The Study On Early Diagnosis And Mechanism Of Bladder Cancer Based On Long Non-coding RNAs Of Urinary Exosomes

purposes This project extracts exosome from the urine of bladder cancer patients using differential ultracentrifugation technology,and to explore its clinical value and molecular mechanism by detecting lnc RNAs in exosomes.Method 1.Collect urine Preoperative patients diagnosed with bladder cancer confirmed by histopathology were selected,and 45 patients who were not receiving any preoperative treatment were selected to collect morning urine.According to the gender and age of bladder cancer patients,25 normal morning urine matched normal people were selected as the control group.All urine samples were stored in a-80 ° C refrigerator.2.Extraction and identification of exosomes Extraction: The method of alternating low speed centrifugation and ultracentrifugation(differential ultracentrifugation)was used to extract exosomes in urine.Identification:(1)Transmission Electron Microscopy(TEM)to identify the shape of exosome;(2)Detection of the concentration and size of exosome by nanoflow detection;(3)Western Blot detected exosome specifically labeled eggs CD81,CD9 and CD63;(4)Exosome specific antibody CD63 was detected by flow cytometry.3.Selection,extraction,detection and data analysis of Lnc RNA According to the literature,five lnc RNAs(UCA1,HOTAIR,BLACAT2,LINC00477)related to bladder cancer were screened,q RT-PCR was used to verify their expression,and lnc RNAs(HOTAIR,BLACAT2,LINC00477)with significant differences were selected.Extraction: Use RNAsimple Total RNA Kit to extract total RNAs from exosome,use a microplate reader to measure the OD value of total RNAs,and check their concentration and purity.Detection: Evo M-MLV reverse transcription kit was used to reverse transcribe RNA into c DNA,and then real-time quantitative PCR technology was used to detect the relative expression of UCA1,HOTAIR,BLACAT2 and LINC00477.Analysis: The expression differences of UCA1,HOTAIR,BLACAT2 and LINC00477 in urinary exosomes of bladder cancer patients and healthy volunteers were compared.Combined detection of HOTAIR,BLACAT2,LINC00477 for early diagnosis of bladder cancer.4.Lifetime analysis The relationship between BLACAT2,HOTAIR,LINC00477 and prognosis of bladder cancer was analyzed using TCGA database.5.Analysis of related mechanisms of BLACAT2,HOTAIR,LINC00477 By starbase,mi RCODE predicts micr RNA that may be combined with BLACAT2,HOTAIR,LINC00477.Through mi Randa,Targetscan,and Pic Tar jointly predict the target phase genes of mi RNA,and then use KEGG and GO to analyze the pathways and biological functions that BLACAT2,HOTAIR,and LINC00477 may affect(p <0.05).Result 1.Extraction by differential ultracentrifugation.The extract was identified by TEM,NTA,western blot and flow cytometry.The results showed that the extract contained a large amount of exosomes.2.q RT-PCR results showed that HOTAIR,BLACAT2,OCT4,and LINC00477 were highly expressed in urine exosomes of bladder cancer patients compared with healthy volunteers,and the differences were statistically significant.3.Through multivariate logistic regression model analysis and drawing ROC curve,it was found that HOTAIR,BLACAT2,LINC00477 combined detection AUC was 0.917.4.Compared with patients with Ta-T1 stage bladder cancer,LINC00477 was up-regulated in T2-T4 stage bladder cancer and related to the invasion of bladder cancer(P = 0.032).The area under the corresponding ROC curve was AUC of 0.721.5.Analysis of TCGA database revealed that high expression of HOTAIR was significantly associated with poor prognosis of bladder cancer.6.The bioinformatics database predicts micr RNAs combined with the lnc RNAs,and targets genes for those micr RNAs.Then analyze the pathways and biological functions that BLACAT2,HOTAIR and LINC00477 may affect.Conclusion 1.The expression of lnc RNAs(HOTAIR,BLACAT2,OCT4,LINC00477)in urine exosomes of bladder cancer patients will be different from that of healthy people.2.Through multivariate logistic regression model analysis and drawing ROC curve,it was found that the combined detection of HOTAIR,BLACAT2,and LINC00477 is promising as a method for early diagnosis of bladder cancer.