| Objectives: Bladder cancer is one of the most common malignancies of the urinary system.It is characterized by a tendency to relapse and complicated etiology.It has both internal genetic factors and external environmental factors.The risk factors are long-term exposure to aromatic substances,smoking,chronic bladder infection,bladder stones and urinary tract obstruction.However,the mechanism of its occurrence and development is still unclear.Noncoding RNA are a class of RNAs that are ubiquitous and cannot be translated into proteins.Which length is more than 200 nt LncRNA(long non coding RNA,LncRNA)and 18 ~ 24 nt microRNA(miRNA,miRNA)proved in regulating tumor cell biology functions play an important role in the process,and non-coding RNA interaction between the regulation of tumor development is becoming a hot spot of research,but to non-coding RNA bladder tumor control mechanism is unclear.Therefore,this study intends to explore the mechanism of LncRNA/miRNA/ key protein pathway in bladder cancer cells,provide new molecular markers for the diagnosis of bladder cancer,and provide new ideas for the treatment of bladder cancer.Methods:1.The identification of key proteins in bladder cancer and the screening and testing of miRNA on the upstreamThe upstream miRNA in the study of fascin1 was searched by Targetscan,DIANA and other online databases.In addition,fascin1 and miRNA were searched in online databases such as GEPIA,oncoLnc and TCGA to examine the patients' survival and expression in bladder cancer in the database,and the miRNA to be studied was determined based on the literature.Fascin1 and miRNA expression were tested by qPCR in 60 patients with pathologically diagnosed bladder cancer and 23 patients with adjacent bladder cancer from the laboratory specimen base.The correlation between fascin1 and miRNA was tested by correlation analysis.Bladder cancer cells of multiple cell lines were cultured,RNA was extracted,and miRNA expression was detected by qPCR.2.Validation of miRNA regulation of downstream target genes and its function in bladder cancer cell linesMiRNA mimics and negative controls/inhibitors of mimics and negative controls of inhibitors were designed and constructed to transfect bladder cancer cell lines,respectively.Changes in the expression of downstream target proteins were detected by qPCR and western blot.The binding sites were determined by dual luciferin reporter system to verify the binding of miRNA and fascin1 mRNA.In this study,the regulatory effect of miRNA on fascin1 expression was further verified by designing the western blot regression test.The effect of miRNA on invasion and migration ability of bladder cancer cells was detected by transwell assay.In this study,transwell regeneration assay was designed to further verify the effect of miRNA on fascin1 expression and its influence on the invasion and migration ability of bladder cancer cells.The cycle changes of Propidium Iodide labeled cells were detected by flow cytometry.The proliferation of 5-ethynyl-2 '-deoxyuridine(EdU)-labeled cells was detected by fluorescence microscopy.Real-time unlabeled cell analyzer(RTCA)was used to detect cell proliferation.3.Screening and detection of miRNA upstream LncRNAOnline databases such as miRwalk and DIANA were used to find the upstream LncRNA of miRNA.LncRNA was retrieved through online databases such as GEPIA,oncoLnc and TCGA,and the survival status and expression in bladder cancer of patients in the database were checked.The LncRNA to be studied was determined from the comprehensive literature.Ten pathologically diagnosed bladder cancer cases and their corresponding paracancerous tissues were selected from the laboratory specimen library.RNA was extracted and LncRNA expression was detected by qPCR.The expressions of LncRNA were further detected by qPCR technology after RNA was extracted from the tissues of 60 cases of bladder cancer and 23 cases of para-bladder cancer with clear pathological diagnosis.The correlation between LncRNA and miRNA,and between LncRNA and fascin1 was tested by correlation analysis.Bladder cancer cells of multiple cell lines were cultured,RNA was extracted,and LncRNA expression was detected by qPCR.Subcellular localization of LncRNA in bladder cancer was detected by in situ hybridization,and the nuclear and cytoplasmic distribution of LncRNA in bladder cancer was further detected by nucleoplasmic RNA separation.4.Verification of the regulatory effects of LncRNA on downstream miRNA and fascin1 and its application in bladder cancer cellsAn inhibitor of LncRNA and its negative control/overexpressed plasmid and its negative control were designed and constructed to transfect bladder cancer cell lines,respectively.The changes in the downstream miRNA and fascin1 RNA levels were detected by qPCR,and the changes in the fascin1 protein level were detected by western blot.The regulatory effect of LncRNA on downstream fascin1 was further verified by designing the western blot test.Transwell assay was performed to detect the effect of LncRNA on invasion and migration of bladder cancer cells.Transwell response assay was designed to further verify the effect of LncRNA on the invasion and migration ability of bladder cancer cells after the regulation of miRNA expression.Annexin v-fitc and Propidium Iodide labeled apoptosis were detected by flow cytometry.The cycle changes of Propidium Iodide labeled cells were detected by flow cytometry.The proliferation of 5-ethynyl-2 '-deoxyuridine(EdU)-labeled cells was detected by fluorescence microscopy.Real-time unlabeled cell analyzer was used to detect the proliferation of cells.The tumorigenic experiment of nude mice was conducted to further verify the effect of LncRNA on cell proliferation by constructing cell lines with stable overexpression of LncRNA.The regulation of fascin1 in nude mice was further verified by immunohistochemical experiments in tumor tissue of nude mice.5.Verification of the role of the LncRNA/miRNA/fascin1 signaling axis in the regulation of cell invasion and migration by the TGF-?1 related signaling pathwayThe changes in the levels of LncRNA,miRNA and fascin1 were tested by qPCR in bladder cancer cell lines treated with TGF-?1.The fascin1 protein level was tested by western blot.The role of LncRNA and miRNA in the regulation of fascin1 by TGF-?1 was tested by designing the western blot test.The role of LncRNA and miRNA in the regulation of TGF-?1 cell invasion and migration was further verified by the design of transwell recovery assay.Results:1.According to the database screening and the survival analysis and the expression correlation analysis,the final determination of miR-200 b was the upstream miRNA of fascin1 for the study.The survival analysis results showed that the survival time of patients in the high-expression group was lower than that in the low-expression group,and the survival time of patients in the high-expression group of miR-200 b was higher than that in the low-expression group of miR-200 b.The expression of fascin1 in the tissue was higher than that in the adjacent tissue by qPCR,while the expression of miR-200 b was not significantly different between the two groups.Fascin1 in the tissue was negatively correlated with the expression of miR-200 b.Fascin1 was highly expressed in the highgrade bladder cancer cell line,while miR-200 b was under expression in the high-grade bladder cancer.2.The expression of fascin1 decreased under the upregulation of miR-200 b and increased under the down-regulation of miR-200 b in bladder cancer cells.The dual luciferase reporter system showed that miR-200 b could bind specifically to the 3 'UTR of fascin1 and down-regulate the expression of fascin1.Western blotting showed that the inhibitor of miR-200 b could inhibit the knockdown of fascin1 and up-regulate the expression of fascin1.Transwell assay showed that upregulation of miR-200 b resulted in decreased migration and invasion ability of bladder cancer cells,while down-regulation of miR-200 b resulted in increased migration and invasion ability of bladder cancer cells.Transwell fascinations assay showed that miR-200 b may affect the migration and invasion of bladder cancer cells by regulating the expression of fascin1.Flow cytometry cycle test results showed that up-regulated miR-200 b had a certain inhibitory effect on the cell cycle of bladder cancer,while down-regulated miR-200 b had a certain promoting effect on the cell cycle of bladder cancer.The results of the EdU experiment showed that up-regulation of miR-200 b inhibited the proliferation of bladder cancer cells,while down-regulation of miR-200 b promoted the proliferation of bladder cancer cells.Real-time unlabeled cell analyzer showed that up-regulated miR-200 b inhibited the proliferation of bladder cancer cells,while down-regulated miR-200 b promoted the proliferation of bladder cancer cells.3.ZEB1-AS1 was finally determined to be the upstream LncRNA of miR-200 b to be studied through database screening and expression correlation analysis.Overall survival rate results showed that the survival time of bladder cancer patients in the ZEB1-AS1 high expression group was not significantly different from that in the ZEB1-AS1 low expression group,but the disease-free survival rate results showed that the disease-free survival time of bladder cancer patients in the ZEB1-AS1 high expression group was lower than that in the ZEB1-AS1 low expression group.The expression of ZEB1-AS1 in tissues detected by qPCR showed that the expression of ZEB1-AS1 in cancer tissues was higher than that in adjacent tissues,and the expression difference was significant.The expression of ZEB1-AS1 and miR-200 b in the tissue was negatively correlated,while the expression of ZEB1-AS1 and fascin1 was positively correlated.ZEB1-AS1 was highly expressed in high-grade bladder cancer cell lines.The results of fluorescence in situ hybridization showed that ZEB1-AS1 was present in the nucleus and cytoplasm.The results showed that the contents of ZEB1-AS1 in the nucleus and cytoplasm were approximately the same.4.Following the down-regulation of ZEB1-AS1,the expression of downstream miR-200 b increased,while the expression of downstream fascin1 decreased.The expression of downstream miR-200 b decreased and the expression of downstream fascin1 increased after the upregulation of ZEB1-AS1.The Western blot test showed that overexpression of ZEB1-AS1 could antagonize the inhibitory effect of miR-200 b on fascin1.Transwell assay showed that down-regulation of ZEB1-AS1 resulted in decreased migration and invasion ability of bladder cancer cells,while up-regulation of ZEB1-AS1 resulted in increased migration and invasion ability of bladder cancer cells.Transwell response AS1 showed that ZEB1-AS1 could further affect the migration and invasion ability of bladder cancer cells by regulating the expression of miR-200 b.Flow cytometry apoptosis test results showed that down-regulation of ZEB1-AS1 could promote apoptosis of bladder cancer cells to a certain extent,while up-regulation of ZEB1-AS1 could inhibit apoptosis of bladder cancer cells to a certain extent.Flow cytometry cycle test results showed that down-regulation of ZEB1-AS1 had a certain inhibitory effect on bladder cancer cell cycle,while up-regulation of ZEB1-AS1 had a certain promoting effect on bladder cancer cell cycle.The results of the EdU experiment showed that down-regulation of ZEB1-AS1 inhibited the proliferation of bladder cancer cells,while up-regulation of ZEB1-AS1 promoted the proliferation of bladder cancer cells.Real-time unlabeled cell analyzer showed that down-regulation of ZEB1-AS1 inhibited the proliferation of bladder cancer cells,while up-regulation of ZEB1-AS1 promoted the proliferation of bladder cancer cells.The tumorigenic experiments of nude mice showed that the tumors in the ZEB1-AS1 overexpression group grew faster and larger than those in the negative control group.Immunohistochemical results of tumor tissue in nude mice showed that the tumor expression levels of fascin and ki67 in the ZEB1-AS1 overexpression group were higher than those in the negative control group.5.After treatment with TGF-?1,qPCR results showed increased expression of ZEB1-AS1,decreased expression of miR-200 b,increased expression of fascin1 and increased expression of fascin1 by western blot.Western blot results showed that knockdown of ZEB1-AS1 could inhibit the expression of fascin1 induced by TGF-?1,while overexpression of miR-200 b could inhibit the expression of fascin1 induced by TGF-?1.The results of Transwell response experiment showed that ZEB1-AS1 knockdown could antagonize the promotion of TGF-?1 on the invasion and migration of bladder cancer cells,and overexpression of miR-200 b could antagonize the promotion of TGF-?1 on the invasion and migration of bladder cancer cells.Conclusion:1.miR-200 b may down-regulate the expression of fascin1 in bladder cancer to inhibit the migration and invasion of cells,and miR-200 b may arrest the cell cycle and inhibit cell proliferation to some extent.2.ZEB1-AS1 may bind with miR-200 b in bladder cancer to indirectly up-regulate the expression of fascin1 and promote cell migration and invasion,and ZEB1-AS1 may inhibit apoptosis,promote cell cycle and promote cell proliferation to a certain extent.3.The ZEB1-AS1 / miR-200 b /fascin axis is regulated by the TGF-?1 related signaling pathway in the process of affecting cell migration and invasion. |
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