The Mechanism Of Hsa₀006332 In Affecting The Biological Behavior Of Bladder Cancer Cells

Bladder cancer is one of the most common cancers in urinary system,with an estimated 430,000 new cases and 165,100 deaths worldwide each year.75% patients with high-risk of bladder cancer recur,progress,or die within 10 years,no more than 6% patients with high-grade or muscle-invasive bladder cancer survive for 5 years.Bladder cancer may result from a series of genetic changes caused by certain chemicals,pathogens,or physical stimuli.Smoking is a major risk factor for bladder cancer,and smokers are with 2-6 times higher risk in developing the disease.However,the specific pathogenesis of bladder cancer is still unclear,and ideal biomarkers are lack in the diagnosis,treatment and prognosis of the disease.Therefore,the screening of pathogenic genes and the study on the mechanism are of great significance for the diagnosis and treatment of bladder cancer.Total transcriptome analysis of bladder cancer based on high-throughput sequencing technology is of great significance for the study of the occurrence and development mechanism in bladder cancer.Due to its conserved nature,high expression abundance and tissue specificity,circRNAs are expected to be ideal biomarkers for some diseases,especially cancer.Recent studies have shown that circRNA are involved in the pathogenesis,progression and prognosis of bladder cancer.Therefore,the whole transcriptome analysis,screening,detection and research circRNA will contribute to the diagnosis and treatment of bladder tumors.The study was divided to three section.Part ? Transcriptome sequencing of bladder cancerObjective: Bladder cancer tissues and adjacent tissues of 5 patients were analyzed by whole-transcriptome high-throughput sequencing to identify new transcripts and detect differentially expressed transcripts,so as to find new biomarkers for diagnosis,treatment and prognosis of bladder cancer and screen disease-causing genes.Methods: Tissue of total or partial resection of bladder cancer was collected,Trizol was used to extract RNA,agarose electrophoresis was used to test RNA integrity,,Hiseq ? was used to sequence after building sequencing library and performing quality control.Then we analyze the data to construct the gene expression patterns,genetic variations through cuttdiff screening,then perform GO function analysis and KEGG pathway enrichment for significant differently expressed RNAs,and construct correlation express network and map ceRNA networks by using online bioinformatics software CircInteractome,miRwalk and TargetScan.Results: The 5 coupled tissues included in the study were qualified for quality control.The sequencing results showed that a total of 118,304 transcripts were assembled,including 68795 mRNA,38606 lncRNA,3376 circRNA,6155 pseudogenes and 1372 other results.mRNA with differential expression was screened for GO functional enrichment.The GO results showed that mainly enrichment terms of high expression gene were nucleosome aggregation,mitosis,cell cycle,cell division,nucleosome and chromatin DNA binding.The mainly enrichment terms of low expression gene were interstitial migration,negative regulation of protein phosphorylation and cyclic adenosine phosphate kinase inhibitor activity.KEGG pathway analysis showed that up-regulated gene mainly enriched in pathways of glutathione metabolism,DNA replication and cell division.The downregulated gene mainly enriched in pathways of calcium ion pathway,arginine,proline metabolism and cyclic guanosine phosphate protein kinase pathway.RNAs with correlation greater than 0.99 or less than-0.99 were selected to draw the co-expression network of ncRNA-RNA,and the top 20 ncRNAs of significant differentially expression were selected to draw the ceRNA network.Conclusion:New transcripts found in sequencing might be used as biomarkers for the diagnosis and prognosis of bladder cancer.Co-expression analysis and ceRNA network are helpful to understand the function of non-coding RNA.Functional enrichment of mRNA is helpful for the study of pathogenesis of bladder cancer and the search of therapeutic targets.Part ? The expression of hsa_circ₀018069 in bladder cancer and its clinical significanceObjective: Differentially expressed circRNA,lncRNA and RNA may contribute to the diagnosis,treatment and prognosis of bladder cancer,and may play a role in the pathogenesis and progression of bladder cancer.Therefore,we screened seven differentially expressed RNAs from them,to detect their expression level by qRT-PCR,and analyzed their expression significance.Methods: RNA was extracted from coupled bladder cancers and adjacent tissues of 29 patients by Trizol method for realtime-PCR verification.The expression levels of 7 selected RNAs were detected.One of the circRNA,hsa_circ₀018069,were analyzed about its expression and clinical significance.The relationship between mRNA and hsa_circ₀018069 expression in the sequencing results was screened by the Pearson correlation coefficient,GO and KEGG analyses were performed for mRNA with high correlation,ceRNA network was plotted based on the prediction in bioinformatics online software,and the mRNA in the network was selected for GO and KEGG analysis.GO and KEGG analysis of selected mRNA were helpful for determining the possible function of hsa_circ₀018069 in bladder cancer.Results: Among the 7 selected RNAs,circRNA-IGHG2 was not amplified,circRNAPGM5,circRNA-KIAA1462,lncRNA IGFBP-AS1 was down-regulated in bladder cancer,and lncRNA GATA3-AS1 and LINC00885 and mRNA DAP were up-regulated.The expression trend of selected RNAs was consistent with the sequencing results.Hsa_circ₀018069 was significantly low expressed in bladder cancer?P <0.01?,and its low expression is closely related to tumor invasion?P=0.046?,which can be used as a biological marker for the early diagnosis of bladder cancer?AUC: 0.708,sensitivity 90%,specificity 53%?.Through bioinformatics analysis,we found that hsa_circ₀018069 might target miR-23 c,miR-34a-5p,miR-181b-5p,miR-454-3p and miR-3666 to affect the invasion of bladder cancer through the focal adhesion pathway.Conclusion:The expression trend of the screened RNA in bladder cancer tissues was basically consistent with the sequencing results.Hsa_circ₀018069 is down-regulated in bladder cancer,and its low expression is related to T stage and muscular invasion,which can be used as a marker for early diagnosis of this disease.Bioinformatics predicted that hsa_circ₀018069 might play a role in tumor inhibition through the focal adhesion pathway.Part ? The mechanism of hsa_circ₀006332 in bladder cancerObjective: We screened the data and found that hsa_circ₀006332 was also a significantly differentially expressed circular RNA,which may be involved in the pathogenesis and progression of bladder cancer.Therefore,we performed a further study for its mechanism in bladder cancer through analyzing its ring characterization,expression and function.Methods: The spliced junction of hsa_circ₀006332 were analyzed by direct Sanger sequencing,online database was used to compare its sequnce,and exonuclease digestion were used to determine its ring characteristics.Subcellular localization of hsa_circ₀006332 was performed by fluorescence in situ hybridization and nucleoplasmic separation PCR.The expression level of hsa_circ₀006332 in bladder cancer was detected by realtime-PCR,and the single-factor chi-square test was used for its clinical correlation analysis.The expression hsa_circ₀006332 was interfered in T24,UM-UC-3 cell lines,then CCK-8,EdU,clonal formation experiment were conducted to determine the effect of hsa_circ₀006332 on cell proliferation in bladder cancer.Transwell was used for invasion detection.Double luciferase reporter gene assay was used to verify the binding sites and binding effects of hsa_circ₀006332 and predicted miRNA,as well as the predicted miRNA and MYBL2.Xenotransplantation model in nude mice was used to confirm the effect of circ₀006332 on the growth of tumors.Results: Hsa_circ₀006332 was formed by the 8 and 9 exon of MYBL2 gene,with a length of 554 nts and a splicing junction of GC,which was resistant to exonuclease digestion and located in cytoplasm of bladder cancer cells.Its high expression in bladder cancer tissue?P<0.001?contributes to the early diagnosis of bladder cancer?AUC: 0.86,sensitivity: 81.3%,specificity: 80.2%?,and is associated with TNM stage?P= 0.014?and muscular invasion?P=0.029?.After silencing circ₀006332,bladder cancer cell viability,DNA synthesis ratio,cloning ability and invasion ability were inhibited.When circ₀006332 was overexpressed,DNA synthesis ratio,cloning ability and invasion ability of bladder cancer cells were promoted.Circ₀006332 was positively correlated with the expression of MYBL2 in tissues and cell lines,and it competitively bind with miR-143 and miR-1182 to affect the expression of MYBL2 to further affect the proliferation and invasion of bladder cancer cells.Circ₀006332 affected the growth of transplanted tumors in nude mice.Concusion:Circ₀006332 is a valuable biomarker in the early diagnosis of bladder cancer.Circ₀006332 can sponge miR-143 and miR-1182 to affect the expression of MYBL2,and plays an important role in the proliferation and invasion of bladder cancer.