Long Non-coding RNA(LncRNA)Expression Profile And Function In Bladder Cancer

BackgroundAccording to the latest data from our cancer center,there are 805,000 new bladder cases nationwide each year,and 32,900 cases of bladder cancer.The bladder cancer is divided into non-muscle invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(MIBC)based on infiltration depth.The recurrence rate of NMIBC range from 50%to 70%within 5 years,and up to 17-45%of NMIBC may develop malignant progression.Currently,high-grade invasive bladder cancer has no ideal therapies,but to choose the radical cystectomy and chemotherapy.Therefore,it is still imperative to explore the underlying potential mechanisms of malignant progression of bladder cancer.Long non-coding RNAs(lncRNAs)are>200nt transcripts in length and are types of RNAs that not translated into proteins.More than 80%of the mammalian genome is composed of lncRNAs.Accumulating evidence supported that lncRNAs participated in the malignant processes in cancer,such as tumorigenesis and metastasis.During the past years,few dysregulation lncRNAs have been discovered and played important role in bladder cancer.For examples,lncRNA UCA1 promote the invasion and metastasis of bladder cancer by raising hexokinase(HK).At the same time,according to data from clinical specimens and The Cancer Genome Atlas(TCGA)data,lncRNA HOTAIR expression increased in recurrent and high-graded tumors and associated with poor prognosis.It can be predicted that the new molecular targets that control the malignant progression of bladder cancer can be further explored from the perspective of lncRNA,and the search for potential therapeutic targets must become a research hotspot.Objective1.To obtain the expression profiles of lncRNA and mRNA genes in bladder cancer and adjacent tissues,then predicted their biological functions.2.To validate the result of high-throughput sequencing in more bladder cancer and adjacent tissues.3.To detect the expression of IncRNA in different bladder cancer cell lines and normal urothelial cells by RT-PCR.4.To investigate the effect of lncRNA LUCAT1 on the progression of bladder cancer cell lines in vitro.Methods1.Three pairs of bladder cancer and adjacent tissues were selected from patients who underwent radical cystectomy at the Nanjing Drum Tower Hospital.The differentially expressed genes were obtained by using the IlluminaHiSeq 4000 sequencing technology.The cut-off criteria were P<0.05 and |log2fold change|>1.2.To identify the biological functions of the aberrantly expressed lncRNAs,GO and KEGG analyses were applied to determine the signaling pathways.The lncRNA data were imported into the Database for Annotation,Visualization and Integrated Discovery(DAVID)(david.abcc.ncifcrf.gov/tools.jsp).Gene ontology(GO)analysis was applied to analyze the main function of the differential expression genes according to the Gene ontology project.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis was used to analyze the biological pathways,involving the differentially expressed lncRNAs.P<0.05 was the standard for choosing to indicate the statistically significant difference3.Expand the clinical sample to 20 pairs of bladder cancer and adjacent tissues.Then use RT-PCR to verify the expression reliability of the sequencing results4.The bladder cancer cell and urothelial cell lines,T24,J82,5637,and SV-HUC-1,were selected and the lncRNA expression were obtained as the background values by RT-PCR.5.By constructing the siRNA,the si-LUCAT J82 stably expressed cell line were obtained,and their biological functions were verified through CCK8 assay,transwell assay,Wound Ilealing assay.Result1.By high-throughput sequencing,269 differentially expressed IncRNAs were obtained,of which 172 were up-regulated,97 were down-regulated.What’s more,1,215 mRNAs were differential expression.of which 554 were up-regulated and 661 were down-regulated.2.By GO and pathway analysis,we found that differential IncRNAs are involved in cell adhesion,cell cycle,and may involve Ras signaling pathways.Through protein co-expression network analysis,we found that LUC ATI may be associated with CCNB1 in bladder cancer.3.In the 20 pairs of cancerous and paired samples,we verified the expression of seven IncRNAs.and the results were consistent with the sequencing in direction.4.Cell function experiments showed that the proliferation,migration and invasion of J82 were significantly attenuated after interference with LUCAT1 expression.Conclusion1.There are a large number of differentially expressed lncRNAs and mRNAs in bladder cancer and adjacent tissues.2.LUCAT1 is highly expressed in bladder tumor tissues relative to adjacent tissues.3.Overexpression of LUCATI promotes the migration,invasion and proliferation of bladder cancer cell lines.4.LUCAT1 and CCNB1 may be related to each other in the occurrence of bladder cancer.