The Mechanism Study Of Extranuclear APE1 And MiRNAs In Osteosarcoma Chemotherapy Resistance

BackgroundOsteosarcoma?OS?is a frequent primary cancer of bone in adolescents.Neoadjuvant chemotherapy followed by surgery has reached a 60-70%5-year survival rate.However,the survival rate has not been further improved over the last four decades,mainly due to the resistance of patients to chemotherapy.Apurinic/Apyrimidinic endonuclease 1?APE1?is involved in various tumors chemoresistance.APE1 is primarily localized the nucleus,but nuclear/cytoplasmic co-expression patterns are common in various tumors,and markedly high cytoplasmic APE1 expression is associated with poor prognosis.We found that APE1 is mainly localized in the nucleus of osteosarcoma cells,and nuclear/cytoplasmic co-expression is associated with chemotherapy resistance and poor prognosis^[1-2].Further research found that APE1 has a mitochondrial localization sequence and that mitochondrial translocation could inhibit ROS-induced cell apoptosis.In addition to cell cytotoxicity,many chemotherapeutic drugs can also produce Reactive Oxygen Species?ROS?.As an anti-tumor role,ROS is involved tumor cell apoptosis.Angkeow found that cytoplasmic APE1 could regulate ROS-mediated apoptosis and oxidative stress by regulating Rac1 to coordinate mitochondrial NADPH oxidase^[3].The above studies suggest that cytoplasmic APE1 may be associated with chemotherapy resistance in osteosarcoma,and the molecule mechanism of APE1 regulat ROS in mitochondrial may be one important mechanism.This study was dedicated to investigate the role of cytoplasmic and mitochondrial APE1in osteosarcoma chemotherapy resistance.We committed to verify whether the changes in APE1 expression level and subcellular distribution could be an indicator for predicting osteosarcoma chemosensitivity,and to explored the molecule mechanism of APE1 regulat ROS in mitochondrial enhance drug resistance in osteosarcoma chemotherapy.In addition,we also performed whole transcriptome sequencing of CDDP-resistant and sensitive human osteosarcoma cell lines.We found that miR-767-5p was significantly under-expressed in CDDP-resistant cell lines.We confirmed that miR-767-5p overexpression could promote the sensitivity of osteosarcoma cells to radiochemotherapy and at the same time promote the migration ability of osteosarcoma cells.Which may be achieved by the following three target genes:PEG10,PLAC8,AHR.We take the increased expression of cytoplasmic APE1 in cisplatin-resistant cell lines as a clue,point on the role of mitochondrial APE1 regulating ROS,and combine the function of miR-767-5p in osteosarcoma chemosensitivity to explore the mechanism of cytoplasmic APE1 and miR-767-5p in osteosarcoma chemoresistance from a new perspective.Provide a new model for clinical treatment.Methods1.Tumor samples and clinical data of osteosarcoma patients who were treated with platinum-based drugs in Army Medical Center from 2013 to 2018 were collected.The expression of cytoplasmic APE1 was analyzed in tumor samples from 35 osteosarcoma patients using immunohistochemistry.Through evaluating the efficacy of platinum-based therapy,patients were divided into:?1?complete response?CR?:All target lesions disappeared completely for at least 4 weeks;?2?partial response?PR?:The sum of the maximal diameters of baseline lesions decreased by more than 30% for at least 4 weeks;?3?progressive disease?PD?:Increase by more than 20%or new lesions in the sum of the maximum size of the baseline lesions.To analysis the correlation between cytoplasmic APE1and the therapeutic effect of platinum-based drugs in osteosarcoma.2.Nuclear,cytoplasmic proteins were isolated from HOS,9607,U2OS,SAOS2 and U2OS?R?cells?CDDP-resistant cells?by nuclear and cytoplasmic protein extraction kits.Expression of APE1 in osteosarcoma cell lines were detected by Western blot.3.Overexpression of cytoplasmic APE1 cell models were established by transfecting cytoplasmic APE1 targeting sequence-fused mutation vector into 9607 and U2OS cells.CCK8 and flow cytometry were used to demonstrate that overexpression of cytoplasmic APE1 could decrease the sensitivity of osteosarcoma cells to cisplatin.4.Nuclear,cytoplasmic and mitochondrial proteins were isolated and purified from U2OS and U2OS?R?cells by nuclear and cytoplasmic protein extraction kit and mitochondria isolation kits.The expression of APE1 was detected by Western blot.5.APE1 deficiency cell models were established by transfecting APE1 knock-down lentivirus into U2OS,U2OS?R?and SAOS2 cells.Western blot,immunofluorescence and CCK8 assay showed that knock-down APE1 could restore the sensitivity of cisplatin-resistant osteosarcoma cell to cisplatin.6.Overexpression of mitochondrial APE1 cell models were established by transfecting targeting mitochondrial APE1 lentivirus into U2OS and SAOS2 cells.Western blot,immunofluorescence and CCK8 assay were used to demonstrate that mitochondrial APE1overexpression could decrease the sensitivity of osteosarcoma cells to cisplatin.7.Using cisplatin or hydrogen peroxide to treat APE1 deficiency and mitochondrial APE1 overexpression osteosarcoma cell models,and measured the production of ROS;N-Acetyl Cysteine?NAC?was used to reexamine the production of ROS in cells.Mitochondrial APE1 overexpression could inhibited ROS production.8.Western blot was used to determined that the high expression of mitochondrial APE1could regulate ROS production by phosphorylated Rac1?P-Rac1?protein and inhibit cell apoptosis induced by cisplatin.9.Western blot and immunohistochemistry were used to detect the expression of APE1and?-H2AX in 7 tissue samples of osteosarcoma,and analysis the relationship between the APE1 expression and the prognosis of chemotherapy.10.Detecting miRNA abnormal expression in U2OS and U2OS?R?cells by whole transcriptome gene sequencing.11.Detecting the different effects of miRNA on chemotherapy,radiotherapy and migration of osteosarcoma by CCK8,clone formation and Tranwell analysis.12.RT-PCR and Western blot were used to identify the miRNA downstream target genes by sequencing data,TargetScan.org,miRDB.org,microRNA.org.Results1.Clinical data showed that the level of cytoplasmic APE1 was negatively associated with sensitivity to combination chemotherapy of cisplatin in osteosarcoma patients.26 and 9cases of the 35 tested osteosarcoma samples showed low and high expression of cytoplasmic APE1,respectively.In the cytoplasmic APE1 high-expression group,only 33.3%of osteosarcoma patients responded to combination therapy of cisplatin,whereas the remaining66.7%of patients experienced either recurrence or new metastasis.In contrast,76.9%of osteosarcoma patients in the cytoplasmic APE1 low-expression group responded to combination therapy of cisplatin,whereas the remaining 23.1%patients did not respond.2.Nuclear and cytoplasmic APE1 expression present in all of osteosarcoma cells.The nuclear expression levels were similar in cell lines,while cytoplasmic expression levels were different.As CDDP-resistant osteosarcoma cell lines,U2OS?R?showed the highest cytoplasmic localization among all the tested osteosarcoma cell lines.APE1 was transported from the nucleus to the cytoplasm after CDDP treatment in all of the OS cells.3.CCK8 assay confirmed APE1 overexpression could decrease the sensitivity of osteosarcoma cells to cisplatin.We chose U2OS and 9607 to transfer the vector,named U2OSsh APE1 and 9607sh APE1.CCK8 assay showed that the IC50 of cisplatin for U2OSsh CON,U2OSsh APE1,9607shCON,and 9607sh APE1 was 9.885,12.879,2.43 and3.39?g/ml,respectively.4.Elevated mtAPE1 and cytoplasmic APE1?cAPE1?levels were observed in U2OS?R?cells.Interestingly,a slight decrease in nuclear APE1?n APE1?expression was observed in U2OS?R?cells,which indicated that APE1 translocated from the nucleus to mitochondria and was overexpressed in U2OS?R?cells.5.Knocking down APE1 could restore the response to cisplatin in U2OS,U2OS?R?and SAOS2 cells,especially in cisplatin-resistant cells.Apoptosis with more DNA damage was observed after cisplatin treatment in APE1-deficient cells.6.Overexpression of mitochondria APE1 could reduce cisplatin-induced apoptosis in U2OS and SAOS2 cells,and the effect was comparable to that in U2OS?R?cells.We chose U2OS and SAOS2 to transfer the vector,named U2OSmt APE1 and SAOS2mtAPE1.CCK8assay showed that the IC50 of cisplatin for U2OSscr,U2OSmtAPE1,SAOS2scr,and SAOS2mt APE1 was 3.5,7.6,4.7 and 6.3?g/ml,respectively.7.Mitochondrial APE1 overexpression significantly inhibited ROS production and apoptosis by cisplatin.ROS production was significantly high in APE1-deficient cell models.NAC could inhibit the production of ROS,thereby reducing cisplatin-induced apoptosis.Overproduction of ROS was a key factor in cisplatin-induced osteosarcoma cell apoptosis.8.High expression of mitochondrial APE1 downregulated the ROS level by phosphorylated Rca1 further promoted drug resistance in osteosarcoma chemotherapy.9.APE1 and?-H2AX expression were negatively correlated in osteosarcoma tissues,and osteosarcoma patients with high expression of APE1 were more prone to cisplatin resistance.10.Transcriptome gene sequencing results showed that compared to U2OS cells,miR-767-5p is under-expressed in U2OS?R?cell lines.The differential micro RNA expression changes more than 2 times and P Value?0.05.11.miR-767-5p could promote the sensitivity of osteosarcoma cells to chemotherapy,radiotherapy and enhance the migration ability of tumor cells.12.We compared the transcriptome sequencing results with TargetScan.org,miRDB.org,microRNA.org databases,then detected 8 downstream target genes of miR-767-5p,which were verified by PCR:The downstream target genes were:BASP1,FOXE1,AHR,PEG10,RPP25,ARHGEF5,SEMA3C,PLAC8.miR-767-5p may promote chemosensitivity,radiosensitivity and cell migration capacity of osteosarcoma through PLAC8,PEG10 and AHR respectively.Conclusions1.The cytoplasmic localization of APE1 was increase with cisplatin-resistance osteosarcoma cells,and patients with high expression of cytoplasmic APE1 were more susceptible to chemotherapy resistance.2.Mitochondrial APE1 played a major role in osteosarcoma cisplatin resistance.3.The high expression of mitochondrial APE1 could down-regulate ROS production by P-Rac1 protein,further reduce cisplatin-induced DNA damage and cell apoptosis.4.As a tumor suppressor gene,miR-767-5p was significantly under-expressed in cisplatin-resistant osteosarcoma cell lines.Overexpression of miR-767-5p promoted the sensitivity of osteosarcoma cells to chemotherapy and radiotherapy,as well as the migration ability of tumor cells.For the osteosarcoma patients with high expression of miR-767-5p,radiotherapy and chemotherapy may increase the risk of distant metastasis,which is helpful to guide clinical treatment and regular follow-up.