MDA-MB-231 cells. Effects of miR-421 are on the S-phase checkpoint and radiation CH5424802 sensitivity of ATM dependent Dependent. A single microRNAis predicted 200 targets for modulating the expression of proteins. Tofurther determinewhether the effects of miR-421 control points The cell cycle and radiosensitivity of ATM are mediated, we used a morpholino antisense oligonucleotide to the recognition sequence of ATM Block 3 UTR. Treatment of cells with ATM HeLa/miR-421 3 UTR target site-specific AMO led to abrogationofmiR421 ofATMexpression regulation mediated, as shown by both Western blot and ELISA. This effect was not observed when cells were treated with controlled Clouded the AMO. Following IR, ATM processing AMO has also signed up Born erh Ht pS966-SMC1 in HeLa/miR-421 cells.
Blocking with AMO ATM also reflect the position of the contr The S-phase of the cell cycle and radiosensitivity theHeLa/miR-421 survive as demonstrated by an examination of radioresistant DNA synthesis and clonogenic analysis. Taken together, these experiments indicate that ATM AMO contr effect of miR-421 PF-04217903 on the position The S phase of cell cycle and radiation sensitivity mediated by the ATM. N-Myc transcription factor regulates miR-421 expression above. MiR-421 is located on human chromosome Xq13 in intergenically. It is interesting that another microRNA, miR-374b, is located only 85 bp proximal of miR-421 and forms a microRNA cluster is entered Born from a single promoter. The function of miR-374b is still unknown.
The factors that influence the transcription of miR-421 expression to determine k To nnten, we performed an in silico analysis of Figure 3 ATM mediates the effect of miR-421 on cell cycle S-phase checkpoint and radiation sensitivity. Schematic model of working ATM 3 TR was targeted by an antisense-OMA. AMO-ATM is designed to correspond to the miR-421 recognition site 3 ATM of TR and specifically block the downregulation of ATM by preventing the binding of mature miR-421. Immunoblot of ATM expression in HeLa cells / Emergency and HeLa/miR-421 cells with or without ATM AMO treated for 5 days. The times change In ATM expression is shown below the immunoblot. ELISA was used to determine the concentration of ATM. Immunoblot pSMC1 in HeLa cells / Emergency and HeLa/miR-421 with AMO AMO Schram or ATM for 5 days with 10 Gy IR followed treated.
The Ver Indicated change in the time pSMC1 level below the immunoblot. Note the increase in pSMC1 HeLa/miR-421 cells with AMO-treated ATM. The analysis of the cell cycle S phase checkpoint after treatment with AMO. HeLa / Emergency and HeLa/miR-421 cells were treated with AMO AMO Schram ATM or treated for 5 days and exposed to increasing doses of radiation. DNA synthesis was monitored by BrdU incorporation and analyzed by FC. The percentage of BrdU + cells in the S phase at the starting point has been set arbitrarily to 50%, and all other data were normalized to this point. This site is repr Sentative for three independent Independent experiments. The arrow indicates that AMO ATM processing stores the default settings HeLa/miR-421 cells. Colony survival fraction of the exposure to the AMO.
HeLa / Emergency HeLa/miR-421 cells with AMO and AMO-Scram or ATM were treated for 5 days, and 500 cells were coated three times, cells were irradiated with increasing doses of radiation and surviving colonies were collected after 2 week achieved. The surviving fraction at each radiation dose was normalized to that of the non-irradiated control. The arrow indicates that the OMA has HeLa/miR-421 cell radiosensitivity saved. Fig. 4th miR-421 is upregulated by N-Myc overexpression in HeLa cells. Chromosomal location miR-374b/miR-421 cluster on chromosome Xq13, which is in the same promoter. The promoter region, an E-box was in the luciferase construct pGL3-Basic to create cloned pGL3-PR421 and entered No transcription of firefly luciferase. The luciferase activity t of