Comprehensive Analysis Of Aberrantly Expressed Profiles Of LncRNAs And MiRNAs With Associated CeRNA Network In Muscle-invasive Bladder Cancer

Background and ObjectivesBladder cancer is one of the most common tumor occurring in the urinary system.It is seriously harmful to human health.The recurrence and mortality of muscle-invasive bladder cancer is higher than non-muscle-invasive bladder cancer,so it becomes the focus of human research.Long non-coding RNAs(LncRNA)are non-protein coding transcript longer than 200nt(nucleotide).Although initially thought to be transcriptional noise,IncRNA are gaining increased attention in human cancers as its diversity function.Researches show that it play an important role in carcinogenesis and cancer metastasis.For urinary tumors,the study of IncRNA related to prostate cancer starts early,and researchers have found many IncRNA related to prostate cancer,such as PCGEM1,DD3/PCA3,PRNCR1,etc.But the study of bladder cancer is not sufficient.The interaction of lncRNA and miRNA play an important role in the cause and development of tumor.Based on this fact,the hypothesis of competing endogenous RNA(ceRNA)was raised,and was confirmed by numbers of studies.In this hypothesis,lncRNAs,mRNAs and other RNAs could regulate each other functionally.Normally,all kinds of RNAs in ceRNA network keep a dynamic balance.Downregulation and upregulation would break the balance,and lead to the occurrence of related illness subsequently.IncRNA-miRNA-mRNA ceRNA network has been constructed in gastric cancer,hepatocellular cancer,breast cancer and pancreatic cancer.However,similar studies are rare in bladder cancer.The Cancer Genome Atlas(TCGA)which aimed to construct a complete atlas of genome change related to cancer,was composed of NCI and NHGTI initially.It has collected approximately 10000 patient samples together with clinicopathological information across more than 30 cancer types.The database not only contains clinical data,but also contains the gene sequencing results.At present,more than 50 thousand lncRNA have been cloned,but it is difficult to find the functional IncRNA related to tumor.The TCGA is a rich resource for data mining and biological discovery.With the aid of bioinformatics tools and related sequencing statistical tools,we can abandon the irrelevant information and find potential gene locus.Research methodBioinformatics is a cross discipline that involves computer science,mathematics and biology.It depends on the basis of computer science,engineering and applied mathematics and the storage of experiment and derivative data.In our research,we extracted level 3 RNAseq and miRNAseq from 341 samples through TCGA database.And the data were classified into two cohorts:322 muscle-invasive bladder cancer and 19 nomal samples.After combining the tumor sample and normal sample data,the RNA and miRNA which are not expressed were filtered out.Then,the original data were processed by DESeq.So we got the differentially expressed RNAs(DERNAs)and differentially expressed miRNAs(DEmiRNAs)between the bladder cancer and normal tissue.We use the GENCODE IncRNA annotation(V22)to identify the DERNAs further.The interaction of IncRNA and miRNA was predicted by miRcode database,and the target mRNAs of miRNA was searched by miRTarBase database.Then,we construct the IncRNA-miRNA-mRNA ceRNA network successfully.To prove the reliability of differentially expressed lncRNA obtained from the TCGA database,we selected 3 lncRNAs(LINC00162?AATBC?UCA1)predicted to be up-regulated and 4 lnRNAs(MIR100HG?MIR143HG?C20orf166-AS1?HAND2-AS1)predicted to be down-regulated to verify.These lncRNAs were detected by real-time PCR in 32 pairs of muscle-invasive bladder cancer tissues and paracarcinoma tissues.And the HAND2-ASI not reported in bladder cancer was choosed for further study including detecting the expression level of HAND2-ASI in SV-HUC-1,5637,RT4 and J82 cell lines,and detecting the influence of HAND2-ASI on the proliferation,invasion and migration of bladder cancer cell by MTT,wound scratch assay and Transwell migration assay.Results1.Comprehensive analysis of aberrantly expressed profiles of lncRNAs and miRNAs with associated ceRNA network in muscle-invasive bladder cancer1.1 Differentially expressed RNAs(DERNAs)in muscle-invasive bladder cancerIn our research,we investigated RNA expression levels in 322 muscle-invasive bladder cancer tumor tissues(Cohort T)and 19 normal tissues(Cohort N).Genes with absolute log 2 fold change greater than 1 and corrected P value less than 0.01 were considered as differentially expressed.As a result,a total of 472(34.08%)up-regulated and 913(65.92%)down-regulated genes were identified(Table 1).Meanwhile,to better understand the mechanisms of the occurrence of muscle-invasive bladder cancer,functional characterization of the DERNAs was performed KEGG analysis through KOBAS 2.0.The results showed that 27 DERNAs,including 21 up-regulated and 6 down-regulated,were involved in cell cycle.1.2 Differentially expressed lncRNAs(DElnRNAs)in muscle-invasive bladder cancerAccording to the cut-off criteria of |log2FoldChange|>1.5 and corrected P value<0.01,11 lncRNAs were considered as aberrantly expressed in muscle-invasive bladder cancer tissues.The expression of 4 lncRNAs(LINC00162,AATBC,UCA1,EPR1)was significantly up-regulated.And the expression of 7 lncRNAs(MIR2HG,MIR100HG,BRE-AS1,MIR143HG,C20orf166-AS1,HAND2-AS1,PGM5-AS1)were down-regulated.We divided the samples into several subgroups according to TNM stages(T3+T4 vs.T1+T2,N2+N3 vs.N0+N1,M1 vs.M0)as well as with or without lymphovascular invasion(Yes vs.No).After comparison analysis for IncRNA expression profiles,we identified 8 IncRNAs of high level(LINC00221,UCA1,ZFHX4-AS1,LINC00284,LOC283731,LINC00698,CNTFR-AS1,and LOC284379)and 4 IncRNAs of low level(LINC00390,SSTR5-AS1,HECTD2-AS1,and LOC400696)involved in the progression of muscle-invasive bladder cancer.1.3 Differentially expressed miRNAs(DEmiRNAs)in muscle-invasive bladder cancerTo construct the lncRNA-miRNA-mRNA ceRNA network,we also compared the miRNA expression difference between tumor tissues and normal tissues.11 DEmiRNAs,including 2 up-regulated and 9 down-regulated,were identified.We also investigate the overall survival related to DEmiRNAs in muscle-invasive bladder cancer patients through Kaplan-Meier curve analysis.5 out of these 11 DEmiRNAs,including let-7c,mir-100,mir-143,mir-1-2 and mir-145,were proved to have negative effects on patient survival.1.4 The establishment of ceRNA network in muscle-invasive bladder cancerWe established a disordered lncRNA-miRNA-mRNA ceRNA network based on above data.Results showed that 32 DERNAs were involved in the network.And some of them have been reported as cancer-related genes such as CDC25A,CDKNIA,MMP1,MMP13 and SOX4.Moreover,the regression analysis between the expression levels of DElncRNAs and DERNAs showed a perfect correlation between them.We also analyzed the signal pathways indirectly regulated by these 32 DERNAs,and found 10 KEGG pathways were significantly enriched,including 4 cancer-related pathways-MicroRNAs in cancer,Bladder cancer,Cell cycle and Chronic myeloid leukemia.2 Preliminary functional verification of Differentially expressed lncRNAs(DElnRNAs)in muscle-invasive bladder cancer and functional research of HAND2-AS12.1 Real-time PCR results of lncRNAs predicted to be aberrantly expressed in bladder cancerWe selected 3 lncRNAs(LINC00162?AATBC?UCA1)predicted to be upregulated and 4 lnRNAs(MIR100HG?MIR143H?C20orf166-AS1?HAND2-AS1)predicted to be downregulated from the DElnRNAs for preliminary functional verification.qRT-PCR was performed to detect the expression level of these 7 IncRNAs in 32 paired bladder cancer tissues and para-carcinoma tissues.The results show that the expression of AATBC and UCA1 which are predicted to be up-regulated,MIR100HG?MIR143HG?C20orf166-AS1 and HAND2-AS1 which are predicted to be up-regulated are consistent with prediction results.While the expression level of LINC00162 which is predicted to be up-regulated is down-regulated actually.The overall coincidence rate of these 7 lncRNAs is 85.7%.2.2 Real-time PCR result of HAND2-AS1 in bladder cancer tissueqRT-PCR was performed to detect the expression level of HAND2-AS1 in SV-HUC-1,5637,RT4 and J82 cell lines.The results show that HAND2-AS1 is down-regulated in bladder cancer.2.3 Influence of HAND2-AS1 on the proliferation,invasion and migration ability of bladder cancer cellsiRNA was designed to down-regulate the expression of HAND2-AS1.The cell proliferation curve shows that HAND2-AS1 knockdown resulted in increase of cell proferation.Meanwhile,HAND2-AS 1 is proved to inhibit the proliferation,invasion and migration of bladder cancer cell by wound scratch assay and Transwell migration assay.Conclusions1.Based on the analysis of large sample of muscle-invasive bladder cancer from TCGA database,we identified the DElnRNAs and DEmiRNAs between the bladder cancer and normal tissue.Then,the IncRNA-miRNA-mRNA ceRNA network was constructed.And related pathway analysis proved that during the occurrence and development of bladder cancer,various lncRNAs and miRNAs involve in the ceRNA nerwork,and take part in multiple signal pathways together.2.Based on the TCGA database,we obtained the expression profiles of aberrantly expressed lncRNAs in muscle-invasive bladder cancer.qRT-PCR revealed that the expression of AATBC and UCA1 were significantly up-regulated in muscle-invasive bladder cancer,while the MIR100HG?MIR143HG?C20orf166-AS1 and HAND2-AS1 were down-regulated,which accorded with the data analysis results previously.In addition to these 7 lncRNAs,there are several lncRNAs need to be validated.3.Based on the TCGA database and preliminary experimental verification,we found that the lncRNA-HAND2-AS1 play an important role in tumor inhibition for the first time.The following experiment showed that HAND2-AS 1 could inhibit the prolifera-tion,migration and invasion of cancer cell.But the construction of over expression plasmid and related animal experiments need to be performed.