| Objective:1.Based on the results of high throughput sequencing of the whole transcriptome,to screen lncRNAs which express obviously differently between bladder tumor tissues and relative adjacent tissues2.To verify the differential expression of lncRNA-n346372 between bladder cancer tissues and relative adjacent tissues,and of various bladder cancer cells,as well as to explore the relationship between the expressive level of lncRNA-n346372 and clinical pathologic results3.To explore the roles what lncRNA-n346372 plays for bladder cancer cells in proliferation,migration,invasion,and apoptosis4.Further analysis was performed to verify the correlation between lncRNAn346372 and mRNA Cyclin B1,and to predict the possible regulatory networkMethods:1.A new generation of sequencing technology was used to survey the whole transcriptome of ten pairs of bladder cancer tissues and adjacent normal tissues.Wilcoxon rank sum test,DESeq2,and EdgeR were used to analyse the results as statistical methods.P values were inferior to 0.05,fdr values were inferior to 0.05,and Fold Change values were more than 2,mading up together the screening strategy,which assisted to screen lncRNAs with obviously different expression,and in order to narrow the range for the following certification.2.Total RNA was dissolved and extracted from forty pairs of bladder cancer tissues and adjacent normal tissues and five kinds of bladder cancer cells,and then was transcribed reversely to cDNA.The relative expression of lncRNA-n346372 of previous screening results was furtherly verified at tissues and cells level through Real Time qPCR technology and FISH technology,while the differential expression of lncRNA-n346372 between high and low grade tumor tissues were identified using Student’s t test,clarifying the relevance.3.To construct the T24 cells transfected with lentivirus containing sh RNA of lncRNA-n346372 and the cells called 5637 overexpressed lncRNA-n346372,and with the assistance of technology such as CCK-8,Migration Assay,and Flow Cytometry,we had observed the change of function of variety cells after being managed compared with control groups.4.On the basis of numerous lncRNAs of differential expression screened by high throughput sequencing,and according to correlation analysis from bioinformatics,the mRNAs which possibly related with lncRNA-n346372 were predicted,the correlation of both which were certified at tissues and cells level through Real Time qPCR,Immunohistochemistry,and Weston Blot,which could provide evidences for the following studies,including the correlation of function and regulating mechanism available.Results:1.According to previous strategy,we had totally screened out thirty-two lncRNAs whose expression level in bladder cancer tissues were over two times than that in adjacent normal tissues,and nine of which have fold change over three.lncRNA-n346372 with the highest fold change was 3.96 times,the mean expression level in tumor group was 4.29,and that in control group was 0.20.2.Ninety per cent of paired tissues(36/40)had been certified the existence of differential expression,the expression level of lncRNA-n346372 in bladder cancer tissues was higher,which also confirmed by FISH experiment in tissues.Moreover,the expression level of lncRNA-n346372 in high grade bladder cancer tissues exceeded which in low grade bladder cancer tissues,such level in T24 bladder cancer cells was higher than that in 5637 bladder cancer cells.3.We had successfully constructed two kinds of bladder cancer cells transfected with lentivirus,and have observed that the function of proliferation,migration,and invasion of bladder cancer cells would descended striking after interfering the expression of lncRNA-n346372.However,when it was overexpressed in 5637 cells,such function would enforce correspondingly.4.The results from bioinformatics showed that the significant correlation existed between lncRNA-n346372 and cyclin B1,and the coefficient of correlation,R,was 0.76,which also had been confirmed at nucleic acid level in tissue.We also found that the expression of cyclin B1 in bladder cancer tissues was actually higher than that in normal bladder tissues through Immunohistochemistry and Weston Blot experiments.The expression level of cyclin B1 in cells interfered lncRNA-n346372 was significant under control groups.Conclusion:1.The expression level of LncRNA-n346372 in bladder cancer tissues striking exceeded adjacent normal tissues,and the more pernicious the pathological results,the more higher the expression level.The significant correlation existed between the expression level of lncRNA-n346372 and the pathological grading.2.LncRNA-n346372 takes part in the process of proliferation,migration,and invasion of bladder cancer cells.3.The correlation existed in expression level between LncRNA-n346372 and Cyclin B1,possibly,lnc RNA-n346372 takes part in the function of bladder cancer cells through influencing the expression of cyclin B1 by targeting regulation. |
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