We investigated the possibility that PGE 2 may mediate the enhanced expression of Myeov in CRC. Consequently, the objectives of our study were two-fold; firstly, to assess the role of Myeov gene knockdown on CRC cell migration in vitro; secondly, to evaluate the effect of PGE 2 on Myeov mRNA expression in CRC. Materials and methods Cell culture The T84 cell line obtained PP2 manufacturer from the European collection of cell cultures
was used in this study as it is an established in vitro experimental model of colorectal carcinoma. The cell were cultured in Dulbecco’s modified Eagle’s medium-F12, with 1 U/ml penicillin, 1 lg/ml streptomycin, and 10% fetal bovine serum under standard conditions. siRNA knockdown The functional Selleck IACS-010759 role of Myeov was assessed using gene knockdown with small interfering RNA (siRNA) designed and synthesized for Myeov knockdown (Qiagen Inc., CA, USA). The siRNA had the following sequences: Myeov sense, 50-GGA UGU AAG UUA UCA ACU A-30; Myeov antisense, 50-UAG UUG AUA ACU UAC AUC C-30. A chemically synthesized
non-selleck inhibitor silencing siRNA duplex with the following sequence; sense, 50-UUC UCC GAA CGU GUC ACG U-30; antisense, 50-ACG UGA CAC GUU CGG AGA A-30 that had no known homology with any mammalian gene was used to control for non-specific silencing events. Gene knockdown was achieved in T84 cells. Briefly, 4 × 10 4 cells were incubated under standard conditions overnight. 5 μg of each siRNA was then mixed with 30 μl of RNAifect (Qiagen) and was added drop wise. Cells were incubated for 48 h again under standard conditions before being assayed. RNA preparation and PCR TRIzol (Sigma-Aldrich, Ireland) was used to extract RNA from cells. Reverse transcription was achieved using AMV reverse transcriptase (Invitrogen Ltd., UK). Real-time RT-PCR was performed using a Rotor Gene (Corbett Research, Australia). GAPDH, which
was amplified in parallel with the genes Paclitaxel manufacturer of interest, served as a housekeeping gene. All measurements were performed in triplicate. The oligonucleotide primers and probes employed in this study were: MYEOV forward primer: CCT AAA TCC AGC CAC GTC AT, reverse primer; GAC ACA CCA CGG AGA CAA TG, GAPDH forward primer: GAA GGT GAA GGT CGG AGT TC, reverse primer GAA GAT GGT GAT GGG ATT TC. Cell migration ‘Scratch Assay’ Following Myeov knockdown, a “”scratch”" was placed in a confluent T84 cell monolayer using a 10 μl micropipette tip [10]. Cell migration over this wound scratch was monitored by photographing at 1, 6, 12, 24 and 36 hours. Subsequent image analysis involved measuring scratch width at 5 random points. Average scratch width and standard deviation was calculated for each time point. Cells were photographed using a × 10 objective lens. Carnoy software (Biovolution) was used to measure the pixel width of the scratches. The effects of PGE 2 on Myeov expression T84 CRC cells were treated with increasing concentrations (0.00025 μM, 0.