| Background and objectiveRadiation therapy plays an important role in the multidisciplinary managelent of breast cancer.It not only provides local control and reduces relapse of tumor,but also increases patients’ long-term survival and decreases their mortality.Radioresistance is a major challenge and an important reason for the failure of breast cancer radiotherapy.Therefore,it is necessary to investigate the molecular mechanisms of radioresistance and develop effective radiosensitizers.LncRNAs are a large class of non-protein-coding transcripts with a length>200 bases.Recently,lncRNAs were suggested to have an important role in various biological processes related to cancer,including proliferation,genomic stability,invasion,and metastasis.For instance,IncRNA HOTAIR interacts with Polycomb Repressive Complex 2 to reprogram chromatin and thus promotes breast cancer invasion and metastasis.In addition,IncRNA NBAT-1 controls neuroblastoma progression by regulating cell proliferation and neuronal differentiation.Furthermore,IncRNA MEG3 is considered to be a tumor suppressor,since it often shows low expression in cancer and its overexpression inhibits the proliferation and metastasis of various tumors.Therefore,IncRNAs represent a wide range of potential targets for cancer treatment.However,the role of IncRNAs in radioresistance remains largely unknown.Recent studies have shown that miRNAs are able to interact with lncRNAs to regulate IncRNAs levels.For example,miR-211 inhibits lncRNA loc285194 expression;miR-17-3p directly targets IncRNA GCASPC and decreases its half-time;and miR-193b suppresses IncRNA MIR3IHG expression.MiR-200c is a well-studied miRNA that is involved in sternness,epithelial-mesenchymal transition,chemoresistance,radioresistance,and invasion,metastasis of various cancer cells.It has been reported that miR-200c sensitizes cancer cells to radiation by targeting TBK1 and VEGF-VEGFR2.Our previous study showed that miR-200c inhibits radiation-induced autophagy and scnsitizes breast cancer cells to radiation.These findings suggest a tumor radiosensitizer role for miR-200c.Given that miR-200c enhances the radiosensitivity of breast cancer cells and its contribution on IncRNA expression has not been assessed,we wonder whether miR-200c could affected IncRNA expression,thereby sensitizing breast cancer cells to radiation.In this study,we used microarray analysis to delineate the alterations of IncRNA expression induced by miR-200c.We identified a IncRNA LINC02582 was required for radioresistance.Mechanistically,LINC02582 interacts with USP7 to deubiquitinate and stabilize CHK1,thereby promoting radioresistance,while miR-200c binds directly to LINC02582 and acts as a negative regulator of LINC02582.Thus,our study suggests IncRNA LINC02582 is a potential target for the radiotherapy of breast cancer.Methods1.Real-time RT－PCR analysisTotal RNA extracted from breast cancer tissues or cells using TRIzol Reagent(Invitrogen),following the manufacturer’s instructions.For miRNA quantification,cDNA was generated using PrimeScriptrtRT reagent Kit(Takara,Dalian,China).For IncRNA and mRNA,cDNA was generated using PrimeScriptRT Master Mix(Takara,Dalian,China);Real-time PCR was performed using TB GreenT?Premix Ex TaqTM(Takara,Dalian,China).2.Oligonucleotides,siRNA and plasmid transfectionThe miR-200c mimic,inhibitors,USP7 siRNA and CHK1 siRNA were purchased from RiBoBio(Guangzhou,China).The USP7 and CHK1 expression plasmid were from FulenGen(Guangzhou,China).Transfection was performed using Lipofectamine3000 reagent(Invitrogen)according to the manufacture’s protocol.After 48 h of transfection,the cells were used for functional assays.For efficient inhibition of the miR-200bc/429 cluster,equivalent amounts of miR-200c and miR-429 inhibitors were combined.3.Lentiviral construction and transductionThe lentiviral vectors encoding full-length human LINC02582 gene sequence and lentiviral vectors encoding LINC02582 silencing short hairpin RNA were constructed by Genechem Company(Shanghai,China).To generate clones stably overexpressing LINC02582,MCF-7 and BT474 cells were infected with a lentiviral vector encoding LINC02582.To knocked down LINC02582,MDA-MB-231 and BT549 cells were infected with lentiviral vectors encoding LINC02582 silencing short hairpin RNA.Stably clones were selected for 2 weeks using puromycin and LINC02582 expression was detected by qRT-PCR.4.Clonogenic survival assayEqual numbers of cells were seeded in six-well culture plates(pretreated with siRNA or oligonucleotide,plasmid transfection for 48 h)in triplicate and exposed to the indicated doses of irradiation using 6-MV X-rays from linear accelerators(Varian2300EX,Varian,Palo Alto,CA)at a dose rate of 5Gy/min.After incubation at 37℃ for 14 to 21 days,the plates were fixed with 100%methanol and then stained with 1%crystal violet.Colonies containing>50 cells were counted by microscopic inspection.The surviving fraction(SF)was calculated.A multitarget single-hit model was fitted to data for generating survival curves using the following formula:SF=1-(1-e-D/D0)N.5.Immunofluorescence assayImmunofluorescence assay was performed by counting the yH2AX foci at 24 h after irradiation.Breast cancer cells were seeded in 24-well culture plates and exposed to 6Gy irradiation.After 24 h,the cells were fixed in 4%paraformaldehyde and permeabilized in 0.1%Triton X-100(Sigma).Then,the cells were blocked in 1%goat serum and incubated with primary ΥH2AX antibody(Cell Signaling Technology),Subsequently,the primary antibody was washed off and the cells were incubated with a secondary antibody conjugated to fluorescein isothiocyanate(FITC).Finally,the cells were then incubated with DAPI to stain the nuclei.ΥH2AX foci was visualized with a fluorescence microscope(Olympus BX51,Tokyo,Japan).The ΥH2AX foci was count at least 50 cells for each group.6.Antibodies and Western blot analysisCell pellets were lysed with RIPA buffer(Cell Signaling Technology)containing proteinase and phosphatase inhibitor cocktails(Sigma-Aldrich),electrophoresed and transferred to a nitrocellulose melbrane(Bio-Rad).Membranes were blocked with]5%BSA for 1 h before incubation with a primary antibody overnight at 4℃,followed by a HRP-coupled secondary antibody and developed with ECL Western blotting substrate(Pierce,Rockford,IL,USA).7.Microarrays and computational analysisMiR一200c一overexpressing MDA-MB-231 cells and control group MDA-MB-231 cells were selected for microarray analysis.Briefly,total cellular RNA was extracted with TRIzol Reagent(Invitrogen,Carlsbad,CA)and purified with a RNeasy Mini Kit(Qiagen,Valencia,CA).Then,complementary DNA was synthesized and labeled before microarray hybridization(Arraystar,Rockville,MD).The slides were washed and then scanned using an Agilent DNA Microarray Scanner(Agilent,Santa Clara,CA).Data were extracted using Agilent Feature Extraction software and further analyzed using Agilent GeneSpring GX,version 12.1,software.Volcano plot filtering(fold change>2.0;P<.05)between miR-200c-overexpressing MDA-MB-231 cells and control group MDA-MB-231 cells was performed to identify significantly different expressions of incRNAs and mRNAs.Hierarchical clustering was performed with Cluster Treeview software(Stanford,CA).8.Bioinformatic analysis for predicting miR-200c target IncRNAWe identified potential targets of miR-200c based on three publicly available databases(TargetScan and miRBase)and then intersected the results from these databases.9.Lueiferase assayLuciferase activities were performed using the Luciferasc Assay Kit(Promega),according to the manufacturer’s instructions.Briefly,cells were first transfected with corresponding plasmids.Then,the cells were harvested and lysed for luciferase assay 48 h after transfection.Firefly luciferase was used for normalization.10.Tumor radiosensitivity studyAll the animal experiments were carried out in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University(Guangzhou,People’s Republic of China).Suspensions of 1×107/0.2 ml LINC02582 deleted or control MDA-MB-231 cells were inoculated subcutaneously into the right hind limbs of 4-week-old female BALB/c-nu/nu nude mice.Mice were randomly assigned to no irradiation or irradiation groups consisting of 4 mice per group.Irradiation treatment was initiated when tumors grew to approximately 150 mm3.Mice in the irradiation groups were irradiated with 2 Gy every other day for five treatments.Tumor sizes were calculated every two days using the formula:(length×width2)/2.11.RNA pull downRNA pull-down and deletion mapping were performed as previously described.23 Briefly,biotinylated LINC02582 or antisense RNA were mixed and incubated with MDA-MB-231 cell protein extracts,which were then targeted with streptavidin beads and washed.The eluted proteins were detected by Western blot analysis.Specific bands were excised and analyzed by mass spectrometry.12.RNA ImmunoprecipitationRNA immunoprecipitation(RIP)experiments were carried out according to the manufacturer’s protocol of Magna RIP RNA-Binding Protein Immunoprecipitation Kit(Millipore,Bedford,MA).The coprecipitated RNAs were analyzed by reverse-transcription(RT)-PCR.13.In vivo ubiquitination assayFor the in vivo ubiquitination assay,transfected MCF-7 cells were treated with 10μM proteasome inhibitor MG 132(Selleck,Shanghai,China)for 16 h.The cell extracts were subjected to immunoprecipitation and western blot analysis with the indicated antibodies.14.Detection of the expression of CHK1 in breast cancer tissueImmunohistochemical(IHC)staining was performed to detected the expression of CHK1 in breast cancer tissue.15.Detection of the expression of miR-200c and LINC02582 in breast cancer tissueFor detection of the expression of miR-200c and LINC02582 in breast cancer tissue,in situ hybridization(ISH)was perforned.16.Statistical analysisAll data are expressed as the mean ± SDs and statistical analysis of data was performed using the Student’s t test or analysis of variance(ANOVA).Spearman rank correlation was used to analyze correlations between miR-200c and LINC02582.Correlation between CHK1 and miR-200c or LINC02582 was analyzed using x2 test.Relapse-free survival curves were plotted using the Kaplan-Meier method and compared with the log-rank test.All analyses were completed using SPSS 19.0 software(SPSS,Chicago,IL,USA)and p-values<0.05 were considered statistically significant.Results1.The expression level of miR-200c of breast cancer cellsFirst,the expression level of miR-200c was determined in several breast cancer cell lines.the expression of miR-200c was lower in basal phenotype breast cancer cells(MDA-MB-231 and BT549)compared with luminal phenotype breast cancer cells(MCF-7 and BT474).Importantly,compare with high expression of miR-200c cell lines(MCF-7 and BT474),nmiR-200c low expressed cell lines(MDA-MB-231 and BT549)have higher clonogenic survival ability after irradiation.These results imply that miR-200c levels correlated with radiosensitivity of breast cancer cell.2.MiR-200c sensitizes breast cancer cells to radiationKnockdown of miR-200c increased the survival fraction of MCF-7 and BT474 cells after irradiation.Conversely,overexpression of miR-200c reduced the survival fraction of MDA-MB-231 and BT549 cells subjected to irradiation.Irradiation caused double-stranded DNA breaks(DSBs)with formation of yH2AX foci,which indicate delayed repair and correlated with radiosensitivity.Indeed,miR-200c overexpression led to persistence of yH2AX loci in MDA-MB-231 cells at 24 h after irradiation.These results confirm that miR-200c sensitize breast cancer cells to radiation.3.MiR-200c inhibits epithelial-mesenchymal transition of breast cancer cellsIn addition,MDA-MB-231 cells transduced with miR-200c showed multiple biochemical and morphological changes,resulting in transformation from a mesenchymal to an epithelial phenotype.Consistent with the phenotypic changes,expression of the epithelial marker E-cadherin was significantly increased and the mesenchymal markers N-cadherin,Fibronectin,Vimentin were markedly decreased.These results indicate that miR-200c inhibits the epithelial-mesenchymal transition of breast cancer cells.4.Overexpression of miR-200c lead to widespread alterations in lncRNA expressionIn order to identify miR-200c-associated IncRNAs,differential expression of IncRNAs between MDA-MB-231 cells stably overexpressing miR-200c and control cells was assessed by microarray analysis.Hierarchical clustering showed variations of IncRNA expression between miR-200c-overexpressing cells and control cells.We found that 422 lncRNAs were upregulated and 522 lncRNAs were downregulated(>2-fold change,P<0.05),which means that the IncRNA expression profile are different between two group cells.We investigated the downregulated IncRNAs that werepredicted to be targets of miR-200c by Targetscan and Miranda software,and identified a potential regulatory network.Taken together,our results show that overexpressxon of miR-200c lead to widespread alterations in IncRNA expression.5.LINC02582 promotes radioresistance of breast cancer cellsLINC02582 expression was higher in the radioresitant breast cancer lines,including MDA-MB-231 and BT549 with low miR-200c level,than in the radiosensitive cell lines,including MCF-7 and BT474 with high miR-200c level.These data indicate a negative correlation between LINC02582 expression and miR-200c expression level.We found that inhibition of LINC02582 had no effect on the viability of breast cancer cells.Interestingly,LINC02582 inhibition led to persistence of ΥH2AX foci in MDA-MB-231 cells and BT549 cells at 24 h after irradiation,suggests that knockdown of LINC02582 impairs DNA strand break repair.Furthermore,inhibition of LINC02582 reduced the surviving.fraction of MDA-MB-231 and BT549 cells after irradiation.Conversely,overexpression of LINC02582 increased the surviving fraction of MCF-7 and BT474 cells after irradiation.These results mean that LINC02582 promotes radioresistance of breast cancer cells.Subsequently,to further investigate the effects of LINC02582 in vivo,MDA-MB-231 cells with LINC02582 knockdown were used to create a xenograft model.When the tumor volume reached 150 mm3,local tumor irradiation was performed with a 2Gy fractionated dose every other day for 8 days.Knockdown of LINC02582 had no effect on the growth of tumors without irradiation,while LINC02582 knockdown caused tumor growth inhibition after irradiation.Taken together,these results demonstrate that LINC02582 promotes radioresistance of breast cancer cells both in vitro and in vivo.6.LINC02582 is a direct target of miR-200cTo investigate whether miR-200c regulated the expression of LINC02582,we suppressed miR-200c expression in MCF-7 and BT474 cells.Interestingly,suppression of miR-200c led to a significant increase of LINC02582 level.Also,ectopic expression of-miR-200c resulted in a significant reduction of LINC02582 in MDA-MB-231 and BT549 cells.However,we found no significant difference of the miR-200c level between LINC02582 knockdown or overexpression.Subsequently,the existence of interactions between miR-200c and LINC02582 was predicted by TargetScan and miRBase software.To confirm the binding between LINC02582 and miR-200c,we performed dual-luciferase assays.These assays showed that miR-200c decreased the luciferase activity of the LINC02582 expression vector,but not the mutant LINC02582 vector.MiRNAs are known to bind their targets and lead to translational repression or RNA degradation in Ago2-dependent manner.To test whether miR-200c regulate LINC02582 in such manner,we conducted RIP experiment in MDA-MB-231 cells using Ago2 antibody.The results showed that LINC02582 and miR-200c were enrich in Ago2 immunoprecipitates relative to IgG immunoprecipitates,which confirmed direct binding between miR-200c and LINC02582.Moreover,ectopic expression of miR-200c shortened the half-life of LINC02582.7.The expression of miR-200c and LINC02582 are negatively correlated in human breast cancer tissueClonogenic survival assay demonstrated that overexpression of LINC02582 reversed the induction of radiosensitivity by ectopic expression of miR-200c.Finally,qRT-PCR revealed a negative correlation between miR-200c and LINC02582 expression in 42 paired samples of breast cancer tissue.These results suggest that miR-200c binds directly to LINC02582 and acts as a negative regulator of LINC02582..8.LINC02582 interacts with USP7Many studies have shown that some IncRNAs perform their functions by interacting with specific proteins.To evaluate whether LINC02582 acts via this mechanism,we performed an RNA pull-down assay to identify proteins interacting with LINC02582 and then carried out mass spectrometry analysis of the specific protein band for LINC02582.Among the proteins identified by mass spectrometry,USP7 was also detected by western blotting.Then we performed RIP experiment with an antibody directed against USP7 using extracts from MDA-MB-231 cells.We observed enrichment of LINC02582(but not GAPDH lRNA)using the USP7 antibody versus a nonspecific IgG control antibody.Moreover,deletion mapping analysis identified a 422-789 nt region that was required for interaction between LINC02582 and USP7.Taken together,these results indicate that a specific interaction occurs between USP7 and LINC02582.9.USP7 promotes radioresistance of breast cancer cells by upregulated CHK1Recent studies have shown that USP7 promotes radioresistance of breast cancer cells through control CHK1 protein stability by direct deubiquitination.Consistently,inhibition of USP7 reduced the CHK1 protein level and decreased radiosensitivity,while overexpression of USP7 elevated the CHK1 protein level and promoted radioresistance.CHK1 is a critical effector kinase in DNA damage response,it facilitates DNA damage repair and promotes radioresistance of breast cancer cells.As expected,knockdown of CHK1 sensitized MCF-7 cells to radiation and overexpression of CHK1 resulted in radioresistance of MDA-MB-231 cells10.LINC02582 deubiquitinate and stabilize CHK1 by interacting with USP7We confirmed that knockdown of LINC02582 diminished CHK1 protein level,while ectopic expression of LINC02582 increased CHK1 protein level.Importantly.LINC02582 inhibition or ectopic expression altered the CHK1 protein level,but not the CHK1 mRNA level.To assed the effect of LINC02582 on CHK1 stability,we treated MCF-7 cells with cyclolcximidc(CHX)to inhibit protein synthesis and detected the level of remaining CHK1 by western blot.The portions of CHK1 remained in LINC02582 overexpressing cells were relative higher than in the control cells.suggesting that CHK1 protein more stable upon LINC02582 overexpression.Moreover,treatment of MDA-MB-231 cells suppressed LINC02582 with the proteasome inhibitor MG132 resulted in increased endogenous CHK1 protein level than in control cells,suggesting that the ubiquitin-proteasome pathway may play a critical role in the LINC02582-mediated upregulation of CHK1 protein.Indeed,the ubiquitination of CHK1 was markedly decreased in cells overexpressing LINC02582 in comparison to control cells,but knockdown of USP7 blocked the deubiquitination of CHK1 induced by LINC02582 overexpression.Collectively,these results indicate that LINC02582 interact with UPS7 to deubiquitinate and stabilize CHK1 protein.11.The effect of LINC02582 on radioresistance was dependent on USP7 and CHK1Interestingly,we observed that the reduction of CHK1 due to inhibition of LINC02582 was reversed by USP7 overexpression,while the upregulation of CHK1 due to LINC02582 ectopic expression could be reversed by USP7 knockdown.Importantly,induction of radiosensitivity by inhibition of LINC02582 could be blocked by USP7 overexpression,but radioresistance induced by ectopic expression of LINC02582 could be blocked by USP7 knockdown.Similarly,ectopic expression of CHK1 in LINC02582-knockdown cells rescued radioresistance,knockdown of CHK1 reversed LINC02582 induced radioresistance.These results indicate that the effect of LINC02582 on radioresistancc was at least partly dependent on USP7 and CHK1.12,MiR-204c/LINC02582/CHK1 pathway regulating the radiosensitivity of breast cancer cellsWe found that suppression of miR-200c elevated CHK1 protein level and ectopic expression of miR-200c decreased CHKl protein level.However,either knockdown of LINC02582 or USP7 blocked the CHKI upregulation induced by miR-200c suppression,either LINC02582 or USP7 ectopic expression rescued the CHKI downregulation induced by miR-200c ectopic expression.Furthermore,knockdown of LINC02582 or USP7,CHK1 prevented the induction of radioresistance due to inhibition of miR-200c,while upregulation of LINC02582 or USP7,CHK1 reversed radiosensitivity due to overexpression of miR-200c.Taken together,these findings demonstrate that the miR-200c/LINC02582/CHK1 pathway is a new molecular mechanism regulating the radiosensitivity of breast cancer cells.13.MiR-200c is negatively correlated with CHK1 expression in breast cancerTo confirm the association between miR-200c and CHK1 in breast cancer specimens,we performed in situ hybridization or immunohistochemistry staining to examine the expression of these molecules in 136 breast cancer tissue samples from breast cancer patients who had received radiotherapy.We found that decreased expression of miR-200c was associated with elevated CHK1 expression.In brief,69.7%(53/76)of the turmors with low miR-200c expression exhibited high CHKI expression.MiR-200c is negatively correlated with CHKI expression in breast cancer.14.LINC02582 is negatively correlated with CHK1 expression in breast cancerTo confirm the association between LINC02582,and CHKI in breast cancer specimens,we performed in situ hybridization or immunohistochemistry staining to examine the expression of these molecules in 136 breast cancer tissue samples from breast cancer patients who had received radiotherapy.A positive correlation between expression of LINC02582 and CHK1 was observed in these breast cancer specimens.In brief,72%(55/76)of the tumors with high LINC02582 expression showed high CHK1 expression.Conclusion1.Overexpression of miR-200c enhances radiosensitivity of breast cancer cells2.MiR-200c inhibits epithelial-mesenchymal transition of breast cancer cells3.Overexpression of miR-200c lead to widespread alterations in lncRNA expression4.LINC02582 promotes radioresistance of breast cancer cells5.LINC02582 is a direct target of miR-200c6.MiR-200c are negatively correlated with LINC02582 in breast cancer7.LINC02582 interacts with USP78.USP7 promotes radiorcsistancc through unregulated CHK1 protein9.LINC02582 deubiquitinate and stabilize CHK1 by interacting with USP710.The effect of LINC02582 on radioresistance was dependent on USP7 and CHK111.Expression of LINC02582 blocked miR-200c induces radiosensitivity12.Expression of USP7 blocked miR-200c induces radiosensitivity13.Expression of CHKI blocked miR-200c induces radiosensitivity14.MiR-200c are negatively correlated with CHK1 in breast cancer15.LINCO2582 are positively correlated with CHK1 in breast cancer. |
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