SiRNA profiling of human ezrin-knockdown ec109 cells(PSE1) and control(PS)

ABSTRACT: Ezrin belongs to the ezrin¨Cradixin¨Cmoesin (ERM) family proteins, which cross-link actin cytoskeleton and plasma membrane, and is actively involved in regulating the growth and metastatic capacity of cancer cells. Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies in the world and the expression of ezrin in ESCC has been described only recently, but its roles and mechanism still remained unclear. we have used the pSUPER RNAi system to stably suppress the expression of the ezrin gene in EC109, an esophageal squamous carcinoma cell line, and then cDNA microarray was performed to explore some changed genes with ezrin silence. Experiment Overall Design: he mammalian expression vector, pSUPER.neo.circular (OligoEngine), was used for expression of siRNA in EC109 cells. Briefly, two primer pairs were synthesized, one pair encoding nucleotides 274¨C292 (TCCACTATGTGGATAATAA) followed by a 9 base ¡®¡®loop¡¯¡¯ (TTCAAGAGA) and the inverted repeat (PSE1), and the second encoding nucleotides 265¨C283 (ACTTTGGCCTCCACTATGT) again followed by the loop and inverted repeat (PSE2). And pSUPER.neo vector of nonspecific siRNA was taken as negative control (PSC). The primer pairs were annealed and inserted into the BglII and HindIII sites of pSUPER.neo and transformed into JM109 competent cells (Promega). Positive clones were verified and transfected into EC109 cells using FuGENE 6 transfection reagent (Roche) according to the manufacturer¡¯s instructions. G418 (400 ¦Ìg/ml, Calbiochem) was added into the culture medium after 24 h. Stable G418-resistant clones were obtained in 7¨C9 days. The expanded cells were then used for subsequent studies.

ORGANISM(S): Homo sapiens

SUBMITTER: Enmin Li 

PROVIDER: E-GEOD-6233 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress