Digestive Diseases and Sciences (2018) 63:676–686
Hypermethylation of NF‑κB‑Activating Protein‑Like (NKAPL) Promoter
in Hepatocellular Carcinoma Suppresses Its Expression and Predicts
a Poor Prognosis
Patrick Kwok Shing Ng
· Carol Po Ying Lau
· Emily Kai Yee Lam
· Sheila Sai Kam Li
· Vivian Wai Yan Lui
· Yuen Keng Ng
· Paul Bo San Lai
· Stephen Kwok Wing Tsui
Received: 10 September 2017 / Accepted: 11 January 2018 / Published online: 20 January 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Background and Aim Hepatocellular carcinoma (HCC) is a complicated disease with low survival rate partially due to
frequent recurrence and no eﬃcient therapy. Promoter hypermethylation of tumor suppressor genes has been demonstrated
as one of the molecular mechanisms contributing to tumorigenesis and progression in HCC. This study aims to investigate
regulation of NKAPL expression by promoter methylation and its clinical relevance as a biomarker for HCC.
Methods We measured mRNA expression of NKAPL in 5 HCC cell lines and a cohort of 62 pairs of primary HCC tumor
and their adjacent non-cancer liver tissues. NKAPL protein expression on HCC cell lines and clinical samples was assessed
by Western blot and immunohistochemistry, respectively. Association analyses between NKAPL expression and clinico-
pathologic characteristics in the cohort were conducted. Methylation statuses of NKAPL promoter in 18 pairs of tumor and
adjacent non-tumor HCC samples were studied using methylation-speciﬁc PCR. Biological functions of NKAPL in HCC
were investigated by ectopic expression of NKAPL in HCC cells, and cell viability and cell cycle analyses were performed.
Results Our present study showed suppressed expression and promoter hypermethylation are common events in HCC.
Demethylation experiment in HCC cells demonstrated that the NKAPL expression was regulated by promoter methylation.
In addition, high methylation level of NKAPL and its low expression predict poor outcome. Furthermore, ectopic expression
of NKAPL in the HCC cells inhibited cell growth.
Conclusions Our ﬁndings suggest that methylation of NKAPL is a frequent event and is a potential prognosis biomarker in
Keywords Hepatocellular carcinoma · NKAPL · Promoter hypermethylation · Prognosis · The Cancer Genome Atlas
Hepatocellular carcinoma (HCC) is the third most common
cause of cancer death worldwide [1, 2] associated with a
number of risk factors, including chronic liver disease, cir-
rhosis, hepatitis B or C virus infection , heavy alcoholism
, and aﬂatoxin B exposure . Late diagnosis, frequent
Electronic supplementary material The online version of this
article (http s://doi.org/10.1007 /s106 20-018-4929 -3) contains
supplementary material, which is available to authorized users.
Patrick Kwok Shing Ng and Carol Po Ying Lau have contributed
equally to this work.
* Stephen Kwok Wing Tsui
School of Biomedical Sciences, The Chinese University
of Hong Kong, Room 524, Lo Kwee-Seong Integrated
Biomedical Sciences Building, Area 39, Hong Kong, China
Department of Clinical Oncology, The Chinese University
of Hong Kong, Hong Kong, China
Department of Surgery, The Chinese University of Hong
Kong, Hong Kong, China
Department of Orthopaedics and Traumatology, The Chinese
University of Hong Kong, Hong Kong, China
Present Address: Institute of Personalized Cancer Therapy,
University of Texas MD Anderson Cancer Center, Houston,