Novel aptasensor-based assay of sonic hedgehog ligand for detection of portal vein invasion of hepatocellular carcinoma.

Affiliation

Li HY(1), Yin FF(2), Li XY(1), Jia WN(1), Ding J(3), Zhang L(1), Wang ZH(1), Hu QQ(4), Zuo JL(5), Jia HL(5), Wang LH(6), Fan CH(7), Gu HZ(8), Wu J(9).
Author information:
(1)Department of Medical Microbiology, MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
(2)Division of Physical Biology, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201800, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
(3)Department of Gastroenterology, Shanghai Jing'an District Central Hospital, Fudan University, Shanghai, 200040, China.
(4)Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.
(5)Department of General Surgery, Huashan Hospital of Fudan University, Shanghai, 200041, China.
(6)Division of Physical Biology, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201800, China; Shanghai Synchrotron Radiation Facility, Zhangjiang Laboratory, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, 201210, China.
(7)Frontiers Science Center for Transformative Molecules, School of Chemistry and Chemical Engineering, Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, China. Electronic address: [Email]
(8)Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China. Electronic address: [Email]
(9)Department of Medical Microbiology, MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China; Dept. of Gastroenterology & Hepatology, Zhongshan Hospital of Fudan University, Shanghai, 200032, China; Shanghai Institute of Liver Diseases, Fudan University Shanghai Medical College, Shanghai, 200032, China. Electronic address: [Email]

Abstract

The high expression of sonic hedgehog ligand (SHh) is closely correlated to the metastasis, drug resistance and poor prognosis of hepatocellular carcinoma (HCC). Therefore, sensitive, specific and efficient detection methods for SHh are needed for the early diagnosis and assessment of prognosis. Herein, an aptamer, AP32 that specifically binds to SHh (KD = 25.7 ± 4.1 nM) was obtained by SELEX technology with further optimization. In vivo experiments confirmed that AP32 has the potential to be an imaging probe for Huh-7 cell-derived xenograft. The interaction mode in 3-dimensional configuration between the aptamer and SHh was established by molecular simulation and confirmed by mutations at key sites of the aptamer. An aptasensor-based assay was successfully developed by conjugating Texas-Red-labeled AP32 to microbeads, and was used to analyze SHh content in hepatoma cell lysates, serum and HCC specimens. The method exhibited a broad detection range from 0.07 to 62.5 nM with a low detection limit of 69 pM, and a recovery rate of 104.6 ± 3.9% in serum. When the assay was used to measure SHh content in tissue lysates, the results demonstrated that it possessed 57.1% positivity, 100% specificity in distinguishing 28 HCC specimens from normal tissues, and was compensatory for detection of HCC in AFP-negative cases. Moreover, elevated SHh levels are indicative of portal vein invasion at 77.8% positive rate. This novel aptasensor-based SHh assay may offer a reliable means in predicting early metastasis and poor prognosis in hepatocellular carcinoma.