ystem, a functional putative binding site was identified by simply measuring luciferase activ http://www.selleckchem.com/products/VX-770.html ity. In LS174T cells, only the upstream binding site responded to miR 145 over e pressed e o genously, and in normal colon cells endogen ously over e pressing miR 145. Specific targeting of the DFF45 putative binding site by miR 145 To test the specificity of miR 145 at the 854 876 site, we co transfected LS174T cells with luc 854 and the miR 145 mimic at various abundances, and found that the inhibition of the luciferase activity by miR 145 was dose dependent. In normal colon cells trans fected with the miR 145 inhibitor, the luciferase activity was increased significantly compared to the inhibitor control at 24 hours and 36 hours. To further demonstrate the importance of the putative binding site, a substitution mutation was gen erated to test its activity.
In the DFF45 854 Mutation vector, seven nucleotides were replaced with ctcgGcct. We cloned the entire region of DFF45 downstream of the repor ter. As e pected, down regulation of reporter activity was detected in the construct that contains the entire region of DFF45. Correspondingly, we demonstrated that the mutation in the putative binding site abolished the miR 145 mediated inhibition of the repor ter gene. Taken together, these data suggest that the miR 145 binding site present in the DFF45 is critical for miR 145 mediated gene regulation. MiR 145 regulates DFF45 at the translational level To identify whether DFF45 potentially regulated by miR 145, we measured the e pression levels of DFF45 by quantitative polymerase chain reaction and Western blotting after treatment with the miR 145 mimic in LS174T cells.
Ectopic e pression of miR 145 signifi cantly reduced the level of DFF45 protein at 24 hours and 48 hours. However, we did not detect the inhibition of DFF45 at the mRNA level, as measured by qRT PCR and real time PCR. These Brefeldin_A results suggest that miR 145 targets DFF45 by function ing at the level of translational regulation. Detection of apoptosis by DNA fragmentation DNA fragmentation is the typical biochemical inde of cell apoptosis. These ladders of DNA fragments are the size of integer multiples of the length of a nucleosome. In DNA ladder assays, cells trans fected with miR 145 mimic siRNA DFF45 were e posed to staurosporine. DNA isolated from LS174T cells showed the characteristic ladder pattern of apop tosis in a time dependent manner.
As time went on, the ladder selleck chemicals llc showed up more obviously in the miR 145 mimic siRNA DFF45 treated group. However, the time dependent changes were not seen in DNA samples e tracted from normal colon cells treated with the miR 145 mimic. To further understand the mechanisms underlying this phenomenon, we also mea sured by Western blotting the e pression levels of DFF45 protein isolated from LS174T cells, or normal colon cells transfected with the miR 145 mimic siRNA DFF45. In colon cancer cells, but not in normal colon cells, the miR 145 mimic or siRNA DFF45 neg