| Background and objectiveAs one of the most common malignancies worldwide,breast cancer(BC)is also the leading cause of cancer-associated deaths in females.Despite the striking breakthrough in the diagnosis and treatment of BC in the past 20 years,the 5-year survival rate of some BC patients is still very dismal,mainly because of its heterogeneity and the fact that most patients are in the advanced clinical stage at the time of diagnosis.Therefore,an in-depth exploration of the pathogenesis of BC is strongly needed and will provide novel targets for the clinical diagnosis and treatment,thereby improving the prognosis of patients with BC.With the completion of the Human Genome Project,scientists have surprisingly found that only about 2%of the genes in the human genome are capable of translating proteins,while the remaining 98%of genes are noncoding RNAs(ncRNAs).A large number of ncRNAs have long been considered as by-products of genome transcription that have no definitive biological functions.However,this is not the case.Recently,multiple lines of evidence showed that ncRNAs are widely involved in the regulation of various important life activities,including the occurrence and development of cancers.As a member of ncRNAs,circular RNA(circRNA)is mainly derived from gene exons(accounting for 80%)with a covalently closed loop structure.CircRNA has different biological functions,of which the most studied is as a molecular sponge of microRNAs(miRNAs),namely,competitive endogenous RNA(ceRNA)mechanism.The most prominent is CDR1as,which was identified to have more than 60 conserved binding sites of the ancient miR-7.Subsequently,a wealth of studies suggests that circRNAs can act as a ceRNA to control tumorigenesis.For example,circ-BACH2,circ-FNDC3B,cire-VANGL1,circ-HIPK3,circ-FAT1and circ-EIF3I were proposed to participate in the development and progression of papillary thyroid carcinoma,gastric cancer,bladder cancer,colorectal cancer,osteosarcoma and non-small cell lung cancer by sponging miR-139-5p,miR-515-5p,miR-605-3p,miR-7,miR-375 and miR-361-3p/miR-615-5p,respectively.However,the role of ceRNA and circRNA in BC remains largely unclear.In the current study,we screened and identified a novel circRNA,circ-UBE2D2,which was highly expressed in BC cell lines and tissues and was closely related to aggressive clinical features and dismal prognosis.Small interfering RNA(siRNA)mediated circ-UBE2D2 silencing notably inhibited the proliferation,migration and invasion of BC cells,whereas circ-UBE2D2 overexpression displayed opposite effects.Mechanistically,circ-UBE2D2 was able to simultaneously function as molecular sponges of miR-1236 and miR-1287 to regulate the expression of their respective target genes.Moreover,circ-UBE2D2-induced tumor-promoting effects could be effectively blocked by miR-1236 or miR-1287 in BC cells.More importantly,therapeutic delivery of cholesterol-conjugated si-circ-UBE2D2 oligonucleotides significantly delayed tumor growth in vivo.Materials and methods1.The circRNA expression profiling chip GSE101123 was downloaded.Then the differential circRNAs were screened for between tumor tissues and normal tissues of breast cancer patients.The miRNAs bound to circRNAs were predicted by using CSCD database.The target genes of miRNAs which were screened for to construct ceRNA network were predicted by using miRDB database and Targetscan database.The String database was used to build a protein interoperability network to identify the core functional genes that influence the development of breast cancer.The biological functions and enrichment pathways of target proteins were analyzed by using GO and KEGG database.2.We collected clinical information,postoperative tumor tissue samples and paracancerous normal tissue samples of breast cancer patients admitted to the First Affiliated Hospital of Zhengzhou University.Real-time quantitative PCR was used to detect the expression of circ-UBE2D2 in breast cancer tumor tissues and paracancerous normal tissues,normal breast epithelial tissue cell lines and five breast cancer cell lines(MCF-7,T47D,BT-474,BT20 and MDA-MB-231).Combined with the clinical information of breast cancer patients,the relationship between the expression level of circ-UBE2D2 and the clinical characteristics of breast cancer patients was analyzed.3.Using gene transfection technique,si-circ-UBE2D2#1 and si-circ-UBE2D2#2 of silent cir-UBE2D2 were transfected into BT-474 and MDA-MB-231.Breast cancer cell proliferation was measured by CCK method,and apoptosis was detected by Annexin V-FITC/PI flow cytometry,and cell migration was detected by scratch experiment,and cell invasion was detected by Transwell chamber experiment.MDA-MB-231 cells were injected subcutaneously in the right lower limb of BALB/c nude mice,and the effect of si-circ-UBE2D2#1 and si-NC on graft tumor production was observed.4.We utilized double luciferase assay to verify the binding of circ-UBE2D2 to miR-1236 and miR-1287.QPCR was applied to detect the expression level of miR-1236 and miR-1287 in breast cancer cells after overexpression and knockdown of circ-UBE2D2.Furthermore,the mechanism of circ-UBE2D2 regulating miR-1236 and miR-1287 was further verified by functional experiments.The proliferation of breast cancer cells was determined by CCK assay,apoptosis was detected by Annexin V-FITC/PI flow cytometry,cell migration was detected by the scratch assay,and cell invasion was detected by the Transwell chamber assay.Results1.We screened 40 differential circRNAs via GSE101123 data chip,of which 24 were up-regulated and 16 were down-regulated,and the expression difference of circ-UBE2D2 in breast cancer was the most significant.2.The CSCD database predicted that the 3’-UTR regions of miR-1236 and miR-1287 was the potential binding site of circ-UBE2D2.3.Genetics analysis showed that the core target genes of miR-1236 included HOXB7,MTA2,ZEB1,KLF8,AFP and p21,and the core target genes of miR-1287 included PIK3CB,AngPT1,GAGE1,CD105,ATF6α and EGFR.4.Circ-UBE2D2 is highly expressed in BC and linked to poor prognosis.5.Circ-UBE2D2 is a circRNA structure.6.Silencing circ-UBE2D2 can significantly inhibit the proliferation,anti-apoptosis,migration and invasion of breast cancer cells.Silencing circ-UBE2D2 inhibited the growth of xenograft tumor and the level of Ki-67 in the tumor in nude mice.Silencing circ-UBE2D2 increased the expression levels of miR-1236 and miR-1287,while overexpression of circ-UBE2D2 decreased the expression levels of miR-12 36 and miR-1287.7.Circ-UBE2D2 regulates downstream genes by sponging miR-1236 and miR-12 87.8.When transfecting tumor cells with circ-UBE2D2 and miR-1236 mimics or miR-1287 mimics simultaneously,the promoting effects of circ-UBE2D2 on the proliferation,anti-apoptosis,migration and invasion of breast cancer tumor cells were inhibited.9.When knocking down circ-UBE2D2 and inhibiting miR-1236 or miR-1287 simultaneously,the inhibitory effect of circ-UBE2D2 on proliferation,anti-apoptosis,migration and invasion of breast cancer tumor cells was restored.Conclusions1.High expression of circ-UBE2D2 is associated with poor prognosis in breast cancer patients,and is a new prognostic marker.2.C irc-UBE2D2 promotes the proliferation,anti-apoptosis,migration and invasion of breast cancer by sponging miR-1236 and miR-1287. |
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