| With over 2 million newly diagnosed cases and more than 626,000 deaths annually,breast cancer is a common type of human cancers among women globally.The treatment of breast cancer includes comprehensive treatments such as surgery,radiotherapy,chemotherapy,endocrine therapy,molecular targeted therapy,and immunotherapy.As local treatment methods,surgery and radiotherapy play a very important role in controlling the local recurrence of breast cancer.Among them,as an adjuvant therapy after surgery and a late rescue therapy,radiotherapy can significantly reduce the risk of breast cancer recurrence,thereby improving the overall survival of patients.However,not all breast tumor tissues are sensitive to radiation,and about5-10% of them still have recent recurrence after adjuvant radiotherapy.Therefore,finding radiosensitivity targets,exploring radiosensitivity measures for breast cancer,and improving the efficacy of radiotherapy are urgent clinical problems.Multiple research studies reported that non-coding RNAs(ncRNAs)played vital roles in the progression of various cancers.Circular RNAs(Circ RNAs)are a special class of endogenous ncRNAs and featured by the continuous covalently circular structure without 3’-end poly A tail and 5’-end cup.Currently,accumulating evidence also suggested the regulatory effect of some Circ RNAs on breast cancer development,while there were few researches on the involvement of Circ RNAs in breast cancer radiosensitivity.A previous research uncovered that hsa＿Circ＿0008500(Circ＿0008500;Position: chr3:196831773-196846401),a novel circ RNA,was found to be upregulated in breast cancer samples.However,the critical role of Circ＿0008500 and its potential underlying mechanism in the radiosensitivity and development of breast cancer are yet to be studied.Herein,we determined the level of circ＿0008500 in breast cancer tissues and cells.Moreover,we also explored the functional role of circ＿0008500 and its molecular mechanism in breast cancer radiosensitivity.Herein,we determined the level of circ＿0008500in breast cancer tissues and cells.Moreover,we also explored the functional role of circ＿0008500 and its molecular mechanism in breast cancer radiosensitivity.Part One The Circ＿0008500 suppression the radiation ensitivity and enhance cell proliferation in breast cancer cellsObjective: Observe the expression of Circ＿0008500 in breast cancer cells.To explore the relationship between Circ＿0008500 on the proliferation and radiosensitivity of breast cancer cells.Methods:1.Expression of Circ＿0008500 in breast cancer tissues and cells with different molecular subtype were measured by RT-qPCR.2.For agarose gel electrophoresis assay,genomic DNA(g DNA)was isolated from the cells using the mammalian genomic DNA extraction kit.Identification of Circ＿0008500 by use agarose gel electrophoresis,RNase R digestion experiments,etc.3.Small interfering RNA(si RNA)specifically against Circ＿0008500(si-Circ＿0008500:5’-AACCTCTTTCAGGCTTTAATAGA-3’)and its si RNA control(si-NC)was transfected into MCF-7 and MDA-MB-468 cells using cell transfection.4.The data of RT-qPCR represented a successful knockdown efficiency of si-Circ＿0008500 in MCF-7 and MDA-MB-468 cells.5.After giving 4Gy radiation treatment,cell colony formation,CCK-8and Ed U were used to determined cell proliferation,the enhanced BAX protein expression and the reduced BCL2 protein expression of cell proliferation were determined by Western blot,and the effect of Circ＿0008500on the radiation sensitivity and cell proliferation of breast cancer cells in vitro.Results:1.RT-qPCR was performed to gauge the expression of Circ＿0008500 in breast cancer tissues and cells.The increase of Circ＿0008500 was observed in breast cancer tissues(n=50)compared with the adjacent normal tissues(n=50).Our data showed that Circ＿0008500 was lowly expressed in triple-negative breast cancer(TNBC;n=24)tissues compared with the non-TNBC tissues(n=26)Additionally,the expression of Circ＿0008500 was highly expressed in ER/PR-positive BC tissues,while there was no remarkable difference between HER2-negative and HER2-positive BC tissues.2.The expression of Circ＿0008500 was higher in breast cancer cells(MCF-7 and MDA-MB-468 cells)than that in MCF-10 A cells.3.According to the annotation of Circ Base2,we speculated that Circ＿0008500 was derived from exons 13-15 of the DLG1 gene,located atchr3:196831773-196846401,thus we synthesized a circular transcript of 381 base pairs.The results of agarose gel electrophoresis assay showed that Circ＿0008500 was amplified only from c DNA using the divergent primer,while Circ＿0008500 could not be amplified from g DNA.In RNase R treatment assay,we found that the expression of DLG1 was inhibited by RNase R treatment,whereas Circ＿0008500 was more stable than DLG1 and resistant to RNase R.These experiments confirmed the existence of Circ＿0008500.4.4 Gy radiation induced a significant decrease of Circ＿0008500 in MCF-7 and MDA-MB-468 cells.5.The data of RT-qPCR represented a successful knockdown efficiency of si-Circ＿0008500 in MCF-7 and MDA-MB-468 cells.6.CCK-8 assay indicated that cell viability was significantly inhibited by Circ＿0008500 knockdown in breast cancer cells.Moreover,Circ＿0008500downregulation aggravated radiation treatment caused inhibition on cell proliferation in breast cancer cells.7.The results in colony formation assay suggested that Circ＿0008500knockdown exacerbated the suppressive impact of radiation treatment on the number of colonies in MCF-7 and MDA-MB-468 cells.In addition,the promotion effect of radiation treatment on cell apoptosis was facilitated by Circ＿0008500 knockdown.8.The enhanced BAX protein expression and the reduced BCL2 protein expression induced by radiation treatment were intensified by transfection of si-Circ＿0008500 in MCF-7 and MDA-MB-468 cells.Conclusions:1.The expression of Circ＿0008500 in breast cancer tissues is higher than that in paracancerous tissues;and it is associated with poor tissue differentiation.2.After successfully knocking down Circ＿0008500,radiotherapy significantly enhanced the inhibition rate of MCF-7 and MDA-MB-468 cells and promoted cell apoptosis.Part Two The Circ＿0008500 regulates proliferation and radiosensitivity of breast cancer cells in vitro though targeting the miR-758-3p-PFN2 axisObjective:1.To observe the expression of miR-758-3p in breast cancer tissues and cells.To Confirm that Circ＿0008500 contains the recognition and binding site of the miR-758-3p complex.To explore the targeted regulation mechanism of Circ＿0008500 through miR-758-3p.2.To observe the expression of PFN2 in breast cancer tissues and cells.Confirmation of the regulatory effect of miR-758-3p on the expression of its potential target PFN2.To explore the downstream mechanism of Circ＿0008500 regulating the proliferation and radiosensitivity of breast cancer cells in vitro.Methods:1.Circ＿0008500 localization: Subcellular localization experiments were used to isolate RNA in the cytoplasm and nucleus,and RT-qPCR was used to detect the expression of Circ＿0008500 in the cytoplasm and nucleus,respectively.2.The binding site between Circ＿0008500 and miR-758-3p was predicted by Circ Bank and Circinteractome database using Venn diagram.3.The wild-type(WT)segments of Circ＿0008500 harboring the complementary sites of miR-758-3p was individually cloned into the p GL3vector(Promega,Madison,WI,USA)to construct the WT-circ＿000850luciferase reporter vectors.The mutated segments of Circ＿0008500 containing the mutant target regions of miR-758-3p was used to form the mutant(MUT)vectors(MUT-Circ＿0008500).Mi RNA NC or miR-758-3p mimic was co-transfected with each reporter construct into the cells.The luciferase activity was determined using the Dual-Glo(?) Luciferase Assay System(Promega).4.Cell transfection: Circ＿0008500(si-Circ＿0008500;small interfering si RNA at 5’)and its si RNA control(NC),micro RNA-758-3p(miR-758-3p)mimic and control(miRNANC)and miR-758-3p inhibitor(miR-758-p inhibitor)and its negative control(inhibitor NC)constructs were transfected into MCF-7 and MDA-MB-468 cells.5.After giving 4Gy radiation treatment,cell colony formation,CCK-8and Ed U were used to determined cell proliferation,the enhanced BAX protein expression and the reduced BCL2 protein expression of cell proliferation were determined by Western blot,and to explore the mechanism of Circ＿0008500 and miR-758-3p targeting regulation.6.Bioinformatics analysis: The potential target genes of miR-758-3p were predicted using the Starbase database.The wild-type(WT)segments of PFN2 harboring the complementary sites of miR-758-3p were individually cloned into the p GL3 vector to construct the WT-PFN2 3’UTR luciferase reporter vectors.The mutated segments of PFN2 containing the mutant target regions of miR-758-3p was used to form the mutant(MUT)vectors(MUT-PFN23’UTR).Mi RNA NC or miR-758-3p mimic was co-transfected with each reporter construct into the cells.The luciferase activity was determined using the Dual-Glo(?) Luciferase Assay System.7.Cell transfection: Construction of small interfering RNA(si RNA)and its si RNA control(si-NC)specifically targeting Circ＿0008500(si-Circ＿0008500);miR-758-3p analog and control(miRNA NC),miR-758-3p inhibitor(miR-758-p inhibitor)and its negative control(inhibitor NC),and PFN2 overexpression(pc-PFN2)and negative control(pc-NC).The above plasmids and oligonucleotides were transfected into MCF-7 and MDA-MB-468 cells.8.After giving 4Gy radiation treatment,cell colony formation,CCK-8and Ed U were used to determined cell proliferation,the enhanced BAX protein expression and the reduced BCL2 protein expression of cell proliferation were determined by Western blot.To observe the regulatory mechanism of miR-758-3p and PFN2 targeting.Results:1.The results showed that Circ＿0008500 was mainly enriched in the cytoplasm.2.Venn diagram displayed that one overlapped miRNA was predicted by circ Bank and circinteractome.Circ Bank predicted the binding sites between Circ＿0008500 and miR-758-3p.3.RT-qPCR analysis represented a successful overexpression efficiency of miR-758-3p mimic in MCF-7 and MDA-MB-468 cells.4.Dual-luciferase reporter and RIP assays were performed to determine the correlation between Circ＿0008500 and miR-758-3p.The results of dual-luciferase reporter assay revealed that miR-758-3p mimic remarkably reduced the luciferase activity in WT-Circ＿0008500 group,while the luciferase activity in MUT-Circ＿0008500 group was not impacted.5.RIP assay showed that Circ＿0008500 and miR-758-3p were enriched in anti-Ago2 group in comparison to anti-Ig G group.6.The clinical data disclosed that miR-758-3p was significantly downregulated in breast cancer tissues.The results suggested that miR-758-3p was elevated in TNBC tissues compared with the non-TNBC tissues.Furthermore,miR-758-3p was significantly decreased in ER/PR-positive BC tissues,but not in HER2-positive BC tissues.7.In vitro cell experiments data showed that miR-758-3p was lower in MCF-7 and MDA-MB-468 cells relative to that in MCF-10 A cells.As described in Figure 4P,the expression of miR-758-3p was upregulated by Circ＿0008500 knockdown in MCF-7 and MDA-MB-468 cells.8.The data of RT-qPCR analysis indicated that miR-758-3p inhibitor markedly suppressed the expression of miR-758-3p in MCF-7 and MDA-MB-468 cells.Further analysis displayed that miR-758-3p inhibitor strikingly counteracted the inhibitory effect on cell viability caused by Circ＿0008500 absence in breast cancer cells.9.EdU and colony formation assays demonstrated that the inhibition of miR-758-3p significantly blocked Circ＿0008500 downregulation-induced suppressive impact on cell proliferation.Besides,Circ＿0008500 knockdown led to the enhanced cell apoptosis,which was harbored by miR-758-3p inhibitor.Furtherly,upregulation of BAX and downregulation of BCL2 protein expression caused by Circ＿0008500 knockdown were rescued by inhibition of miR-758-3p in MCF-7 and MDA-MB-468 cells.10.Starbase predicted the potential binding sites between miR-758-3p and PFN2.11.Dual-luciferase reporter assay indicated that the luciferase activity was remarkably reduced by miR-758-3p mimic in WT-PFN2 3’UTR group,but not in MUT-PFN2 3’UTR group.Moreover,the relationship between miR-758-3p and PFN2 was also verified by RIP assay.12.RT-qPCR analysis revealed that PFN2 was highly expressed in breast cancer tissues(n=50)compared with the normal tissues(n=50).Moreover,PFN2 was decreased in TNBC tissues in comparison to the non-TNBC tissues.The upregulated PFN2 expression was observed in ER/PR-positive breast cancer tissues,while the expression of PFN2 showed no significant difference between HER2-positive and HER2-negative breast cancer tissues.13.Western blot showed that the protein expression of PFN2 was significantly increased in breast cancer tissues and cells.Mi R-758-3p mimic markedly repressed PFN2 protein expression in MCF-7 and MDA-MB-468 cells.Additionally,Circ＿0008500 knockdown suppressed the protein expression of PFN2 by targeting miR-758-3p.14.The results from western blot showed that the protein expression of PFN2 was significantly upregulated in MCF-7 and MDA-MB-468 cells transfected with pc-PFN2.Functionally,overexpression of PFN2 strikingly blocked miR-758-3p mimic-induced repression on cell viability and proliferation in MCF-7 and MDA-MB-468 cells.In addition,miR-758-3p mimic-caused promotion on cell apoptosis was reversed by PFN2 overexpression,as evidenced by the decreased BAX and the increased BCL2.Conclusions:1.Circ＿0008500 was mainly enriched in the cytoplasm.2.Circ＿0008500 can directly bind to miR-758-3p,which is the target gene of Circ＿0008500.3.Circ＿0008500 negatively regulates the expression of miR-758-3p.4.Circ＿0008500 downregulation repressed cell proliferation and elevated cell apoptosis by targeting miR-758-3p in breast cancer cells.5.PFN2 was a target of miR-758-3p and Circ＿0008500 regulated PFN2 expression through targeting miR-758-3p.6.Mi R-758-3p inhibited cell proliferation,and enhanced cell apoptosis by regulating PFN2 in breast cancer cells.Part Three Circ＿0008500 knockdown inhibited tumor growth and elevated the radiosensitivity in breast cancer in animal experimentsObjetive:Validation of the functional role and mechanism of Circ＿0008500 in breast cancer in animal experiments.Methods:1.Cs-137 irradiator was utilized for irradiation in tumor formation assay.2.16 BALB/c nude mice(4-week-old)to establish the murine xenograft model of breast cancer.The mice were divided into sh-Circ＿0008500,sh-NC,sh-NC + radiation,and sh-Circ＿0008500 + radiation groups(n = 4 per group).The tumor volume(length × width2 × 0.5)was recorded weekly.4 weeks later,the mice were euthanatized and tumor weight was detected.3.The tissue sections acquired from xenograft model mice were fixed with formalin and embedded with paraffin.The sections were incubated with anti-PFN2 and the secondary antibody.Following the staining using the diaminobenzidine(DAB)kit,the positive protein PFN2 expression of tissue sections was observed and photographed under a microscope.Resuts:1.Tumor volume and weight were significantly repressed by Circ＿0008500 downregulation or radiation treatment,and a more remarkable suppression of tumor volume and weight was found in sh-Circ＿0008500transduction+ radiation treatment group compared with the radiation treatment group.2.Circ＿0008500 knockdown enhanced miR-758-3p expression and inhibited Circ＿0008500 and PFN2 expression in the tissues extracted from the mice.3.Circ＿0008500 knockdown aggravated the inhibition effect of radiation treatment on Circ＿0008500 and PFN2 expression,as well as promotion effect on miR-758-3p expression4.IHC assay suggested that PFN2 was reduced in sh-Circ＿0008500 or radiation treatment group,and Circ＿0008500 knockdown enhanced the inhibition impact of radiation treatment on PFN2 expression.Conclusions:Circ＿0008500 knockdown inhibited tumor growth and e levated the radiosensitivity in breast cancer in vivo. |
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