| Part One Diagnostic value of plasma hsa＿circ＿0000615 and the relationship between expression level of hsa＿circ＿0000615 and clinical features in breast cancer patientsObjective: To explore the expression level of hsa＿circ＿0000615 in peripheral blood samples and evaluate its diagnostic value for breast cancer patients.Methods:1.Through Pubmed search,45 circRNA molecules that were differentially expressed in breast cancer tissues and adjacent tissues and whose function and mechanism had been verified by in vitro cell experiments were screened out.2.Plasma of 95 pathologically confirmed preoperative breast cancer patients and 95 gender-age-matched healthy subjects were collected in the Fourth Hospital of Hebei Medical University.The expression level of hsa＿circ＿0000615 was detected by real-time qPCR and its diagnostic value was analyzed by ROC curve.The stability of hsa＿circ＿0000615 as serological biomarkers in plasma was analyzed.Results:1.Comparison of plasma samples of 95 pairs of breast cancer patients and healthy subjects show that hsa＿circ＿0000615 expression level of breast cancer patients the was 4.8 times higher than healthy subjects,the difference was statistically significant(P < 0.001).The expression level of hsa＿circ＿0000615 in 38 pairs of breast cancer tissues was 6.2 times higher than that in adjacent tissues and the difference was statistically significant(P<0.001).2.The high expression of hsa＿circ＿0000615 in plasma was significantly correlated with tumor stage(P=0.01),lymph node metastasis(P=0.001),and grade of recurrence risk(P=0.012),but no significant correlation with age,Ki-67,tumor diameter and molecular typing.3.ROC curve showed that the AUC value of plasma hsa＿circ＿0000615for the diagnosis of non-metastatic breast cancer was 0.904(95%CI:0.863-0.944),with a sensitivity of 76.8% and specificity of 88%.CA153(AUC:0.482,95% CI:0.399-0.564)14.7%,92.6%.CA125(AUC:0.513,95% CI: 0.430-0.595)41.1%,67.4%.CEA(AUC:0.498,95% CI:0.415-0.580)15.8%,91.6%.The AUC value of early stage(stage 0-1)breast cancer was0.799(95%CI 0.713-0.885),the sensitivity was 79.2%,and the specificity was68.7%.CA153(AUC:0.383,95% CI: 0.269-0.497)14.6%,91.7%.CA125(AUC:0.472,95% CI: 0.356-0.589)14.6%,91.7%.CEA(AUC:0.475,95% CI:0.358-0.592)81.3%,25.0%.4.The stability assay showed that there was no difference between RNA extraction at 0 hour and 24 hours in terms of the expression of hsa＿circ＿0000615(P = 0.327).Conclusions:1.The hsa＿circ＿0000615 can be used as a stably potential plasma biomarker for differential diagnosis of breast cancer.The diagnostic value is better than CA125,CEA and CA153.2.High expression level of hsa＿circ＿0000615 in plasma of breast cancer patients means more aggressive,and can indirectly predict the risk of breast cancer recurrence after surgery.Part Two Study on secretion pattern of plasma exosome hsa＿circ＿0000615 in breast cancerObjective:To analyze the secretion pattern of hsa＿circ＿0000615 in breast cancer plasma by in vitro experiment and plasma detection,and to provide a theoretical basis for hsa＿circ＿0000615 as a plasma biomarker by exploring the exocrine mode of breast cancer cells in vitro.Methods:1.To analyze the expression level of hsa＿circ＿0000615 in plasma and plasma exosomes of breast cancer patients by real-time PCR,and the expression level of hsa＿circ＿0000615 in exosomes of breast cancer cells supernatant fluid and cells.2.Observe the morphological characteristics of exosomes in supernatant of breast cancer cells by transmission electron microscopy.3.Use western blot to detect specific antibodies of exosome membrane proteins.Results:1.We randomly selected and collected the blood samples of 14 breast cancer patients to compare the expression level of hsa＿circ＿0000615 in plasma exosomes and plasma,and the results showed that there was no expression difference between the two groups(P = 0.243).2.Compared with normal ductal epithelial cells,the expression levels of exosomal hsa＿circ＿0000615 in MCF-7 cells,Sk-br-3 cells and BT-549 cells are markedly upregulated(P < 0.001).3.We collect the exosomes in the culture medium of breast cancer cells and plasma of breast cancer patients,the transmission electron microscopy revealed that isolated exosomes had a disk-like appearance with a diameter of40–150 nm vesicles.4.Western blot analysis showed the presence of exosome markers CD9 and CD81 in exosomes extracted from culture solution of breast cancer cells.Conclusions:1.In vitro cell experiments confirm that exosomal hsa＿circ＿0000615 in breast cancer cells and their exudate of culture medium higher than those in normal mammary duct epithelial cells,providing theoretical basis for their use as a biomarker.2.Breast cancer cells secrete hsa＿circ＿0000615 into plasma by secreting exosomes. |
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