Bladder Cancer Related Circular RNA-2(BCRC-2)Sponges MicroRNA-558 And Suppresses Heparanase To Inhibit Migration,Invasion And Angiogenesis Of Bladder Cancer Cells

Bladder cancer is one of the most common malignacy of urinary system,with a rising tendency of incidence.Although considerable progress has been made in surgical techniques and adjuvant chemotherapies,the mortality of bladder cancer has not sharply decreased due to the characteristic of easily recurrence,invasion and metastasis of bladder cancer.CircRNA is a type of non-coding RNA that forms a covalently closed continuous loop,which was misinterpreted as by-products of splicing errors.With the advent of high-throughput sequencing and bioinformatic analysis,thousands of circRNAs have been successfully identified in multiple cell lines and across various species,CircRNAs are predominantly found in cytoplasm,with the specificity of cells,tissues,development stage and diseases,which exert crucial functions in regulating gene expressions.To research the expression of circRNAs in bladder cancer tissues and its influence on the biological function of bladder cancer cells,we performed the high-throughput circRNA sequeceing on bladder cancer tissues and paired normal bladder tissues.We selected bladder cancer related circRNA-2(BCRC-2)as the candiated circRNA to study the biological function on bladder cancer cells through in vitro and in vivo assays,and finally study the mechanism of BCRC-2 as 'microRNA sponges'.This research is composed of the following three parts:Part ? CircRNA profiles of human bladder cancer tissues and the expression of bladder cancer related circular RNA-2 in clinical specimens and cell lines of bladder cancerObjective:Detecting the circular RNA(circRNA)profiles of human bladder cancer tissues and paried normal bladder tissues,and subsequently selects the differentially expressed circRNA for further study.Methods:Using high-throughput RNA sequencing and bioinformatic analysis,we detected the circRNAs in human bladder cancer tissues and paired normal bladder tissues,and then selected the differentially expressed the circRNA for further study.Next,we designed divergent primers specifically for the candidate circRNA,and confirmed the existence and expression of this circRNA in bladder cancer tissues and cell lines by reverse transcription PCR(RT-PCR)and quantified RT-PCR.Finally,we performed RNA fluorescence in situ hybridization(FISH)to detect the localization of the candidate circRNA.Results:1.By using high-throughput RNA sequencing on 3 pairs of human bladder cancer tissues and normal bladder tissues,we detected 16,535 circRNAs,most of them origined from exons(88.9%),others from introns,linc RNA,intergenic region,3'UTR and 5'UTR.2.Among all these circRNAs,571 circRNAs are differentially expressed between bladder cancer tissues and normal bladder tissues,and 524 circRNAs are downregulated in bladder cancer tissues(91.2%),others are upreguluated.3.The existence of BCRC-2 in bladder cancer tissues and cell lines was validated by RT-PCR.Among the 44 paires of bladder cancer tissues and normal bladder tissues,BCRC-2 was significantly downregulated in bladder cancer tissues(35/44,79.5%),and also related to high pathological stage(31/33),grade(28/31),lymph node metastasis(16/17)and vascular invasion(15/15).The expression of BCRC-2 also significantly downregulated in bladder cancer cell lines(T24T,UMUC3)compared to SV-HUC-1(p<0.01).4.BCRC-2 is resisitence to RNase R treatement,but the HIPK3 mRNA can be digested by RNase R.5.BCRC-2 is predominantly localized in cytoplasm of bldder cancer cells.Conclusion:CricRNA are abundantly expressed in human bladder cancer tissues,most of them originated from exons and downregulated compared to normal bladder tissues.BCRC-2 is predominatly localized in cytoplasm of bladder cancer cells,which downregulated in bladder cancer tissues and cell lines.The expression of BCRC-2 is correlated with pathological stage,grade,lymph node metastasis and vascular invasion.These results indicate that BCRC-2 may exert potential function in promoting bladder cancer progession.Part II Biological effects of bladder cancer related circular RNA-2(BCRC-2)on bladder cancer cells in vitro and in vivoObjective:To explore the biologicacl effects of BCRC-2 on bladder cancer cell lines.Methods:1.The BCRC-2 overexpression plasmid was designed and transfected into T24 and UMUC3 cells,and evaluated the effectiveness of the plasmid by using qRT-PCR.2.Using wound healing assay,transwell invasion and migration assay to detect the changes in the cells that stably overexpressed BCRC-2.3.The small interference RNAs(siRNAs)targeting BCRC-2 were designed and transfected into T24T and UMUC3 cells to knockdown BCRC-2.After that,wound healing assay,transwell invasion and migration assay were carried out to evaluate the ability changes of bladder cancer cells.4.Using qRT-PCR and western blot to detect the expressions of MMP-9 and VEGF in bladder cancer cella that overpress or knockdown the BCRC-2.5.Stable T24T cells transfected with BCRC-2 overexpression plasmids were injected into the subcutaneous and tail vein of the nude mice,and then detected the effectiveness of BCRC-2 on tumor growth and metastasis.Results:1.Compared to cells that transfected with vector plasmids,after transfected with BCRC-2 overexpression plasmids,the expression of BCRC-2 significantly incerased in T24T(fold change 113.8±12.87)and UMUC3(fold change 195.6±14.77)cells.2.The ability of invasion and migration of T24T and UMUC3 cells were significantly decreased after transfecing with BCRC-2 plasmids;however,after knocking down the BCRC-2,invasion and migration abilities increased.4.After transfecting with BCRC-2,the expression of MMP-9 and VEGF decreased;on the other hand,knockdown the expression of BCRC-2 could increased the expression of MMP-9 and VEGF.5.Overpression of BCRC-2 can significantly inhibite the tumor growth and metastasis in nude mice modles.Conclusion:Overpression of BCRC-2 can significantly inhibited the invasion and migration abilities of bladder cancer cells,and BCRC-2 plays an important role in suppressing bladder cancer progression.Part ? The mechanism of bladder cancer related circular RNA-2(BCRC-2)modulating miR-558 on biologic behaviors of bladder cancer cell linesObjective:To explore the mechanism of regulating function of BCRC-2 on the invasion and migration abilities of bladder cancer cell lines.Methods:1.Through overlapping the prediction results of miRanda,PITA and RNAhybrid database,searching the candidate miRNAs that can bind to BCRC-2.2.We designed biotinylated probe that specific to the backsplice sites of BCRC-2,and then detected the specificity of probe by using RNA pulldown assay,meanwhile,to quantify the miRNA that sponged by BCRC-2.3.Using FISH assay to validate the colocalization of BCRC-2 and the candidate miRNA.4.Detecting the expression of the candidate miRNA in bladder cancer tissues and cell lines,then transfecting the miRNA mimics and inhibitors into T24T and UMUC3 cells to evaluate the effection on invasion,migration and angiogenesis by using wound healing assay,transwell invasion and migration assay and tube formation assay.5.miRNA-target gene was detected by using qRT-PCR and western blot.6.Co-transfecting BCRC-2 plasmids and miRNA mimics into T24T and UMUC3 cells then evaluate the effection on invasion,migration and angiogenesis by using qRT-PCR,western blot,wound healing assay,transwell invasion and migration assay and tube formation assay.Results:1.12 candidate miRNAs were predicted after overlapping the results of miRanda,PITA and RNAhybrid database.2.Biotinylated probes could specifically pulldown BCRC-2.3.BCRC-2 could sponge miR-558 in both T24T and UMUC3 cells,and the colocalization of BCRC-2 and miR-558 in cytoplasm were confirmed by using RNA FISH assay.4.The expression of miR-558 was significantly upregulated in bladder cancer tissues compared to normal bladder tissues,the same results also found in T24T and UMUC3 cells that compared to SV-HUC-1 cells.5.Transfection of miR-558 mimics could increasing the expression of heparanase and its downstream MMP-9 and VEGF,which lead to promote the ability of invasion,migration and angiogenesis of T24T and UMUC3 cells,however,transfection of miR-558 inhibitors could downregulate the expression of heparanase,MMP-9 and VEGF,and suppress the invasion,migration and angiogenesis of T24T and UMUC3 cells.6.Over-expression of BCRC-2 reverses miR-558-induced enhancement of cell migration,invasion and angiogenesis in bladder cancer cells.Conclusion:BCRC-2 could suppress cell migration,invasion and angiogenesis through sponging miR-558 and subsequently inhibiting the expression of HPSE and its downstream MMP and VEGF.