The Mechanism Of LncRNA MIR100HG Promoting The Development Of Mibc By Regulating MiR-218-5p/PDGFRA

ObjectiveThis study aims to find new lncRNAs related to the occurrence and development of bladder cancer through next generation gene sequencing and bioinformatics methods,to evaluate their potential as diagnostic biomarkers of bladder cancer,and to further study the molecular mechanism of their progression in MIBC,as well as explore the role of exosomes in this process.The study revealed whether the lncRNA could be an effective therapeutic target to provide scientific basis.Methods1.Bladder cancer tissues and their corresponding paracancerous normal tissue samples were collected for next generation gene sequencing,and differentially expressed lncRNAs were obtained,defined as dataset 1;Transcriptome data of bladder cancer and paracancerous normal tissues from the TCGA database were downloaded,and differentially expressed lncRNAs were obtained by R software processing,which were defined as dataset 2.LncRNAs differentially expressed from stage I to IV of bladder cancer were obtained from the CRN database,and were defined as dataset 3.Then,lncRNAs were obtained after the intersection of dataset 1,2,and 3,and they were defined as dataset 4.2.The data of Next Generation Gene Sequencing were analyzed again,and lncRNAs differentially expressed in MIBC and NMIBC were obtained,which were defined as dataset 5.Two lncRNAs were obtained by intersecting data sets 4 and 5.Then,MIR100 HG was selected as the target gene for further study.3.RT-qPCR was used to verify the expression of lncRNA MIR100 HG in bladder cancer tissues,cell lines and exosomes derived from cell supernatant.Combined with clinical data,the correlation between the expression of MIR100 HG and the TNM stage and other clinical features of bladder cancer was statistically analyzed.ROC curves were drawn to evaluate the specificity and sensitivity of MIR100 HG as a tumor diagnostic biomarker,and the correlation between MIR100 HG and the survival risk of bladder cancer patients was analyzed.4.MIBC cell lines(T24,J82)and NMIBC cell lines(RT4)were selected to overexpress MIR100 HG,and CCK8,Transwell,cell scratching,cell cycle,cell apoptosis and other experimental methods were used to determine the effect of MIR100 HG on the biological behavior of different bladder cancer cells.5.To explore the role of exosomes in the biological function of MIR100 HG.Exosomes were enriched in the supernatant of T24 cells overexpressing MIR100 HG.After identification by transmission electron microscopy,NTA and Western blot,T24 cells were co-cultured with exosomes.Then,Using a confocal laser microscope to observe the uptake of exosomes,and the changes in the biological behavior of T24 cells were detected.Finally,tumor-forming experiments were carried out in nude mice to verify the effect of MIR100 HG on bladder cancer proliferation in vivo.6.Bioinformatics method was used to analyze the genes and signal pathways related to MIR100 HG,and the regulation network of co-expression of ce RNA was mapped.RT-q PCR,Western blot and luciferase assay were used to further investigate the molecular mechanism of MIR100 HG promoting the progress of MIBC.Results1.Through the next generation gene sequencing of 6 pairs(3 MIBC pairs and 3NMIBC pairs)of bladder cancer and its adjacent normal tissues,and data analysis of TCGA database and CRN database,we obtained a total of 5 lncRNAs in dataset 4,namely,FENDRR,MIR100 HG,RHPN1-AS1,RNF144A-AS1,and DIO3 OS,and 2lncRNAs in dataset 5,respectively,MIR100 HG and FENDR.We finally selected MIR100 HG as the target gene.2.The expression of MIR100 HG was low in bladder cancer tissues,cells and exosomes in their supernatant.Through statistical analysis,the expression of MIR100 HG was positively correlated with the depth of bladder cancer invasion and negatively correlated with the survival rate of patients with bladder cancer(P<0.05).3.Overexpression of MIR100 HG in NMIBC cell lines(RT4)resulted in reduced cell proliferation,invasion and migration(P<0.05);When MIR100 HG was overexpressed in MIBC cell lines(T24,J82),cell proliferation,invasion and migration were enhanced,apoptosis was inhibited,and the cell cycle was mainly stuck in S and G2 phases(P<0.05);Exosomes from the supernatant of T24 cells overexpressing MIR100 HG were co-cultured with T24 cells,and the proliferation,invasion and migration of the cells were also enhanced(P<0.05).Further study on tumor formation in nude mice in vivo confirmed that the growth rate of subcutaneous implant tumors was significantly faster in the overexpressed MIR100 HG group compared with the NC group and the Control group(P<0.05).4.Combined with bioinformatics prediction and verification of experiment of RT-q PCR,Western blot,and dual luciferase assay,the results showed that MIR100 HG regulated the expression level of miR-218-5p/PDGFRA.At the same time,MIR100 HG inhibited miR-218-5p by competitively binding with PDGFRA and activated the ERK/MAPK signaling pathway,ultimately promoting the development of MIBC.ConclusionsMIR100HG affects the survival and prognosis of patients with MIBC and can be used as an ideal biomarker.As a double-sided gene,MIR100 HG acts as a tumor suppressor gene to inhibit the occurrence of bladder cancer.When NMIBC developed into MIBC,MIR100 HG promoted the development of MIBC.In this process,exosome functions as a "carrier",carrying MIR100 HG and delivering it to adjacent MIBC cells.In addition,MIR100 HG promotes the progression of MIBC by regulating miR-218-5p/PDGFRA and can serve as a new potential target for the treatment of MIBC.