DICER-dependent biogenesis of let-7 miRNAs affects human cell response to DNA damage via targeting p21/p27

Nucleic Acids Research DICER-dependent biogenesis of let-7 miRNAs affects human cell response to DNA damage via targeting p21/p27 Bailong Liu 1 2 Min Liu 1 2 Jian Wang 1 Xiangming Zhang 1 Xiang Wang 1 Ping Wang 1 Hongyan Wang 1 Wei Li 0 Ya Wang 1 0 Department of Cancer Center, The First Hospital of Jilin University , Changchun 130021 , China 1 Department of Radiation Oncology, Emory University School of Medicine and Winship Cancer Institute , Atlanta, GA 30322 , USA 2 Department of Radiation Oncology, The First Hospital of Jilin University , Changchun 130021 , China Recently, it was reported that knockdown of DICER reduced the ATM-dependent DNA damage response and homologous recombination repair (HRR) via decreasing DICER-generated small RNAs at the damage sites. However, we found that knockdown of DICER dramatically increased cell resistance to camptothecin that induced damage required ATM to facilitate HRR. This phenotype is due to a prolonged G1/S transition via decreasing DICER-dependent biogenesis of miRNA let-7, which increased the p21Waf1/Cip1/p27Kip1 levels and resulted in decreasing the HRR efficiency. These results uncover a novel function of DICER in regulating the cell cycle through miRNA biogenesis, thus affecting cell response to DNA damage. - INTRODUCTION MicroRNAs (miRNAs) are 22 nucleotide small RNAs that regulate mRNA expression through binding to the 3 untranslated region (3 UTR) of their target mRNAs. During the miRNA biogenesis process from generating premiRNAs to mature miRNAs via digesting the loop off a pre-miRNA, DICER plays a major role (1,2) and a DICER-independent but AGO2-dependent pathway also contributes to the maturation of a few miRNAs, such as miR-451 (35). Most human genes are regulated by at least one miRNA (6). Considering that 1% of the human genome is devoted to miRNA genes (7), and each miRNA may have many mRNA targets, the potential impact of altered miRNA levels is conceivably enormous. Therefore, miRNAs affect multiple functions of cells through their mRNA targets, which includes affecting cell-cycle distribution and DNA damage response in p53-dependent pathway (812) or p53-independent pathway (1319). Recently, it was reported that the DICER-generated small RNA products at the DNA double-strand break (DSB) sites (20) were required for the ATM-dependent DNA damage response (21) and efficient homologous recombination repair (HRR) (22,23), which was independent of miRNA biogenesis. Both HRR and non-homologous end-joining (NHEJ) are the two major pathways in mammalian cells to repair DNA DSBs that generated exogenously or endogenously are a severe threat to cell survival. Deficiency in either one of the DNA DSB repair pathways will result in more killing (24,25). Camptothecin (CPT)-induced DSBs at the DNA replication fork (26) require an ATM signaling response (27) to trigger the HRR pathway (28). The reports that DICER was required for the ATM-dependent DNA damage response (21) and efficient HRR (22,23) suggest that knockdown of DICER should sensitize human cells to DNA DSB inducers, particularly to CPT treatment. However, when we examined the effects of knockdown of DICER on the sensitivities of human cells to CPT, we obtained unexpected results: knockdown of DICER made the human cells more resistant to CPT. Through exploring the underlying mechanism, we discovered that knockdown of DICER affecting cell response to DNA damage is independent of the small RNAs but depends on biogenesis reduction of let-7, which results in overexpression of p21Waf1/Cip1/p27Kip1 and prolonged G1/S transition. Our results provide a new explanation for the effects of DICER on cell response to DNA damage via affecting biogenesis of let-7 to target p21/p27. MATERIALS AND METHODS Cell lines, irradiation and CPT treatment The human cell lines used in this study include immortalized fibroblast cell lines MRC5SV (wild type), 180BRM (Lig4 mutant, NHEJ deficient) and AT5BISV (ATM/, HRR deficient); tumor cells lines HeLa and M059J (DNAPKcs/, NHEJ deficient); HRR reporter cells (obtained from Dr. Jasins lab (29)) and NHEJ reporter cells (obtained from Dr. Panditas lab (30)). The mouse cell lines used in this study include mouse embryo fibroblast wildtype and Ku80/ (NHEJ deficient) cells (verified by western blot). These cell lines were grown in Dulbeccos modified Eagles medium medium supplemented with 10% Fetal Bovine Serum (FBS). The cell irradiation was performed with an X-ray machine (X-RAD 320, North Brandford, CT, USA) at 320 kV, 10 mA with a 2-mm aluminum filter and the dose rate was 2 Gy/min. The cells were treated with CPT that was obtained from NCI at different times before collecting for further detection. Construct of FLAG-DICER resistant to siRNA The plasmid encoding FLAG-hs-DICER was purchased from Addgene. Three rounds of polymerase chain reaction (PCR)-mediated mutagenesis were done on pCAGGSFlag-hs-Dicer to generate siRNA resistant Dicer1 expression plasmid (Figure 1B) with the proper primers (Supplementary Table S2), which is completely resistant to siGENOME SMARTpool siRNA to human Dicer1. The FLAG control vector was obtained by deleting the Dicer cDNA sequence pCAGGS-Flag-hs-Dicer. siRNAs and transfection The control RNA and a pool of siRNA against DICER, p21 or p27 were purchased from Dharmacon Inc. Cells were transfected with the plasmid or siRNA for 4896 h (mouse cells for 3036 h) then collected for further experiments. Cell survival assay Cell sensitivity to CPT or radiation was evaluated for loss of colony-forming ability. For radiation sensitivity, the cells were exposed to radiation with different doses, and then the cells were collected and plated for colony genesis. For CPT sensitivity, the cells were collected, plated (based on a colony genesis condition) and then treated with different concentrations of CPT at different times; the cells were changed with fresh medium for colony forming. Duplicate dishes were prepared for each dose of irradiation or CPT treatment. The cells were incubated for 1014 days and the colonies were stained with crystal violet in 100% methanol solution. Immonoblotting and antibodies used in this study The whole cell lyses were prepared as described previously (31). The antibodies against human DICER, DNAPKcs, Ku70, Lig4, XRCC4, p27/Kip1 (also against mouse p27/Kip1), CHK1, CHK2, Rad51, Rad54, Cyclin E, Cyclin A, HA, Actin, the mouse DICER and p21Waf1/Cip1 were purchased from Santa Cruz Biotechnology Inc. The antibodies against human ATM, Cyclin D1, phosphorylated CHK2 and phospho-histone H3 were purchased from Cell Signaling Technology Inc. The antibodies against autophosphorylated ATM and DNA-PKcs, XRCC2 and XRCC3 were purchased from Abcam Inc. The antibody against Artemis was purchased from Aviva System Biology Inc. The antibody against human p21Waf1/Cip1 was purchased from Thermo Scientific Inc. Foci of phosphorylated ATM HeLa cells plated in dishes containing coverslips were treated with contro (...truncated)