Gankyrin gene deletion followed by proteomic analysis: insight into the roles of Gankyrin in tumorigenesis and metastasis.
BACKGROUND
Gankyrin was originally purified and characterized as the p28 component of the 26S proteasome, and later identified as an oncogenic protein in hepatocellular carcinomas (HCC). It has recently been found to be highly expressed in several other malignancies, and compelling evidence show gankyrin plays important roles in tumorigenesis. However, its mechanism of action remains unclear.


METHODS
In order to further clarify the functions of gankyrin and better understand its molecular mechanisms, we generated a gankyrin null cell line, HCT116 gankyrin-/- , by targeted homologous recombination in human colon cancer cells, and then employed two-dimensional electrophoresis (2-DE) based proteomic approaches followed by MS identification to investigate alterations in the proteome due to the gankyrin knockout. Western blot and qRT-PCR assays were also used to examine the protein and mRNA levels of some identified proteins.


RESULTS
Compared with wild-type control cells, gankyrin null cells were impaired in terms of their proliferation, migration and anchorage-independent growth. A total of 21 altered proteins were identified, which included 18 proteins that had not previously been reported to be related to gankyrin. Notably, eight metastasis-related proteins were identified. Western blot analyses confirmed that the changes in three examined proteins were consistent with 2-DE gel analysis.


CONCLUSIONS
In summary, we have generated a useful cell tool to clarify the functions of gankyrin. Our proteomic data provide novel information to better understand the roles and underlying mechanisms by which gankyrin is involved in tumorigenesis and cancer metastasis.
National Center of Biomedical Analysis, 27 Taiping Road, Beijing, 100850, China.
Xue
X
Luo
Liang
L
Chen
Jiang
J
Dai
Yanfei
Y
Gao
Hongli
H
Wang
Na
N
Wang
Yongqiang
Y
Zhao
Feng
F
Liu
Zhihong
Z
Sang
Jie
J
Wang
Weihua
W
Li
Kun
K
He
Baofeng
B
Jin
Jianghong
J
Man
Wei
W
Zhang
Qing
Q
Xia
D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D045325:HCT116 Cells; D006801:Humans; D009362:Neoplasm Metastasis / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D040901:Proteomics; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*
C403591:PSMD10 protein, human; D011518:Proto-Oncogene Proteins; D046988:Proteasome Endopeptidase Complex