The Role Of Long Noncoding RNA Lnc-RI In Radiation-induced DNA Double-strand Breaks Repair

Purpose:IncRNA(long noncoding RNA)is termed as "noncoding RNA with more than 200 nucleotides in length".Recently,these molecules have emerged as new layer of cell biology as their diverse functions in biological processes.While,up to date,the mechanisms of most of them remain unknown.Lnc-RI(lncRNA radiation induced)is originally identified as a lncRtA which can be upregulated post 60-Co ? radiation exposure in our lab.This paper focuses on the effects of Inc-RI on the radiosensitivity of cancer cells,homologous recombination(HR)repair and regulation of RAD51 gene expression in DNA damage response(DDR).Methods:(1)In the first part,lentivirus pGLV2-U6(shRNA-lnc-RI#1,shRNA-lnc-RI#2,or shRNA-NC)infected Hela cells were exposured to 60Co ? radiation at a dose of 4Gy.Radiosensitivity of cells was measured via clone formation assay,cell cycle analysis and detection of essential components in DDR by western blot.(2)In the second part,four types of cells,including Hela,U20S,MCF-7 and L02,were transfected with specific siRNAs for lnc-RI.DNA double-strand breaks(DSBs)induced by lnc-RI knockdwon were detected by western blot,indirect immunofluorescence assay or comet assay.A well established HR assay(DR-GFP/I-Sce I report system)was employed to measure the effect of lnc-RI depletion on HR efficiency.(3)In the third part,Hela cells stably infected with lentivirus shRNA-lnc-RI#2(or shRNA-NC)were treated with cycloheximide(CHX)or actinomycin D(CHD),respectively.Then the expression of RAD51 was detected by western blot or real-time PCR.Based on a"ceRNA" hypothesis,miRNAs coregulating lnc-RI and RAD51 expression were predicted via bioinformatics analysis.According to the predicted targeting sites,luciferase reporter vectors were constructed by cloning the wild-type(or mutated-type)lnc-RI and RAD51 3'UTR sequences into pGL3M plasmid for a Dual Reporter assay.Results:(1)lnc-RI depletion increased the sensitivity of Hela cells to ionizing radiation through DNA damage repair inhibition.As the results shown,lnc-RI knockdown led to reduced clone formation,G2/M arrest delay and significant decrease of critical regulators for DSBs repair,e.g.LIG4,RAD51,PLK1,RAD50 etc.(2)Knockdown of lnc-RI expression increased DSBs dramatically.Increased ?-H2AX were found in Hela,U20S,MCF-7 and L02 cells after lnc-RI RNAi.Besides,increased y-H2AX foci were analyzed using indirect immunofluorescence assay in lnc-RI depleted Hela or U20S cells.In a further investigation,comet assay analysis shown that lnc-RI deficiency induced an obviously increased percentage of tail DNA.(3)lnc-RI depletion induced DSBs by inhibiting HR repair.lnc-RI knockdown in vitro cells inhibited the expression of essential regulators in HR pathway and resulted in a significant decrease of GFP singnaling following in I-Sce I-induced HR.(4)Lnc-RI knockdown facilitated degradation of RAD51 mRNA.The expression of RAD51 was suppressed at both protein and mRNA level after lnc-RI RNAi.We analyzed the stability of RAD51 mRNA using Hela cells stably infected with lentivirus shRNA-lnc-RI(or control cells)by blocking translation with CHD.Our results indicated that RAD51 mRNA degraded more rapidly in lnc-RI depleted cells.While analysis of RAD51 at protein level post CHX exposure shown no difference between lnc-RI knockdown and control cells(5)Lnc-RI regulated the expression of RAD51 by competitively binding to hsa-miRNA-193a-3p.In our study,hsa-miR-193a-3p was predicted as a coregulator of both lnc-RI and RAD51.According to bioinformatics analysis,a targeting site of hsa-miR-193a-3p was located in lnc-RI at 937bp-959bp region or in RAD51 mRNA 3'UTR at 704bp-724bp region(equal to 1805bp-1825bp within RAD51 mRNA sequence).Via a dual luciferase reporter assay,we identified that hsa-miR-193a-3p can directly interact with lnc-RI and RAD51 through the predicted targeting sites.Conclusion:This study illustrates the effect of lnc-RI on DSBs repair.It proved that lnc-RI is a indispensable supporter for HR repair and it regulates the stability of RAD51 mRNA by competitively binding to hsa-miR-193a-3p as a ceRNA.According to our knowledge,it's the first study to confirm that lncRNA lnc-RI regulates HR repair by targeting RAD51 via lnc-RI/hsa-miR-193a-3p/RAD51 axis.