The Biological Function And Regulation Of LINC00612 In Bladder Cancer

Bladder cancer(BC)is a common type of cancer involving tumors of the urinary system,has the highest published incidence involving malignant urinary system tumors,and poses a serious threat to human health.In recent years,as the development of science and technology,we have a deeper understanding and insight of pathogenesis,occurrence and development in BC,and great progress has been made in treatment.However,the prognosis of BC remains unsatisfied,especially for patients with advanced stage.No more we can do to improve their survival.LncRNAs are non-coding RNAs that are more than 200 nucleotides in length and that affect regulatory gene expression.LncRNAs lack a complete open reading frame and do not have a protein coding function.By using mouse DNA transcripts.Up to 4-9%of the sequences in the mammalian genome sequence can produce LncRNAs.LncRNAs were originally thought to be the "dark matter"or "noise" of genomic transcription and to have no biological function.Later studies demonstrated that LncRNAs are involved in many important cellular functions,such as X chromosome silencing,genomic imprinting,chromatin modification and transcriptional activation or inhibition,and can promote changes in molecular function in related signaling pathways,as well as alter cell life activitiesEMT is a biological process in which epithelial-derived malignant cells transform into mesenchymal cells with increased migration and invasion ability30.The characteristic changes in EMT include the loss of polarity of epithelial cells,degradation of intercellular junctions,changes in cell morphology due to the reorganization of cytoskeletal structures,and downregulation of epithelial gene expression accompanied by upregulation of mesenchymal gene expression.These changes provide the cell with a greater ability to migrate,invade and degrade the extracellular matrixCompetitive endogenous RNA(ceRNA)are a well-known regulatory mechanism of LncRNA.LncRNA inhibits expression of miRNAs by absorbing a variety of miRNAs,thus reducing its regulatory effect on target genes.At present,there have been some studies on LncRNA in BC,but as the pathogenic mechanism of ceRNA in BC,and the role of LncRNA in EMT-related metastasis and invasion are still unclear,which needs to be further studied.This study was divided into three parts.In the first part,LncRNA microarray was used to screen the most differentially expressed LncRNA in BC and paracancer tissues.The expression level of target LncRNA in BC tissues and cells were detected by In Situ Hybridization(ISH)and qRT-PCR.Its biological function in BC was confirmed by in vitro and in vivo experiments.In the second part,through in vivo and in vitro experiments,it was to confirm that LINC00612/miR-590/PHF-14 axis constituted the pathogenic mechanism of ceRNA network regulating BC development.The third part elucidates the mechanism of LINC00612/miR-590/PHF-14 axis regulating epithelial-mesenchymal transformation(EMT)of BC cells through in vitro experiments.Part Ⅰ The impact of LINC00612 on the proliferation and invasion ability of Bladder Cancer[Objective]To determine the expression profile of LncRNA between BC tissues and paracancer tissues,screen the most differential expressed LncRNA,and explore its biological function in BC[Method]The differences of LncRNA expression in 13 BC tissues and 8 paracancer tissues were analyzed by LncRNA microarray analysis.The most differential expressed LncRNA was screened and verified by qRT-PCR and in situ hybridization(ISH)in fresh BC tissues and BC cell lines.RNA isolation of nuclear and cytoplasmic fractions and Fluorescence In Situ Hybridization(FISH)were applied to confirmed its subcellular localization.CCK8,colony formation and Transwell assay confirmed the impact of LINC00612 on the proliferation and invasion in BC cells in vitro.The impact of LINC00612 on the proliferation and invasion of BC cells in vivo was validated by nude mouse xenograft model and Interactive Video Information System(IVIS)bioluminescence imaging system.[Results]The top 60 differential expressed LncRNAs in BC tissues and paracancer tissues were screened(Fold Change>1.5,P<0.01),among which LINC00612 was the most differentially expressed LncRNA.The expression of LINC00612 in BC tissues and BC cell lines was significantly higher than that in paracancer tissues and bladder cells.LINC00612 is mainly located in the cytoplasm.In vitro experiments showed that the overexpression of LINC00612 in BC cells increased cell proliferation and invasion ability,while down-regulated LINC00612 decreased cell proliferation and invasion ability.The results were aslo confirmed in vivo.[Conclusion]LINC00612 is abnormally up-regulated in BC tissues,and plays a role in enhancing cell proliferation and invasion ability as an oncogene in BC.Part Ⅱ LINC00612/mir-590/PHF-14 as ceRNA regulating occurrence and development of bladder cancer[Objective]To identify the downstream miRNA and target genes regulated by LINC00612 and study the mechanism which LINC00612/mir-590/PHF-14 as ceRNA to regulate BC development was clarified.[Method]Target miRNA of LINC00612 was predicted by bioinformatics.RNA immune-precipitation(RIP),RNA pull down and double lucigenase reporter gene assay were used to verify the binding.qRT-PCR was used to verify in BC cells.Downstream target genes that may be regulated by target miRNA were predicted by bioinformatics methods,and the binding between target miRNA and target genes was verified by qRT-PCR,RIP and double luciferase report experiments.The expression of target miRNA in BC cells was determined at mRNA and protein levels.These results were validated by the in vitro and in vivo function recovery experiments(rescue experiment).[Results]miR-590 may be the target downstream miRNA of LINC00612.RIP,RNA pull down and double luciferase report experiments have proved that LINC00612 and mir-590 can directly bind.In BC cells,the changes of miR-590 were negatively correlated with the regulation of LINC00612.It was verified by qRT-PCR,RIP and double luciferase reporting experiments that PHF-14 was the downstream target genes of miR-590.The function recovery experiment showed that LINC00612/miR-590/PHF-14 axis could regulate the proliferation and invasion ability of BC cells,which was further confirmed in vivo experiments.[Conclusion]LINC00612 sponge adsorbs mir-590 and further regulates the expression of downstream target gene PHF-14,thereby affecting the proliferation and invasion ability of BC cells.LINC00612/mir-590/PHF-14 constitutes the ceRNA network involved in the occurrence and development of BC.Part Ⅲ The regulation of epithelial-mesenchymal transformation in bladder cancer cells by LINC00612/miR-590/PHF-14 axis[Objective]To investigate the regulation of EMT in BC by LINC00612/mir-590/PHF-14 axis.[Method]The influence of LINC00612/miR-590/PHF-14 axis on e-cadherin,n-cadherin and vimentin was determined by western blotting and FISH assay.[Results]Down-regulation of LINC00612 enhanced the expression of e-cadherin and weakened the expression of n-cadherin and vimentin.Overexpression of LINC00612 inhibited the expression of e-cadherin,while increased the expression of n-cadherin and vimentin.Regulation of PHF-14 could neglect the effect of LINC00612 on e-cadherin and vimentin.[Conclusion]LINC00612/miR-590/PHF-14 axis has been involved in regulation of EMT in BC cells through the ceRNA mechanism.