shRNA interference for extracellular signal-regulated kinase 2 can inhibit the growth of esophageal cancer cell line Eca109.

ABSTRACT

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.