Effects And Molecular Mechanisms Of MiR-384 Mediated Radiosensitization In NSCLC Cells

Background Lung cancer is the most morality cancer for both men and women all over the world.The efficient repair of DNA DSBs in the tumors makes the tumors radioresistant,which frequently influenced by the DNA damage response(DDR)system.Many studies revealed that deficient in DDR proteins(such as ATM,Ku70,Ku80)can siginificant sensitive tumor cells to IR because of the inefficient of DSBs repair.As a type of endogenous non-coding RNA,microRNAs(miRNA)are responsible for post-transcriptional regulation by combining with target’s 3’UTR and participate in nearly all biological processes.MiR-384 is differentially expressed in a variety of tumors.Recent studies have reported correlated with development and progression in hepatocellular carcinoma and glioma.However,its mechanism in lung cancer is still not clear.In this study,we identified that miR-384 has the ability to regulate DNA damage responses and enhance the radiotherapy sensitivity in NSCLC cells by targeting ATM,Ku70 and Ku80.The present study were tired to explore the effects of miR-384 in DNA damage response pathway,which could potentially be used to predict radiosensitivity in NSCLC.Methods Lentivirus-based infection and CRISPR/Cas9 editing technology were applied for overexpression and depletion of microRNA-384(miR-384)respectively.Clonogenic assay and γ-H2 AX immunofluorescence staining were performed to analyze the effects of miR-384 on radiosensitivity of NSCLC cells.FACS was applied to detect cell death.Luciferase reporter assay,western blotting and q RT-PCR were applied to prove ATM,Ku70 and Ku80 to be direct targets of miR-384.Luciferase reporter assay,Ch-IP and q RT-PCR were applied to demonstrate that NF-κB inhibits miR-384 directly.Result In the current study,we found that the expression of miR-384 positively correlated with radiosensitivity of NSCLC cells.Ectopic overexpression of miR-384 radiosensitized NSCLC cells by inhibiting DNA damage repair and increasing apoptosis,while knockout miR-384 led to radiation resistance with enhanced DNA damage repair and decreased apoptosis.Further investigation revealed that ATM,Ku70 and Ku80 were direct targets of miR-384.Moreover,we identified miR-384 to be a direct downstream gene of NF-κB,and NF-κB inhibits the expression of miR-384.Conclusion 1.MiR-384 was down-regulated in NSCLC cells.2.MiR-384 upregulated the radiation sensitivity of NSCLC cells.3.MiR-384 suppressed ATM through correlating with the 3’UTR of ATM.4.MiR-384 reduced NSCLC cells DNA damage response and IR induced apoptosis subsequently enhanced NSCLC readiosensitivity.This mechanism was regulated by the NF-κb pathway.