Construction Of Invasion And Migration Lncrna Prognosis Signature As Well As The Role And Mechanism Of LncRNA RBM11-4 In The Progression Of Bladder Cancer

PART1 CONSTRUCTION OF INVASION AND MIGRATION LNCRNA PROGNOSIS SIGNATUREObjective:To investigate the predictive role and value of the invasion and migration-associated(EMT-associated)lncRNA prognostic signature on the survival prognosis of bladder cancer patients.Methods:Transcriptome data and clinical data of bladder cancer samples were downloaded and collated in the TCGA database.The EMT gene set was downloaded in the molecular model database.EMT-related lncRNAs were screened by Pearson correlation analysis,and EMT-related lncRNA signatures related to the prognosis of bladder cancer patients were constructed by Cox regression analysis.Risk scores of clinical samples were calculated according to the signature and analyzed for their relationship with clinical characteristics and survival prognosis.The accuracy of the signature was assessed by ROC curves.In addition,the predictive ability of the signature for prognosis of bladder cancer patients was assessed by constructing column line plots.Finally,the possible enrichment of KEGG pathways in different risk groups was analyzed by GSEA.Results:Person correlation analysis of lncRNA transcription data and EMT gene set expression data from bladder cancer samples in the TCGA database screened a total of 525 EMT-associated lncRNAs.a 14-EMT-associated lncRNA prediction signature was successfully constructed by univariate and multifactorial Cox regression analysis.Patients were divided into high-risk and low-risk groups according to the signature risk score.The overall survival time of patients in the low-risk group was significantly longer than those in the high-risk group,and the AUC values of the ROC curves were all greater than 0.73.Based on patient age,gender,stage,and the signature risk score to construct the column line graph,the risk score of our signature had higher predictive efficacy for patient prognosis compared with traditional clinical indicators,which was found to be more accurate by plotting the calibration curve and ROC curve.We further performed GSEA pathway enrichment analysis for patients in the high-risk and low-risk groups.The low-risk group was mainly enriched to some immune-related pathways.In contrast,the high-risk group was mainly enriched to some tumor and tumor progression-related pathways.Conclusion:The invasion and migration-related lncRNA signature has excellent predictive efficacy for the prognosis of bladder cancer patients.PART2 EXPRESSION OF LNCRNA RBM11-4 IN BLADDER CANCER AND ITS ROLE IN BLADDER CANCER PROGRESSIONObjective:To further screen EMT-related lncRNAs that may influence the malignant progression of bladder cancer.to investigate the expression of EMT-related lncRNA RBM11-4 in bladder cancer and its role in the progression of bladder cancer.Methods:405 bladder cancer samples were divided into EMT-up and EMT-down groups based on the expression of 200 EMT genes,and the differentially expressed lncRNAs in the EMT-up and EMT-down groups were screened.in the TCGA database,the tumor tissue group(Tumor-up)and the paracancerous tissue group(Tumor-down)were screened for the differentially expressed lncRNAs were screened for EMT-related lncRNAs that might affect the malignant progression of bladder cancer by taking intersections of high expression lncRNAs in the EMT-up group,high expression lncRNAs in the Tumor-up group,and EMT-person-related lncRNAs.after identifying the target lncRNA RBM11-4 for the study,the lncRNAs were screened by the expression of RBM11-4 in bladder cancer tissues was predicted by TCGA database and verified by RT-qPCR at the tissue and cellular levels,respectively.The effects of RBM11-4 on bladder cancer invasion and metastasis,drug sensitivity,cell proliferation,and tumorigenic ability in nude mice were examined by Transwell cell migration/invasion assay,CCK-8 drug sensitivity assay,plate cloning assay,and tumorigenic ability in nude mice in vitro.Results:In the TCGA database,the differentially expressed lncRNAs in the EMT-up and EMT-down groups were screened,of which 111 lncRNAs were upregulated in the EMT-up group and 57 lncRNAs were upregulated in the EMT-down group.In the TCGA database,we screened differentially expressed lncRNAs in the tumor tissue group(Tumor-up)and normal tissue group(Tumor-down),of which 1701 lncRNAs were up-regulated in the Tumor-up group and 756 lncRNAs were up-regulated in the Tumor-down group.Finally,we found that only the lncRNA RBM11-4 was identified after taking the intersection of the highly expressed lncRNAs in the EMT-up group,the highly expressed lncRNAs in the Tumor-up group,and 525 EMT-person-related lncRNAs.The expression of RBM11-4 in bladder cancer tissues was predicted by TCGA database.High expression of RBM11-4 in bladder cancer tissues was found by analyzing 19 pairs of bladder cancer tissues and paired paracancerous normal tissues.Analysis of 205 bladder cancer tissues revealed that RBM11-4 was more highly expressed in bladder cancer tissues with high stage(Stage Ⅲ-Ⅳ)compared to bladder cancer tissues with low stage(Stage Ⅰ-Ⅱ).The expression of RBM11-4 in bladder cancer tissues and bladder cancer cell lines was verified by RT-qPCR.Compared with normal uroepithelial immortalized cells(SV-HUC-1),RBM11-4 was highly expressed in bladder cancer cell lines T24,5637,and UM-UC-3,with T24 expressing the highest and UM-UC-3 expressing the relatively lowest.Compared with normal bladder tissues,RBM11-4 was highly expressed in bladder cancer tissues.transwell assay revealed that the invasive and metastatic ability of cells was significantly reduced after knockdown of RBM11-4.After overexpression of RBM11-4,the invasive and metastatic abilities of cells were significantly increased.Cell scratch assays revealed a significant decrease in cell lateral healing rate after knockdown of RBM11-4.After overexpression of RBM11-4,cell transverse healing rate was significantly increased.CCK8 drug sensitivity assay revealed that the drug sensitivity of cells to cisplatin was significantly increased after knockdown of RBM11-4.After overexpression of RBM11-4,the drug sensitivity of cells to cisplatin was significantly reduced.Plate cloning assays revealed a significant increase in the number of cell clones in the RBM11-4 overexpression group compared to the control group.Tumor formation assay in nude mice showed a significant increase in both volume and weight of tumors in the RBM11-4 overexpression group compared to the control group.In addition,the growth rate of tumor-forming tumors in nude mice overexpressing RBM11-4 was also significantly increased compared with the control group.Conclusion:EMT-associated lncRNA RBM11-4 is highly expressed in bladder cancer tissues and bladder cancer cell lines,and it promotes bladder cancer cell invasion and metastasis,cell proliferation,affects the sensitivity of bladder cancer cells to chemotherapeutic drugs,and promotes the tumorigenic ability of cells in nude mice.PART3 MECHANISM OF LNCRNA RBM11-4 IN PROMOTING BLADDER PROGRESSIONObjective:To investigate the specific mechanism of EMT-related lncRNA RBM11-4 in promoting bladder cancer progression.Methods:To detect the localization of RBM11-4 in bladder cancer cells by fluorescence in situ hybridization;to detect the mRNA co-expressed with RBM11-4 by RNA-seq technique;to detect the possible proteins bound by RBM11-4 by RNA pull down and RIP techniques.The mRNA co-expressed with RBM11-4 and EMT-related markers were verified by Western Blot;after inhibition of the co-expressed mRNA,the migration/invasion ability of bladder cancer cells and changes in drug sensitivity were detected by Transwell cell migration/invasion assay and CCK-8 drug sensitivity assay.The effect of RBM11-4 on the stability of possible binding proteins was examined by CHX assay.Results:Intracellular localization of RBM11-4 was detected by fluorescence in situ hybridization:RBM11-4 was expressed in both the nucleus and cytoplasm of T24 cells,with higher expression in the cytoplasm.RNA-seq results showed that NLRP3 was co-expressed with RBM11-4.We confirmed that RBM11-4 could regulate NLRP3 expression by RT-qPCR.Western Blot results showed that the expression of NLRP3 and Vimentin was significantly lower and E-cadherin expression was significantly higher in the si-RBM11-4#2 group compared to the si-NC group.After overexpression of RBM11-4,the expression levels of NLRP3 and Vimentin were significantly increased and the expression level of E-cadherin was significantly decreased.In addition,when OE-RBM11-4 cells were treated with NLRP3 inhibitor CY-09,the results of Transwell assay suggested that the number of migrating and invading cells was significantly lower than that of OE-RBM11-4 group.the results of CCK-8 assay indicated that the inhibition of NLRP3 by CY-09 significantly increased the drug sensitivity of OE-RBM11-4 cells to cisplatin.After RNA pull down,proteins bound to RBM11-4 positive and antisense RNA were separated by SDS-PAGE and detected by silver staining.Each different protein band in the cut and solubilized gel and mass spectrometry analysis revealed the presence of Vimentin.In addition,RIP experiments were used to verify specific interactions between Vimentin and RBM11-4.These results suggest that RBM11-4 can bind to Vimentin.To further explore how RBM11-4 affects Vimentin levels,we tested the effect of RBM11-4 on Vimentin stability by CHX assay.The half-life of Vimentin in the OE-RBM11-4 group was significantly longer than the half-life of Vimentin in the vector group,and the degradation levels of Vimentin at different time points were detected by Western Blot after treating the cells with CHX.Conclusion:RBM11-4 can activate the NLRP3/EMT signaling axis as well as maintain the stability of Vimentin by binding to Vimentin,thus promoting bladder cancer progression.