APE1-MicroRNAs Interaction In Osteosarcoma And Its Effect On Radiosensitivity

BackgroundRadiation therapy is one of the most commonly used clinical treatments forosteosarcoma. The resistance of osteosarcoma to radiotherapy remains an important elementinterfering with therapeutic effect and prognosis. Apurinic/apyrimidinic endonuclease/redoxeffector factor (APE1), with dual functions of both DNA repair and redox activity, is the keygene in regulating radioresistance in osteosarcoma. Previous studies have shown thatknocking down APE1expression by siRNA significantly sensitized human osteosarcomacells to radiation. Although the essential function of APE1has been confirmed, knowledge ofits regulation or interactome in mediating radioresistance is still scanty. Through suppressinggene expression by binding to the3’-untranslated region (3’-UTR) of target genes,microRNAs (miRNAs) play a large range of biological roles in tumorigenesis, apoptosis andstress reactions. We postulated that miRNAs may play important roles in APE1regulatorynetwork and interactome. It might also provide a plausible explanation to the APE1-dependent radioresistance in osteosarcoma. Additionally, as a class of endogenous singlestranded non-coding RNA, miRNAs has been considered to be a more effective andpromising target to increase radiosensitivity of cancer. Recently, studies have reported somemiRNAs interaction with many important tumor-associated genes such as p53. However, todate, little report concerning the interaction between APE1and miRNAs are available in thecurrent scientific literature.ObjectiveTo identify the interaction network between APE1and miRNAs in osteosarcoma andinvestigate the APE1-dependent modulation of radiation sensitivity by miRNAs in humanosteosarcoma.Materials and Methods1. Microarray and qRT-PCR were used to confirm the change of microRNAs (miRNAs) in APE1-knockdown osteosarcoma HOS cells followed by analysis with comprehensivebioinformatics-based analysis.2. APE1-transcription factors-miRNAs network was predicted and identified bybioinformatics-based analysis, EMSA, RNAi and qRT-PCR in osteosarcoma HOS cells.3. Then we used luciferase report gene vectors, Western blot, AP site activity assay andEMSA to identify miRNAs are the targeted regulation factor of APE1. At last, we generatedmiRNA overexpression lentiviral vector and used cell proliferation assay, cell apoptosis,immunohistochemisty, Western blot and qRT-PCR to investigate the miRNAs increaseosteosarcoma radiosensitivity by targeting APE1in vitro and vivo.Results1. Both microarray and qRT-PCR demonstrated that13miRNAs were significantlychanged (>2-fold) in APE1-knockdown HOS cells; seven of them (miR-451, miR-1290,miR-765, miR-483-5p, miR-513a-5p, miR-129-5p and miR-31) were up-regulated and theother six (miR-29b, miR-197, let-7b, miR-324-5p, let-7i and miR-484) were down-regulated.Furthermore, pathway analysis showed that these miRNAs and their target genes affected bythe expression of APE1were involved in pathways relating to cell signaling (such as TGF-β,Wnt, MAPK and the p53signaling pathway), cell survival, proliferation, adhesion andcancers.2. APE1could regulate both miRNAs and transcription factors. There were putativebinding sites of NF-κB, p53, HIF-1α, AP-1, PEBP2, ATF, NF-Y, Pax-2, CREB and c-Myb inthe promoters of several downregulated miRNAs, indicating that APE1may regulatemiRNAs via transcription factors. The DNA binding activity of NF-κB was inhibited afterAPE1-knockdown in osteosarcoma cells. And the expressions of let-7b and let-7i weredecreased by NF-κB siRNA.3. MiR-513a-5p and miR-765significantly suppressed APE1protein expression bybinding to the3’UTR of APE1mRNA. Overexpression of miR-513a-5p could inhibit theactivity of APE1in AP site repair and the DNA binding activity of NF-κB、p53and AP-1.Flowing with ionizing radiation, overexpression of miR-513a-5p in osteosarcoma couldinhibit cells proliferation and promote cell apoptosis in vitro and vivo. Besides, the expressionof Bcl-2was decreased in osteosarcoma HOS cells. Conclusion1. Our data provide evidence that APE1appears to have a direct influence on globalmiRNAs expression. According to the bioinformatics-based analysis, APE1may regulatemiRNAs to affect the expression of target genes. These interactions may be involved incancers, cell survival, cells proliferation, adhesion and many cell signal pathways, especiallywith regard to the tumorigenesis, development, invasion and metastasis of osteosarcoma.2. The bioinformatics-based analysis has showed the network associated with APE1,transcription factors and miRNAs. APE1may regulate the expression of let-7b and let-7i viatranscription factor NF-κB.3. APE1is the target gene of miR-513a-5p and miR-765. MiR-513a-5p can suppressDNA damage repair activity and redox activity of APE1, and sensitize human osteosarcomato radiation through suppressing cells proliferation and promoting apoptosis in vitro and vivo.