamer might be through binding to HLJ1 to reverse the original function of HLJ1 in NSCLC.
Rights: 同意授權瀏覽/列印電子全文服務,2021-08-07起公開。
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