The Anticancer Effects Of RNaseH1 In Telomerase-negative Osteosarcoma:Inhibits The Proliferation And Increases The Radiosensitivity

Objective Osteosarcoma is the most common primary malignancy in bone which mainly afflicts children and adolescents.In recent years,the improvement of treatment strategies has seen an increase in the 5-year survival rate of young patients with osteosarcoma,but the overall treatment outcome is not yet satisfactory.Radiotherapy is an important adjuvant therapy for osteosarcoma.However,the efficacy of radiotherapy is limited by radioresistance.Therefore,it’s important to find new methods to inhibit the progression and reduce the radiation resistance in osteosarcoma cells.The nucleic acid endonuclease RNase H1 has been shown to have an inhibitory effect on telomere lengthening in cells using the alternative lengthening of telomeres(ALT)mechanism to maintain the telomere length.Currently,there are no relevant reports on RNase H1 affecting tumor proliferation,cell cycle distribution,radiosensitivity and other phenotypes in China and abroad.In this study,RNase H1 overexpression stable transfected cell lines from telomerase negative cells and telomerase positive cells were constructed,and RNase H1 stable knockdown cell lines of telomerase negative osteosarcoma cells were constructed as well,to investigate the effects of RNase H1 on cell proliferation,cell cycle and radiosensitivity,and to preliminarily explore its mechanism.This study could find new targets for the inhibition and radiosensitization of telomerase negative osteosarcoma and to provide a basis for improving the treatment of osteosarcoma.Methods1.Telomerase negative cell U2 OS and telomerase positive cell 143 B were transfected with lentivirus to construct stable transfected cell lines overexpressing RNase H1.Similarly,the cell line of U2 OS cell with stable RNase H1 knockdown was constructed.Cell proliferation of transfected cells was determined using CCK-8 assay,colony formation assay and immunohistochemistry assay.The cell cycle was determined using flow cytometry respectively.Xenograft models were constructed to detect the effect of RNase H1 on the growth of subcutaneous xenograft.The bioinformatics technique was applied to analyze the relationship between RNase H1 expression and overall survival(OS).The Chi-square test was performed to analyze the association between clinicopathological parameters of osteosarcoma patients and RNase H1 expression.2.A radioresistant cell line model of telomerase negative osteosarcoma cells was established and their RNase H1 expression was detected.Transfected telomerase negative osteosarcoma cells were treated with radiation and their cell radiosensitivity was detected by clone formation assay,cell cycle and apoptosis were detected by flow cytometry,and DNA damage was detected by immunofluorescence assay.3.The relative length of telomeres was detected by quantitative real-time PCR,and the co-location of DNA damage and telomeres was detected by fluorescence in situ hybridization(FISH).The expression levels of proteins related to homologous recombination and ATM signaling pathway in the transfected telomerase negative osteosarcoma cells were examined by Western blotting.And the cell cycle and proliferation ability of telomerase negative osteosarcoma cells overexpressing RNase H1 were examined after treatment with ATM inhibitor KU-55933.Results1.Overexpression of RNase H1 in U2 OS cells significantly inhibited the proliferative and induced G1 cell cycle arrest(P < 0.05),whereas RNase H1 knockdown produced the opposite results.On the other hand,overexpression of RNase H1 did not have an inhibitory effect on telomerase positive 143 B cells.Animal experiment showed that enforced expression of RNase H1 hindered tumor growth.High RNase H1 levels were correlated with prolonged OS of RNase H1 patients.However,RNase H1 expression levels were not significantly associated with the clinicopathological characteristics.2.The level of RNase H1 was significantly lower in the U2 OS radioresistant cell model compared to parental U2 OS cells.And overexpression of RNase H1 leads to enhanced radiosensitivity,increased radiation-induced cell cycle G2 phase block,and increased levels of DNA damage in U2 OS cells(P < 0.05),whereas RNase H1 knockdown produced the opposite results.The radiosensitivity of telomerase positive 143 B cells was unaffected by the overexpression of RNase H1.3.Overexpression of RNase H1 in U2 OS cells significantly shortened the telomere length,decreased the expression of RAD51,BRCA1 and BRCA2,increased phosphorylation levels of ATM and Chk2,and upregulated levels of p53 and p21,whereas RNase H1 knockdown produced the opposite results.Meanwhile,overexpressing RNase H1 in U2 OS cells resulted in increased DNA damage on the telomeres.ATM inhibitor KU-55933 rescued the reduced cell proliferation capacity and cell cycle arrest induced by overexpression of RNase H1 in U2 OS cells.Conclusion RNase H1 induces suppressed proliferative and enhanced radiosensitivity in telomerase-negative osteosarcoma cells by shortening telomeres and activating ATM pathway,which attributed to inhibition of homologous recombination and increased DNA damage on telomeres.