| Background and objectives:Breast cancer is one of the main causes of cancer death among females worldwide.Early diagnosis and treatment for breast cancer are of great importance.MicroRNAs(miRNAs)are small single-stranded non-coding RNAs.Increasing evidence has shown that miRNAs can serve as potential diagnostic biomarkers for several types of cancer.For the past few years,circRNAs have become research hotspots in RNA,especially in the potential as biomarkers.Studies have shown that circRNAs are rich in complementary binding sites of miRNAs,playing a role of miRNAs "molecular sponge",and finally influencing the progression of disease.This study aims to reveal the potential of miRNAs/circRNAs in whole blood as diagnostic biomarkers for breast cancer and the expression,clinical significance,function and potential mechanism of circRNA circ＿0075796 in breast cancer.Methods:1.MiRNA microarray of the blood of breast cancer patients and healthy controls was performed.Candidate differentially expressed miRNAs were further verified by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).2.Bioinformatic analysis was used to predict circRNAs related to the differentially expressed miRNAs.QRT-PCR was performed to detect levels of five circRNAs(circ＿0000501,circ＿0000745,circ＿0001531,circ＿0001640 and circ＿0001978)in breast cancer patients,benign breast tumor patients and healthy controls.The diagnostic accuracy of circRNAs was assessed using the receiver operating characteristic curve(ROC)analysis.A circRNA-miRNA-mRNA network was constructed based on bioinformatic analysis.3.Whole transcriptome resequencing was used to screen differentially expressed circRNAs in breast cancer tissues and adjacent normal tissues.The expression of circ＿0075796 in breast cancer tissues and adjacent normal tissues was detected by qRT-PCR.Cell Counting Kit-8(CCK-8)assay and colony formation assay were conducted for cell proliferation.Transwell assay and wound healing assay were used for cell migration and invasion.Flow cytometry analysis was adopted for cell cycle and cell apoptosis.The cellular localization of circ＿0075796 was determined by fluorescence in situ hybridization(FISH).The circ＿0075796/miR-452-3p/SAMD5 axis was screened out by bioinformatic analysis and verified by qRT-PCR.Methylated RNA Immunoprecipitation(MeRIP)was used to detect the N6-methyladenosine(m6A)modification levels of circ＿0075796.QRT-PCR was used to detect the influence of enzymes(methyltransferases:METTL3 and METTL14;demethylases:FTO and ALKBH5)related to m6A modification and RNA binding proteins(RBPs)(DHX9,ADAR1 and QKI)on the expression of circ＿0075796 in breast cell lines.Results:1.Six upregulated blood miRNAs(miR-26b-5p,miR-106b-5p,miR-142-3p,miR-142-5p,miR-185-5p and miR-362-5p)were identified in breast cancer patients.These six miRNAs could discriminate breast cancer patients from healthy controls,Bioinformatic analysis showed that the six miRNAs were potentially involved in numerous cancer-related pathways.Importantly,two miRNAs(miR-185-5p and miR-362-5p)were used to construct a two-miRNA panel by logistic regression.The two-miRNA panel displayed a better diagnostic performance than each of the miRNAs alone.Additionally,the high expression of the six miRNAs or the two-miRNA panel was associated with poor prognosis of breast cancer.2.Candidate circRNAs(circ＿0000501,circ＿0000745,circ＿0001531,circ＿0001640 and circ＿0001978)were predicted by bioformatic analysis.QRT-PCR validated that circ＿0000745,circ＿0001531 and circ＿0001640 were upregulated in breast cancer,compared with benign tumor and healthy control.ROC curve analysis revealed that circ＿0000745,circ＿0001531 and circ＿0001640 had good diagnostic potential.Notably,a signature comprising the three circRNAs showed better diagnostic potential.And a circRNA-miRNA-mRNA network revealed that the circRNAs could participate in complex regulated network and thus involve in cancer development and progression.3.Whole transcriptome resequencing showed that circ＿0075796 was significantly downregulated in breast cancer tissues compared to adjacent normal tissues.The results of qRT-PCR further verified that circ＿0075796 was downregulated in breast cancer tissues compared with adjacent normal tissues.In addition,circ＿0075796 showed satisfactory diagnostic value to discriminate breast cancer and normal controls.Downregulation of circ＿0075796 was correlated with aggressive clinical features of breast cancer.Overexpression of circ＿0075796 inhibited cell proliferation,migration and invasion in vitro.FISH showed that circ＿0075796 was localized in the cytoplasm and nucleus of breast cancer cells.Bioinformatics analysis and qRT-PCR revealed the potential circ＿0075796/miR-452-3p/SAMD5 axis.Moreover,circ＿0075796 showed lower m6A modification levels in breast cancer tissues compared to adjacent normal tissues.Enzymes(demethylases:FTO;methyltransferases:METTL3 and METTL14)related to m6A modification affected the expression of circ＿0075796 in breast cells.RBPs(DHX9 and ADAR1)could affect the expression of circ＿0075796 in breast cells.Conclusions:In conclusion,our study identified six upregulated miRNAs(miR-26b-5p,miR-106b-5p,miR-142-3p,miR-142-5p,miR-185-5p,and miR-362-5p)and three upregulated circRNAs(circ＿0000745,circ＿0001531 and circ＿0001640)in whole blood of breast cancer patients.These miRNAs,circRNAs and the related diagnostic models could discriminate breast cancer patients from healthy controls with high accuracy and can serve as candidate biomarkers for breast cancer diagnosis.Circ＿0075796 was downregulated in breast cancer tissues compared to adjacent normal tissues,and showed potential diagnostic value for breast cancer.Downregulation of circ＿0075796 was correlated with aggressive clinical features of breast cancer and overexpression of circ＿0075796 inhibited the progression of breast cancer in vitro,indicating that circ＿0075796 may be related to tumorigenesis and development of breast cancer.In addition,Enzymes(demethylases:FTO;methyltransferases:METTL3,METTL14)related to m6A modification and RBPs(DHX9,ADAR1)affected the expression of circ＿0075796 in breast cells,providing directions for researches on mechanisms of differential expression and functions of circ＿0075796 in breast cancer. |
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