shRNA interference for extracellular signal-regulated kinase 2 can inhibit the growth of esophageal cancer cell line Eca109.
BACKGROUND
Esophageal squamous cell carcinoma is one of the most common digestive tract cancers with 5-year survival rate less than 10% owing to its poor prognosis. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway has been mainly involved in the pathogenesis of various cancers. In present study, we investigated the role of ERK2 in human esophageal cancer cell line Eca109.


METHODS
Short-hairpin RNA (shRNA) interference vector targeted ERK2 was constructed using pGeneclip U1 hairpin cloning systems, then transfected into Eca109 cell line. The transfection efficiency was observed by fluorescence microscope and cell growth after transfection with shRNA-ERK2 vector was determined by methylthiazolyl blue tetrazolium (MTT) assay. The ERK2 expression after transfection was detected by western-blotting. The cell apoptosis and cell-cycle was analyzed by flow cytometry. The role of p-ERK2 was confirmed by immunohistochemistry and soft agar colony formation assay.


RESULTS
The growth of Eca109 transfected with shRNA-ERK2 vector was obviously inhibited compared to control group via MTT analysis. The inhibition rate after transfection with shRNA-ERK2 for 96 h was 10.45%, the expression of ERK2 was obviously reduced compared to the control analyzed by western-blot, cell apoptosis was 9.7% (compared to control, P < 0.05), and cell-cycle was arrested at G1 phase.


CONCLUSIONS
In present study we demonstrated for the first time that transfection with shRNA-ERK2 targeted ERK2 into Eca109 cells can inhibit growth of Eca109, inducing cell apoptosis and influencing cell-cycle. Together, these results we obtained suggested that ERK2 plays an important role in cell growth of Eca109.
Huo
Qi
Q
Medical Research Center, First Affiliated Hospital, Xinjiang Medical University, Urumqi, Xinjiang Uygur Autonomous Region, China.
Zheng
Shu-tao
ST
Tuersun
Aerziguli
A
Huang
Cong-gai
CG
Liu
Qing
Q
Zhang
Xue
X
Sheyhidin
Ilyar
I
Lu
Xiao-mei
XM
D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D004938:Esophageal Neoplasms / Q000201:enzymology*; D005434:Flow Cytometry / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D048370:MAP Kinase Kinase 2 / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection
D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; D048370:MAP Kinase Kinase 2; C022616:thiazolyl blue