| ObjectiveCervical cancer(CC)remains one of the most frequent female malignancies worldwide,and it is still the second most common carcinoma and third leading cause of death from cancer among females in developing countries.Radiotherapy is one of the most effective treatments employed for CC and has shown great local control.However,patients with radiotherapy resistance are prone to suffer from local recurrence and/or distant metastasis and have poor outcomes.Therefore,it is warranted to explore manageable prognostic factors related with radiosensitivity for CCtreatment.Non-homologous end joining(NHEJ)is the most dominant pathway of DNA double strand breaks(DSBs)repair in mammalian cells,as well as the main mechanism through which cancer cells escape from death induced by ionizing radiation(IR).Recently,upregulation of IncRNA in non-homologous end joining pathway 1(LINP1)was identified in triple-negative breast cancers(TNBC)that could lead to resistance to chemoradiotherapy.However,the expression and functions of LINP1 in other types of cancer is not well understood.This study aimed to explore the expression,biological function and molecular mechanism of LINP1 in mediating radioresistance of CC.Methods1.LINP1 expression levels in tumor tissues and cell lines of CC were measured with qRT-PCR;2.Interaction between LINP1 and NHEJ proteins(Ku80 and DNA-PKcs)was defined using RNA-immunoprecipitation and RNA-pulldown analysis;3.LINP1 subcellular localization was visualized by performing RNA-FISH assay;4.Cleaved Caspase3 and cleaved PARP were measured by Western blot analysis;5.γ-H2AX foci in the cellular nucleus were visualized using immunofluoresence staining;6.Flow cytometry analysis was performed to examine cell apoptosis;7.Colony formation assay was employed to clarify the effects of LINP1 knockdown on radiation sensitivity.Results1.The expression of LINP1 was identified in both the tissue and cell lines of cervical carcinoma.Three of five specimens derived from CC patents displayed high levels of LINP1 compared with adjacent tissue,and Hela S3 cells were detected with highest levels of LINP1 expression among four different types of CC celllines.2.Complex interaction between LINP1 and Ku80.DNA-PKcs were examined in CC cells.3.IR could induce the change of subcellular localization of LINP1.The results from RNA-FISH indicated that LINP1 distributed mainly in the cytoplasm of normal cells,and could translocate to the nucleus in response to IR treatment.4.LINP1 knockdown could significantly increase radiation induced cell apoptosis.The present results of flow cytometry indicated that,compared with irradiation alone,LINP1 knockdown plus radiation combination treatment could significantly increase apoptosis rates in Hela S3 cells(P<0.05).Western blot analysis revealed that the expression levels of cleaved Caspase3 and cleaved PARP in cells with LINP1 knockdown were significantly increased after irradiation.5.LINP1 knockdown could enhance the radiosensitivity of CC cells.Results of immunofluoresence staining showed that compared with control group,LINP1 knockdown significantly increased the number of y-H2AX foci positive cells 24 hours post radiation(P<0.05).Clonogenic assay in vitro revealed the radiosensitization effects of LINP1 knockdown to Hela S3 cells using different shRNAs,and the sensitizing enhancement ratio(SER)were calculated as 1.44 and 1.37,respectively.These results suggested that LINP1 could promote the progression of DSBs repair and reduce radiotherapy efficiency.ConclusionsLINP1 facilitates DSBs repair through NHEJ pathway and may thus serve as a prognostic factor and a potential target for CC treatment. |
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