Experimental Study Of Detection Exosomal RNA Biomarkers By TCLN Chip For Early Diagnosis Of Prostate Cancer

Prostate Cancer?PCa?is one of the most common malignant tumors in older men in worldwide.Its mortality ranks fifth in male malignant tumors and first in genitourinary tumors^[1].In China,due to the lack of effective early screening methods,most pat ients with prostate cancer are diagnosed with advanced or metastatic.Therefore,it is urgent to explore rapid non-invasive experimental methods for prostate cancer screening and early diagnosis.Exosomes are nano-sized small vesicles secreted by cells,rich in various proteins,nucleic acids and lipids.Among them,the most studied are nucleic acids,and mainly RNA,whose expression can reflect the physiological and pathological state of the body.Studies have shown that exosomes penetrates various stages of tumorigenesis and development,so exosomal RNA has potential as a tumor biomarker.TCLN?Tethered Cationic Lipoplex Nanoparticle?chip is a new exosomes detection technology.It was introduced from the United States in 2015 and obtained Chinese patent?ZL201310073081.3?.Its biggest feature is in-situ-detection of exosomes,without RNA extraction and amplification,so the sample is used in a small amount,the analysis speed is fast,and the detection cost is low.It is a good technical model for the conversion of exosomal RNA into a clinical detection method.Therefore,this study intends to use TCLN technology to detect exosomal RNA biomarkers in blood and urine from prostate cancer patients,establish a diagnostic model,and provide new methods and new ideas for early rapid diagnosis of prostate cancer.Based on this,we conducted the following two parts of research:Part 1 Screening of serum exosomal RNA biomarkers for prostate cancer and establishment of TCLN chip diagnostic methodObjective:To screen exosomal RNA with different expression of PCa and normal controls as a candidate biomarker,perform TCLN chip detection and analyze differences between groups,establish a diagnostic model and evaluate its diagnostic efficacy.Methods:Eight candidate exosomal RNA biomarkers,including 4 microRNAs and 4mRNAs,were selected by literature search combined with previous research.Exosomes were separated from serum by invitrogen precipitating reagent.Morphological characteristics of serum exosomes were observed by transmission electron microscope.Exosomes concentration and particle size were detected by NTA.Exosomes potential was detected by DLS.The expression of candidate exosomal RNA biomarkers were verified by qRT-PCR in 21 cases of PCa,20 cases of patients with benign prostatic hyperplasia?BPH?and 16 normal controls;The expression of the above candidate exosomal RNA biomarkers in 44 cases of PCa,40 cases of BPH patients and 36 normal controls was detected by TCLN chip.The mean comparison between the two groups was perfo rmed using the nonparametric Mann-Whiteney U test.The difference was statistically significant at P<0.05.The ROC curve was constructed to evaluate the ability of each candidate marker to distinguish PCa from normal controls,to construct a diagnostic model and to evaluate the diagnostic efficacy.Results:1.Extraction and identification of serum exosomes:serum precipitates isolated by invitrogen precipitation reagent,particle size of 30¹50 nm,uniform size,maximum particle size distribution of 99 nm,both negatively charged,expressing exosomes specific proteins ALIX and CD63,in line with the exosomes feature.2.The expression of serum exosomal miR-141,miR-375,miR-21 and PCA3 mRNA in the PCa group was significantly higher than that in the normal control group by small sample qRT-PCR.The P values were less than 0.05.3.Using TCLN chip to detect serum candidate exosomal RNA biomarkers,miR-141 was found to be optimal,which can be used to distinguish between PCa and healthy people?P<0.01,AUC=0.709?,and can also distinguish between PCa and BPH?P<0.01,AUC=0.719?.4.Using TCLN chip to detect serum candidate exosomal RNA biomarkers,PCA3 mRNA,PSA mRNA,let-7c and miR-375 were found to distinguish prostate cancer from healthy people?P<0.05,among which AUC<0.7 of let-7c and miR-375?,but can not distinguish between PCa and BPH?P>0.05?.5.The three biomarkers of miR-141,PCA3 mRNA and PSA mRNA were selected to establish a model.The AUC of PCa was diagnosed as 0.800,the sensitivity was 81.8%,and the specificity was 72.4%.Conclusion:TCLN chip was used to detect 8 candidate exosomal RNA biomarkers.After statistical analysis,PCa diagnostic models were established using three RNA biomarkers?miR-141,PCA3 mRNA,PSA mRNA?with significant differences between groups,AUC=0.800,which has potential diagnostic value.Part 2 Screening of exosomal RNA biomarkers in urine from prostate cancer and preliminary study on the TCLN chip diagnostic methodsObjective:To screen exosomal RNA with differential expression of PCa and normal controls as a candidate marker,to detect small sample size by TCLN chip and analyze the differences between groups,and to explore its potential value for PCa diagnosis.Methods:Through the literature search combined with the previous research basis,8 first batch of candidate exosomal RNA biomarkers in urine were selected,including 4microRNAs and 4 mRNAs,and 8 microRNAs in the second batch.The exosomes purification method from urine was optimized,and the method of“precipitation after concentration”was established.The morphology,concentration,particle size and potential of exosomes were detected by transmission electron microscopy,NTA and DLS.After determining the separation of exosomes,the effect of repeated freeze-thaw and freezing time on exosomal RNA content was further clarified.The differential expression of the first batch of candidate biomarkers was verified by qRT-PCR in 12 PCa,12 BPH patients and 15 normal controls The expression levels of the above candidate exosomal RNA biomarkers in 20 PCa,20 BPH patients and 19 normal controls were initially detected by TCLN chip.The nonparametric Mann-Whiteney U test analyzed the differences between the two groups.Results:1.The optimized method was used to separate the urine exosomes of the same source from the traditional sedimentation method.The morphology,particle size and concentration were not significantly different,and the precipitates extracted by Western blot were consistent with exosomes.2.Exosomes of 13 urine samples were extracted by"conc entration plus precipitation" method.The concentration of exosomes was 6.3×10¹⁰(1.8×10¹⁰¹.75×10¹¹) particles/mL by NTA.3.The RNA content of urine exosomes decreased gradually after repeated freezing and thawing.After 5 freeze-thaw cycles,the exosomal let-7c and PSA mRNA contents were significantly lower than fresh exosomes,P values were:0.019,0.018.4.qRT-PCR preliminary verification of the first batch of candidate exosomal RNA biomarkers in urine:1)Compared with the normal control group,the expression of exosomal miR-21,PCA3 mRNA and T1-E2 mRNA in PCa group increased,P values were 0.0303,0.0060,and 0.0480,respectively;2)Compared with the normal control group,the expression of exosomal miR-375,let-7c,and miR-141 in the PCa group decreased,and the P values were 0.0452,0.0042,and 0.0347,respectively;3)There was no significant difference in the expression of exosomal PSA mRNA and T1-E4 mRNA in the PCa group compared with the normal control group.5.TCLN chip detected 20 cases of PCa,20 cases of BPH and 19 healthy controls of the irst batch of urinary exosomal RNA,compared with the normal control group,exosomal miR-375 and let-7c significantly decreased in the PCa group,P value was ess than 0.05,initially showed diagnostic potential;compared with the BPH group,the expression of exosomal miR-375 in the PCa group was decreased,P<0.001.The xpression of the remaining 6 biomarkers was not statistically different between the 3 roups.Conclusion:The optimized method of"concentration plus precipitation"can stably separate exosomes from urine,and the isolated exosomes can be stably maintained at-80°C for a short time,but repeated freeze-thaw should be avoided;The expression of the first batch of urine candidate exosomal RNA biomarkers was detected by TCLN chip.The expression of let-7c and miR-375 in PCa group was decreased,and the diagnostic potential was initially shown.