miR-145 regulates non homologus end joning by targeting DNA-PKC's

Abstract

The DNA double-strand breaks (DSB) are the most lethal lesions which are caused by ionizing radiation and radio-mimetic chemicals, but can also be caused by mechanical stress on chromosomes. The cells have many repair pathways for repairing the damage, the two main pathways are homologous recombination (HR) and non homologous end joining (NHEJ). NHEJ pathway is the one pathway which is used by the cells to repair the damaged DNA at any stage of the cell because unlike HR, NHEJ doesn’t require homologous sister chromatid for repairing the damaged DNA. DNAPKC's is an important paralog of NHEJ repair mechanism. MicroRNAs (miRNAs) are short non-coding RNAs, which are viewed as fundamental regulators of cell function. MiRNAs are approximately 20–25 nucleotides (nt) long which are involved in regulation of cellular mechanism at translational level by mRNA decay or translation repression. MiRNAs regulates various proteins in cell which are involved in various mechanisms it also regulates some proteins which are involved in DNA repair. During the cell stress condition, cells express various miRNA to regulate protein expression and vary the repair mechanism. MiR-145 is a tumor suppressor micro RNA and its expression will be suppressed in many cancer cell types. MiR-145 plays major role in NHEJ (Non Homologous End joining mechanism) by targeting most fundamental protein DNAPKC's. The 3' UTR of DNAPKC's protein has partial sequence complimentary to miR-145 miRNA. Over-expression of miR-145 results in depletion of DNAPKC's which results in suppression of NHEJ activity in cells and DNAPKC's depleted cells show hypersensitivity to IR radiation. We show that the cell expresses miR-145 during stress condition and regulates the cells. This study also provides a new molecular mechanism for controlling the DNA damage repair by micro RNA. |DNA-PKcs 단백질은 이중나선절단에 의한 DNA손상 자극시 비상동말단결합 활성에 중요한 역할을 하는 단백질중의 하나이다. DNA-PKcs 단백질의 기능 소실은 DNA손상복구 결함을 야기함으로써 유전체 불안정성을 유도한다. 그러나 이런 DNA-PKcs단백질 자체의 발현 조절기전 연구는 미흡한 실정이다. 본 연구를 통해 DNA-PKcs 발현을 조절하는 microRNA로 miR-145를 동정하였다. miR-145는 DNA-PKcs의 3`UTR에 결합하여 DNA-PKcs발현을 억제하고 이중나선절단에 따른 DNA-PKcs 활성을 저해함을 comet assay와 γ-H2AX foci 형성을 통해 확인하였다. 또한 miR-145 저해제와 DNA-PKcs cDNA 과발현을 통해 miR-145가 DNA-PKcs의 발현과 활성을 조절함을 재확인하였다. 이상의 결과를 토대로 miR-145가 DNA-PKcs 단백질 발현을 조절함으로써 비상동말단 결합에 관여함을 증명하였다. 또한 본 연구는 DNA 손상복구를 조절하는 새로운 분자적 기전을 제시한다.