10.6084/m9.figshare.4822939.v1
Taniguchi T.
Taniguchi
T.
Kishi K.
Kishi
K.
Nakagawa T.
Nakagawa
T.
Tanaka H.
Tanaka
H.
Tanaka T.
Tanaka
T.
Tomonari T.
Tomonari
T.
Okamoto K.
Okamoto
K.
Sogabe M.
Sogabe
M.
Miyamoto H.
Miyamoto
H.
Okahisa T.
Okahisa
T.
Muguruma N.
Muguruma
N.
Kajimoto M.
Kajimoto
M.
Sagawa I.
Sagawa
I.
Takayama T.
Takayama
T.
Supplementary Material for: Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma
Karger Publishers
2017
Des-gamma-carboxy prothrombin
Poly-(ADP-ribose) polymerase-1
Hepatocellular carcinoma
Biomarkers
Prothrombin
2017-04-06 09:45:41
Dataset
https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Poly-_ADP-Ribose_Polymerase-1_Promotes_Prothrombin_Gene_Transcription_and_Produces_Des-Gamma-Carboxy_Prothrombin_in_Hepatocellular_Carcinoma/4822939
<p><b><i>Background and Aim:</i></b> Although des-gamma-carboxy
prothrombin (DCP) is a well-known tumor marker for hepatocellular
carcinoma (HCC), the mechanism of DCP production is unclear. This study
aimed to investigate the mechanism how DCP is produced in HCC cells. <b><i>Methods:</i></b>
Levels of mRNA and DCP were analyzed by real-time polymerase chain
reaction and electro-chemiluminescence immunoassay respectively.
Secreted alkaline phosphatase (SEAP) expression vectors including
deletion mutants of the prothrombin gene promoter were constructed for
reporter gene assay. The transcription factors bound to DNA fragments
were analyzed by mass spectrometry. An electrophoretic mobility shift
assay (EMSA) was performed using a biotin end-labeled DNA. <b><i>Results:</i></b>
The prothrombin mRNA levels in all 5 DCP producing cell lines were
appreciably high. However, those in 2 DCP non-producing cell lines were
below detectable levels. A SEAP vector with -2985 to +27 showed a very
high transcription activity in DCP-producing Huh-1 cells. However,
transcription abruptly decreased when the vector with -2955 to +27 was
transfected, and then remained at the similar levels with larger
deletion mutants, indicating the existence of a cis-element at -2985 to
-2955 (31-bp). Mass spectrometry analysis identified the protein that
bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1).
Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells
induced marked inhibition of prothrombin gene transcription. The EMSA
clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment
in the prothrombin gene promoter. <b><i>Conclusions:</i></b> Our data
suggest that PARP-1 activates prothrombin gene transcription and that
the excessive prothrombin gene transcription induces DCP production in
DCP-producing HCC cells.</p>