| Background&objective:Bladder cancer is highly prevalent in the population and is often accompanied by poor prognosis,which is mainly caused by the limited existing screening and diagnostic methods for patients with bladder cancer,and patients are always diagnosed at an progressed stage.Therefore,a biomarker with high sensitivity and specificity is urgently needed to help clinical screening and diagnosis of bladder cancer,predict the progression of cancer,guide personalized treatment,and improve the prognosis of patients.Methods:1.Screening of differentially expressed LncRNA in bladder cancer:1)Sample source:This study included 50 cancer tissue and adjacent normal tissue samples from bladder cancer patients before and after surgery in Tongji Hospital,Tongji University from June 2014 to June 2018.2)Screening of LncRNA differentially expressed genes;RNA samples from bladder cancer cells and adjacent normal cells were extracted for LncRNA differential expression analysis;3)Data analysis:Gene Spring GXv 11.5.1 software package(AgilentTechnologies)for quantile normalization and subsequent data processing.Differentially expressed LncRNAs and mRNAs that exhibited statistically significant differences were identified by VolcanoPlot filtering,and differentially expressed LncRNAs and mRNAs were identified by fold-change filtering.Second,the exploration of the mechanism of LncRNA CASC11 in bladder cancer:1)the bladder cancer patients used in this section were consistent with the first part,and the bladder cancer cell lines were HT-1197 and HT-1376:2)LncRNA CASC11 and iniR-150 expression was detected using real-time PCR;3)the LncRNA CASC11 overexpression cell line was constructed by cell transfection;4)CCK-8 was used to observe the effect of LncRNA CASC11 overexpression on cell proliferation;5)transwell was used to observe the effect of LncRNA CASC11 overexpression on cell migration and invasion;6)In this study GraphpadPrism6 software was used for all of the statistical analyses.All of the experiments were repeated three times.And their mean ± standard deviation was calculated carefully.Receiver operating characteristic(ROC)curve analysis was used to evaluate the diagnostic potential of plasma levels of LncRNA CASC11 and miRNA-150 for bladder cancer,with bladder cancer patients as true positive cases and healthy controls as true negative cases.Third,the regulatory mechanism of LncRNA CASC11 in urine exosomes in bladder cancer:1)the study subjects were consistent with the above studies,2)the isolation and detection of exosomes were analyzed using transmission electron microscopy,nanoparticle technology analyzer and Western blot;3)the expression level of LncRNA CASC11 in exosomes was detected by real-time PCR;4)the tumor stage and grade described by the WHO 2004 grading scheme were consistent with the tumor-nodemetastasis(TNM)staging system for staging,respectively.Results:1.Screening of differentially expressed LncRNAs in bladder cancer:1)Differentiated RNA expression analysis:the results of RNA extraction showed that the quality of extracted RNA met the needs of subsequent differentiated LncRNA detection;2)Scatter plot was used to visually assess the LncRNA expression variation or reproducibility between two samples or two groups of samples,setting the default fold change to 2.0,and LncRNAs showing at least 2.0-fold difference between the comparison samples or two comparison sample groups.3),the hierarchical clustering was to analyze LncRNA expression;4)the parameter screening values of the analysis mapped log2 fold changes ≥ 2.0 and P values≤0.05,and the two groups were subjected to volcanic heatmap for cluster analysis to screen differentially expressed LncRNAs.5),among the differentially expressed LncRNAs,the differential expression of LncRNACASC11 presented a situation of high expression,with the expression level being the highest among all LncRNAs,an increase of nearly 100-fold.Second,the mechanism of action of LncRNA CASC11 in bladder cancer was explored:1)The target genes of LncRNA CASC11 were predicted,and the data showed that LncRNA C.ASC11 may regulate the expression of mRNA-150,iniR-676-3p,and inicroRNA-302,and in HT-1197 and HT-1376 as well as solid bladder cancer cells,the iniR-150 gene expression level was significantly decreased.The difference of decreased expression was statistically significant,P<0.01,while the expression of miR-676-3p and inicroRNA-302 was not significantly different in bladder cancer cell lines and adjacent normal cells;2)Compared with healthy control patients,the plasma LncRNA CASC11 expression level was significantly increased in bladder cancer patients,while the expression level in the plasma miRNA-150 level bladder cancer patient group were significantly lower than those in the control group;3)ROC curve analysis was uesed to assess the diagnostic value of plasma LncRNA CASC11 and miRNA-150 levels for bladder cancer.For plasma LncRNA CASC11,the area under the curve was 0.8990,a standard error of 0.02478%and a 95%confidence interval(0.8505-0.9476)(P<0.0001;Figure 2A).For plasma miRNA-150,the area under the curve was 0.9147,the standard error of 0.02243%,and the 95%confidence interval(0.8707-0.9586)(P<0.0001;Figure 2B);4)LncRNA CASC11 expression was negatively and significantly correlated with miRNA-150 in bladder cancer patients,but there was not any significant correlation between LncRNA CASC11 expression and miRNA-150 in healthy people controls;5)miRNA-150 expression was inhibited in HT-1197 and HT-1376 bladder cancer cell lilies overexpressing LncRNA CASC11,and overexpression of miRNA-150 failed to significantly affect LncRNA CASC11 expression in cell lines;6)overexpression of LncRNA CASC11 could significantly promote the proliferation of bladder cancer cell lines,overexpression of LncRNA CASC11 could inhibit the expression of iniR-150,and overexpression of LncRNA CASC11 could inhibit the expression of miR-150,which in turn promoted the proliferation ability of cells;7)LncRNA CASC11 and miR-150 had no significant effect on cell invasion and migration.Third,the regulatory mechanism of LncRNA CASC11 in urine exosomes in bladder cancer:1)there was no significant difference in the general clinical information of patients;2)identification of exosome isolation:the effect of exosome isolation was detected using electron microscopy,NTA and westernblot;the above exosome particle size detection results were consistent with those reported in the literature,demonstrating that the exosome extraction was correct and would be performed for subsequent study.3)Analysis of exosome secretion level in patients with different tumor grades:The exosome level in patients gradually increased with the deepening of tumor grade.Exosomes levels in patients with stage I,II,IIIA,IIIB,ⅣA,ⅣB bladder cancer were 0.58 μg/ml,1.24 μgml,1.89 μg/ml,2.65 μg/ml,4.39μg/ml,and 6.67 μg/ml,respectively.4)The results of real-time PCR showed that the expression level of LncRNACASC11 in the urine exosomes of patients increased before surgery,about 3 times higher than the normal level,and its expression level decreased significantly after surgery,returning to the level of healthy patients;5)With the increase of tumor grade,the secretion level of exosomes in the urine of patients increased,while the expression level of LncRNACASC11 in the urine exosomes of patients increased can be detected.6),exosome membranes can protect LncRNAs from degradation,and their excellent stability makes exosome LncRNAs ideal biomarkers for tumor diagnosis.CONCLUSION:The expression of LncRNA CASC11 is significantly up-regulated in bladder cancer cells,which regulates bladder cancer tumorigenesis and development by inhibiting miR-150.Detection of LncRNA CASC 11 secretion levels in exosomes suggests that LncRNA CASC 11 has great potential as a biomarker for bladder cancer detection. |
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