Effect of silencing NEK2 on biological behaviors of HepG2 in human hepatoma cells and MAPK signal pathway.
To investigate the expression level of NEK2 in 40 tissue specimens of primary liver cancer and to search for clues whether the effect of NEK2 depletion plays a role on biological behaviors of HepG2 cells and the relevant molecular mechanism are the objectives of this study. Real-time PCR and immunohistochemistry assessed expression level of NEK2 in specimens of cancerous tissues and carcinoma-adjacent tissues. The NEK2 expression level in HepG2, Huh7, SMMC, and 7402 cells was detected by real-time PCR and western blot to screen experimental cell line. To assess the expression levels of NEK2 mRNA and protein, an effective siRNA transfected into the HepG2 cells was designed. CCK8 and colony-forming assays were performed to verify short-term and long-term proliferative activities, respectively. Capacity of apoptosis and cell cycle changes were assessed by flow cytometry. Ability of transference and invasion was measured by Transwell Chambers. Western blot approach was used to determine the protein expression levels. There was significantly high expression level of NEK2 in cancerous tissues compared to adjacent tissues. The expression of NEK2 was higher in HepG2 cells than other cell lines. Real-time PCR and western blot shown there were obviously down-regulated NEK2 expression in the NEK2-siRNA group compared to control groups. The capacity of amplification and invasion was inhibited distinctly, and FCM revealed the apoptosis rate was increased and G1 phase was arrested in NEK2-siRNA group. Western blot indicated that low expression of NEK2 in HepG2 cells could increase the expression levels of Bax, caspase-3, P21, and TIMP-1, but significantly suppressed the c-myc, c-jun, Bcl-2, cyclinD1, CDK4, MMP2, and MMP9 expression levels and the phosphorylation levels of ERK, JNK, and P38 compared with the control groups. Our findings demonstrated that NEK2 could be a valuable carcinogenic factor and a promising therapeutic target for primary liver cancer; NEK2 may regulate proliferation, apoptosis, and other biological behaviors of HepG2 cells via MAPK signal pathway.
Department of Oncology, Renmin Hospital, Wuhan University, 238 Jiefang road, Wuchang district, Wuhan, 430060, Hubei, People's Republic of China.
Mei-Xia
MX
Zhang
Department of Oncology, Renmin Hospital, Wuhan University, 238 Jiefang road, Wuchang district, Wuhan, 430060, Hubei, People's Republic of China. vipdoctorxu@163.com.
Xi-Ming
XM
Xu
Department of Urology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei, 430071, People's Republic of China.
Peng
P
Zhang
Department of Oncology, Renmin Hospital, Wuhan University, 238 Jiefang road, Wuchang district, Wuhan, 430060, Hubei, People's Republic of China.
Na-Na
NN
Han
Department of Oncology, Renmin Hospital, Wuhan University, 238 Jiefang road, Wuchang district, Wuhan, 430060, Hubei, People's Republic of China.
Jun-Jian
JJ
Deng
Department of Oncology, Renmin Hospital, Wuhan University, 238 Jiefang road, Wuchang district, Wuhan, 430060, Hubei, People's Republic of China.
Ting-Ting
TT
Yu
Department of Oncology, Renmin Hospital, Wuhan University, 238 Jiefang road, Wuchang district, Wuhan, 430060, Hubei, People's Republic of China.
Yuan-Yuan
YY
Gan
Department of Oncology, Renmin Hospital, Wuhan University, 238 Jiefang road, Wuchang district, Wuhan, 430060, Hubei, People's Republic of China.
Xiao-Qin
XQ
He
Department of Oncology, The Fifth People's Hospital, Wuhan, Hubei, 430050, People's Republic of China.
Zhi-Xiong
ZX
Long
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D000072379:NIMA-Related Kinases / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases
Apoptosis; MAPK; NEK2; Proliferation; Small interfering RNA