DICER-dependent Let-7 MiRNAs Affects Tumor Cell Response To Chemo-Radiotherapy

Objective: DICER is a key enzyme responsible for generating mature mi RNA. It’s has close relationship between cancer. This study will identify its role in chemo-radiotherapy induced DNA damage response and reveal the mechanism.Methods: Human MRC5SV(normal fibroblast cell) and tumor cells:He La,M059J(ATM low expression,DNAPKcs-/-, NHEJ deficient), ATM-/-(HRR deficient), 180BRM(Ligase 4 mutant, NHEJ deficient)were used for in vitro experiments. The above cells were incubated with different concentrations of CPT or X-ray irradiation after knockdown of DICER. Colony-forming ability was used to analyze the change of cell sensitivity to CPT or X-ray. Western Blot was used to detect the expression of key proteins directly involved in HRR(phosphorylated ATM, Rad51, Rad54, XRCC2 and XRCC3) and NHEJ(phosphorylated DNAPKcs, Artemis, Ku70, Lig4 and XRCC4) in human cells(MRC5SV and He La). The levels of phosphorylated ATM and the extent of ATM foci formation in MRC5 SV and He La cells with DICER knocked down, at different times after exposure to IR(2 Gy) were further detected. Micro RNA array was used to compare the mi RNA expression profiles of MRC5 SV with or without knockdown of DICER. Flow cytometry was used to detect the cell cycle distribution when cells treated with Ct RNA or DICER. Western Blot was used to detect the expression of key proteins in cell cycle regulation such as Cyclin E、Cyclin A、Cyclin D1 and p21 Waf1 /Cip1 /p27Kip1. Real-Time PCR approach was used to detect the level of p21/p27 m RNA. Relative luciferase activities were detected to find out whether there were binding sites between Let-7 and p21 and p27 3’UTR. U2 OS HRR reporter cells and 293 FT NHEJ reporter cells were used to test HRR and NHEJ efficiency when DICER knock down.Results:(1) Knockdown of DICER causes human cells to become resistant to CPT, a DNA damage inducer We examined the effects of the knockdown DICER on the sensitivities of human normal(MRC5SV) and tumor(He La) cells to CPT. Unexpectedly, knockdown of DICER made the human cells resistant to CPT. To confirm the results we did a rescue experiment by detecting the effects of expressing the si RNA resistant FLAG-DICER on cell sensitivity to CPT when the endogenous DICER was knocked down. The results showed that expression of the si RNA resistant FLAG-DICER when the endogenous DICER was knocked down, the resistance of the cells to CPT was abolished. These results strongly support that deficient DICER is the major reason for the cell resistance to CPT treatment. These surprising results also suggest that knockdown of DICER may not directly affect ATM signaling and the HRR pathway since the ATM pathway is required to protect cells from CPT-induced killing. Additionally, knockdown of DICER made other cell lines such as ATM-/-, 180 BRM, M059 J resistant to CPT.(2) Knockdown of DICER prolongs the G1/S transition through up-regulating p21Waf1/Cip1 and p27/Kip1 We noted that knockdown of DICER transiently reduced the cell number, which supports that DICER is required for cell proliferation. These results led us to further examine the effects of knockdown of DICER on cell-cycle progress since G1 cells are much more resistant to CPT treatment. Knockdown of DICER did induce a G1/S block in CPT-treated cells.We then examined the expression levels of p21Waf1/Cip1, p27Kip1, Cyclin A, Cycin E and Cyclin D1, the proteins that are involved in the G1/S transition. Knockdown of DICER changed the levels of p21, p27, Cyclin E and A, but not the level of Cyclin D1. Knockdown of p21 and p27 reduced the levels of Cyclin A and E, the consistency of the Cyclin E and A levels depend on the presence of p21/p27. More importantly, knockdown of p21 and p27 reversed the extended G1/S transition induced by knockdown of DICER in CPT-treated cells, indicating that the delay of G1/S transition in CPT-treated cells induced by knockdown of DICER is due to an increase in p21 and p27 expression.(3) Knockdown of DICER upregulated p21waf1/Cip1 and p27/Kip1 is due to the reduction of biogenesis of Let-7 Knockdown of DICER. did not change the m RNA levels of p21 and p27, indicating that knockdown of DICER up-regulates p21 and p27 at a post transcriptional stage. These results also suggest that the changes in the p21/p27 level might be due to mi RNA biogenesis. Therefore, we compared the mi RNA expression profiles of human fibroblast cells(MRC5SV) with or without knockdown of DICER. Notably, the expression-reduced mi RNAs in DICER knocked down MRC5 SV cells include the Let-7 family. To determine whether Let-7 could target p21 and p27, we measured the levels of p21 and p27 in Let-7 mimic transfected cells. The p21 and p27 levels were dramatically reduced in the cells treated with Let-7b mimics, suggesting that p21 and p27 are targets of Let-7. We then searched for and found the potential binding sites of Let-7 in the 3’UTR of p21 and p27. A luciferase reporter assay showed no change in activity when cells were transfected with a control RNA, but luciferase activity was significantly inhibited when cells were transfected with Let-7b mimics. This inhibition of luciferase activity was reversed when the key Let-7b binding site at the 3’UTR of p21 or p27 mutated. These results confirm that human p21 and p27 are the direct targets of Let-7b. Aside from Let-7b, other members of the hsa-let-7 family could also target p21, p27 or both, suggesting that the Let-7 family regulates the cell cycle through targeting p21/p27. To test this hypothesis, we examined the G1/S transition in cells treated with Let-7b mimics while DICER was knocked down. The results showed that overexpression of Let-7b abolished the extended G1/S transition induced by knockdown of DICER.(4) Knockdown of DICER didn’t change the radiosensitivity of cells. We examined the effects of knockdown of DICER on sensitivity to ionizing radiation(IR) in 180 BRM and M059J(the NHEJ deficient cell lines), MRC5 SV, He La, Ku80+/+(wild type) and Ku80-/-(NHEJ deficient mouse cell line). Knockdown of DICER does not affect human and mouse cell sensitivity to IR-induced killing. The cell surviving fraction didn’t change of M059 J and 180 BRM cells, after exposure to different doses(1Gy/ 2Gy to M059 J, 2Gy/4Gy to 180 BRM, 4Gy to MRC5 SV and He La, 4Gy to Ku80+/+, 1Gy to Ku80-/-). To explain the CPT resistance in DICER knockdowns, we examined the levels of the key proteins directly involved in HRR(phosphorylated ATM, Rad51, Rad54, XRCC2 and XRCC3) and NHEJ(phosphorylated DNAPKcs, Artemis, Ku70, Lig4 and XRCC4) in human cells(MRC5SV and He La) by knocking down DICER 1 h after exposure to 5Gy of IR. Knockdown of DICER did not change the levels of the examined repair proteins, which rules out alterations in expression of DNA DSB repair proteins as a cause of resistance to CPT when DICER is knocked down. To further verify this conclusion, we examined the levels of phosphorylated ATM and the extent of ATM foci formation in MRC5 SV and He La cells with DICER knocked down, at different times after exposure to IR(2Gy). Knockdown of DICER did not change the levels of phosphorylated ATM or ATM foci formation in these cells, which eliminates the possibility that the CPT-resistant phenotype in DICER knockdowns is because of any change in levels or activities of ATM protein.(5) Prolonged G1/S transition results in decreased HRR and cellular resistance to CPT treatment We examined the effects of overexpressing Let-7b mimics or inhibiting p21/p27 on the sensitivity of the DICER knockdowns to CPT. Either overexpressing Let-7b mimics or inhibiting p21 and p27 in the DICER knockdowns abolished the resistance of the cells to CPT. HR reporter test showed that HR rate decreased when DICER knock down. More importantly, the inhibition of HRR efficiency caused by knockdown of DICER was completely recovered by transfecting the cells with either si RNA against p21/p27 or with Let-7b mimics. NHEJ reporter cell’s result also support our hypotheis. Our results provide a new explanation for DICER in regulating the G1/S transition is the major reason for cellular resistance to CPT, which indirectly affects cell sensitivity and response to DNA damage.Conclusion:(1) A prolonged G1/S transition via decreasing DICER-dependent biogenesis of mi RNA Let-7, which increased the p21Waf1/Cip1/p27Kip1 levels and resulted in decreasing the HRR efficiency is pivotal mechanism of increased cell resistance to camptothecin.(2) DICER is low expressed in tumor cells. Tumor cells exert resistance to S-stage specific chemotherapeutic drug by the above mechanism, uncovering a novel mechanism of topoisomerase I resistance.(3) Knockdown of DICER didn’t change the radiosensitivity of cells. Summarily, 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 mi RNA 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 mi RNA biogenesis, thus affecting cell response to DNA damage.