Aberrant splicing of Hugl-1 is associated with hepatocellular carcinoma progression.
OBJECTIVE
Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC).


METHODS
Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay.


RESULTS
In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short "direct repeat" sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice.


CONCLUSIONS
We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.
Lu
Xuefeng
X
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Feng
Xiujing
X
Man
Xiaobo
X
Yang
Guang
G
Tang
Liang
L
Du
Dan
D
Zhang
Fan
F
Yuan
Haixin
H
Huang
Qin
Q
Zhang
Zhe
Z
Liu
Yinkun
Y
Strand
Dennis
D
Chen
Zhengjun
Z
D000328:Adult; D000368:Aged; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D008969:Molecular Sequence Data; D017354:Point Mutation; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D047368:Tumor Burden
D003598:Cytoskeletal Proteins; C097906:LLGL1 protein, human; D012333:RNA, Messenger