| Background: Bladder cancer(BCa)is the second most common cancer in the urinary system,exhibiting high incidence and mortality globally.In general,BCa is a low-grade papillary non-muscle invasive tumor,known as non-muscle invasive bladder cancer(NMIBC).NMIBC includes tumors which are in stage Ta,T1 and carcinoma in situ.NMIBC is characterized by a high rate of recurrence and a risk of progression to muscular invasion.According to statistics,the recurrence rate of NMIBC is about 50-70%,and the rate of progression to muscle invasion is about10-15%,indicating a good prognosis.High-grade papillary neoplasms and flattened dysplasia can progress to muscle invasive bladder cancer(MIBC).MIBC(T2 stage and above)has a poor prognosis,with a 5-year survival rate of 50% and common metastatic progression.A comprehensive analysis of RNA sequences from tumor samples of NMIBC and MIBC has reported that candidate genes and pathways play important roles in the development and progression of BCa.So far,the treatment of patients with BCa are based on the clinical and pathological parameters.As for the primary drawbacks of these methods,patients may have different reactions to the same treatment even in some cases who share similar histological features in tumors.Therefore,the combination of traditional methods and biomarkers is important to improve the clinical therapeutic effect and prognosis.All novel biomarkers that influence the progression of malignant biological behaviors of BCa may be considered to be useful,and potential mechanisms of these biomarkers may provide new directions and methods for the treatment of BCa.However,the mechanism of these biomarkers remains to be further studied.Long noncoding RNA(lnc RNA)is a class of RNA without protein-coding function.Abnormal expression or mutation of lnc RNA has been widely observed in human diseases,especially in cancers.AGAP2-AS1 is a lnc RNA and plays an important role in human diseases,including cancers and congenital diseases.Overexpression of AGAP2-AS1 has been confirmed to be closely related to the occurrence and development of various cancers,including colon cancer,hepatocellular carcinoma,breast cancer,non-small cell lung cancer and esophageal cancer.However,it still remains unclear with respect to the role of AGAP2-AS1 in the development and progression of BCa.Purpose:To identify the relationship of lnc RNA AGAP2-AS1 with the progression and prognosis of BCa,and to explore the possible mechanism.Methods: 1.The expression of AGAP2-AS1 in BCa tissues or BCa cell line was verified by real-time quantitative polymerase chain reaction(qRT-PCR)and compared with that in paracancer non-tumor tissues or normal epithelial cell.2.The survival date of BCa patients and gene expression information of BCa samples collected from Kaplan-Meier Plotter database was analyzed to determine the relationship of AGAP2-AS1 with the survival status and prognosis of patients.3.Clinical sample data in our hospital was also collected to analyze the relationship of AGAP2-AS1 with the clinical pathological features of BCa patients.4.The plate cloning formation assay and CCK-8 assay were performed to evaluate the effect of AGAP2-AS1 on cell proliferation.The transwell assay was used to evaluate the effect of AGAP2-AS1 on cell migration and invasion.Tube-formation assay was conducted to evaluate the effect of AGAP2-AS1 on cell angiogenesis.5.Subcutaneous tumorigenesis model and tail vein injection model in nude mice were established to evaluate the effect of AGAP2-AS1 on tumorigenesis and metastasis in vivo.6.Subcellular isolation and FISH assay were used to determine the distribution of AGAP2-AS1 in BCa cells.7.Starbase 3.0 and RBPsuite online forecasting tools were used to predict the potential RNA-binding proteins(RBPs)of AGAP2-AS1.8.IF-FISH,RIP and RNA pull down assay were used to identify the RBP of AGAP2-AS1.9.Starbase 3.0 and RBPsuite online forecasting tools were used to predict the potential target genes of RBP.10.RIP and RNA pull down test were used to identify the target gene of RBP.11.Radiomycin test,qRT-PCR and Western Blot were used to explore the potential molecular mechanisms underlying AGAP2-AS1-mediated regulation of target genes.12.The plate clone formation assay and CCK-8 assay were performed to evaluate the effect of target gene LRG1 on cell proliferation.The transwell assay was used to evaluate the effect of LRG1 on cell migration and invasion.Tubeformation assay was conducted to evaluate the effect of LRG1 on cell angiogenesis.Results: 1.The high expression of AGAP2-AS1 in BCa patients was positively correlated with high pathological T stage,high grade and vascular infiltration,but negatively correlated with the survival and prognosis of patients.2.In vitro,AGAP2-AS1 overexpression promoted proliferation,migration,invasion and angiogenesis of BCa cells.In vivo,AGAP2-AS1 promoted tumor growth and metastasis.3.AGAP2-AS1 promoted the stability of LRG1 m RNA by recruiting the RNA-binding protein IGF2BP2 to LRG1.4.AGAP2-AS1 promoted uncontrolled proliferation,migration,invasion and angiogenesis of BCa cells in a LRG1-dependent manner,thus accelerating the malignant progression of BCa.Conclusion:AGAP2-AS1 may be a potential biomarker to predict the prognosis and a potential intervention target of gene therapy of BCa. |
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