| Breast cancer is the most common cancer and the leading cause of cancer death among females.Early diagnosis,appropriate treatment,recurrence and metastasis monitoring are of great importance to reduce breast cancer mortality.Traditional methods of cancer detection such as mammography and needle biopsy,could cause radioactive or invasive damage to patients,have not high sensitivity,and are not comprehensive enough to capture the entire genomic landscape of breast tumors.Liquid biopsy,comprising the noninvasive analysis of circulating tumor DNA,circulating tumor cells,exosomes and tumor-educated platelets in the biological fluid such as blood and urine to obtain abundant information of tumors and achieve continuous real-time analysis,have been used in the early diagnosis of cancer,prediction of tumor progression and treatment response.Among them,exosomes is a subtype of EVs in 50-150 nm size,which encase DNA,mRNA,microRNA(miRNA)and proteinsconstituents of potential biomarkers of malignancy of cancer.Therefore,liquid biopsy based on detection of blood exosomal miRNAs can be developed for breast cancer diagnostics.In this study,we selected dysregulated miR-1246 and miR-21 in breast cancer patients’ plasma exosomes as biomarkers to design and synthesize two fluorescent probes for in situ measuring their expression levels.Based on the quantitative and specific reponse of the probes to the miRNAs,we established liquid biopsy methods for breast cancer diagnosis.These methods are economical,non-invasive,simple,rapid,sensitive,and accurate.It is expected to provide new approaches for breast cancer early screening,recurrence and metastasis monitoring,and therapeutic evaluation.The experimental results are as follows:Part 1:A molecular beacon(mpsMB-1246)composed of 2’-O-methyl RNA backbone and an additional phosphorothioate modified loop domain were designed and synthesized for detecting miR-1246.Compared with unmodified molecular beacons(MB-1246),mpsMB-1246 significantly improves ribonuclease resistance,remaining stable in various RNases including RNase A,T1,I and their mixtures even up to 1000 U/mL.In buffer solution,mpsMB-1246 can quantitatively and specifically detect synthetic miR-1246 by generating fluorescent signal in a linear correlation ranged from 0～10 nM with a coefficient of 0.9997,a sensitivity of 2.18 fold/nM,and a detection limit of 0.11 nM.As to the exosomes isolated from MCF-7 cell supernatant,mpsMB-1246 was proved to generate sensitive and quantitative fluorescent signals by specifically targeting encased miR-1246 in simple incubation with the exosomes for 45 min in the presence of Triton X-100.The conclusion was reached by a series of experiments such as anti-interference experiments,super-resolution optical microscopy,transmission electron microscopy(TEM),negative probe control experiments,and measurements of miR-1246 levels in exosome of normal breast epithelial MCF-10A cell,etc.Moreover,mpsMB-1246 can directly response to human plasma exosommal miR1246 by help of Triton X-100 on the exosomal membrane rupture.Recovery rates of standard additions of MCF-7 exosomes into human plasma specimens were obtained at 91.0～103.0%.By the liquid biopsy assay we set up,fluorescent signals exhibited by mpsMB-1246 incubating with 30 μL of plasma in Triton X-100 for 45 min can distinguish 33 breast cancer patients from 37 healthy individuals.The receiver operating characteristic(ROC)curve revealed a 93.94%sensitivity and 97.30%specificity at the best cutoff value of 2.9,with the area under the curve(AUC)at 0.9828.Part 2:A multi-targeting gold nanoparticles fluorescent probe for simultaneously detecting hsa-miR-1246,hsa-miR-21,and cel-miR-54 was synthesized by the freezing method.The morphology and diameter of the multiplexed probes were characterized by UV-Vis,nanoparticle tracking analysis,TEM imaging.The multiplexed probe can detect the 3 synthesized miRNA targets by generating fluorescent signals in a dosedependent way with the best performance of targeting miR-1246 at detection limit of 0.20 nM,sensitivity of 1.60 fold/nM and a linear range of 0～20 nM.A series of experiments of TEM imaging,anti-interference experiments,self-negative reference,and control of normal MCF-10A cell were carried out to demonstrate that the multiplexed probe can enter the MCF-7 exosomes during 1 h of incubation with the exosomes and generate sensitive and quantitative fluorescent signals by specifically responding to the targets.Further standard addition experiments were performed to show that the multiplexed probe can detect exosomes spiked into human plasma with recovery rate of 100.0～108.0%(calculated by fluorescence signal responding to miR1246 level)and 94.0～109.0%(calculated by fluorescence signal responding to miR21 level).The blood biopsy based on the probe incubating with 40 μL of plasma for 1 h can distinguish 21 breast cancer patients from 24 healthy individuals.The ROC curve reached 100%of both sensitivity and specificity at the best cutoff by any of miR-1246 and miR-21 markers. |
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