The Role And Molecular Mechanism Of LncRNA-LINP1 In The Progression Of Bladder Cancer

BackgroundBladder Urothelial Carcinoma is one of the most ordinary urinary cancers in the world.The high recurrence rate leads to the poor prognosis of bladder cancer.In recent years,long non-coding RNA(LncRNA)has been found to play an important role in the occurrence and development of malignant tumors,and can be used as a detection marker to predict the prognosis of cancer.Indeed,scholars have turned their attention to the relationship between LINP1 and tumors and They found LINP1 significantly influences different tumors.Though the role of LINP in bladder cancer has not been studied,this study hopes to explore the expression of LINP1 in bladder cancer.Its influence on the growth,invasion and progression of bladder cancer,preliminarily explore the molecular mechanism of LINP1 regulating bladder cancer.It also provide theoretical foundation for the diagnosis and treatment of bladder cancer.Methods1.In GEPIA database,we analyze the expression of LINP1 in bladder cancer tissue and normal bladder tissue;2.Using qPCR technology to detect the expression of LINP1,10 bladder cancer patients with fresh bladder cancer tissue and normal adjacent bladder tissues was collected from August 2018 to May 2020 in the Department of Urology,Third Affiliated Hospital of Guangzhou Medical University.3.LINP1 overexpression plasmids transfected 5637 and BIU87,then detecting expression LINP1 by qPCR;4.SiRNA-LINP1 transfected BIU87 and 5637,then detecting expression LINP1 by qPCR;5.Bladder cancer cell lines transfected LINP1 overexpression plasmid and interference fragment SiRNA-LINP1(1)CCK-8 kit detects the change of cell growth curve;(2)Transwell experiment detects changes in cell migration and invasion ability;6.Through high-throughput transcriptome sequencing to compare the expressions of miRNA,LncRNA and mRNA of stable transfer 5637-LINP1 and 5637-NC,we can predicted the ceRNA network and related signal pathway proteins that may interact with LINP1;7.Dual luciferase to verify the binding of LINP1 to hsa-mir-148a-3p;8.The potein of transfected cells was deteced by Western blot.The result of WB were the protein expressions of E-cadherin,vimentin,Wntl and β-catenin;9.Statistical analysis:The difference between the two groups was tested by t test,and the difference between three groups and above was analyzed by variance analysis.ResultThe GEPIA database acknowledges that LINP1 is up-regulated in bladder cancer tissues;qPCR detection found that in 10 pairs of bladder cancer tissues and normal bladder tissues,the expression of LINP1 is higher in bladder cancer tissues than normal bladder tissues.1.LINP1 transfected to overexpress bladder cancer cell lines:BIU87 and 5637,5637 was effectively up-regulated expression levels LINP1,but BIU87 was down-regulated;2.Transfection of interference fragment SiRNA-LINP1 into bladder cancer cell lines:5637 and BIU87,5637 and BIU87 was all down-regulated the expression of LINP1;3.Cell function experiments show that down-regulating the expression of LINP1 can inhibit the proliferation,migration and invasion of bladder cancer cells,while overexpression of LINP1 can enhance the proliferation,migration and invasion of bladder cancer cells.Apoptosis has nothing to do with the expression of LINP1.4.miRNA and mRNA sequencing results:After overexpression of LINP1,the miRNA of the Wnt pathway was significantly increased,and hsa-mir-148a-3p was significantly up-regulated and targeted Wntl protein.5.Dual luciferase test:LINP1 targets hsa-mir-148a-3p.6.WB results showed that up-regulation of LINP1 significantly increased the expression of Wntl protein and β-catenin protein in the Wnt/β-catenin signaling pathway;at the same time,it increased the expression of N-cadherin and Vimentin in the EMT pathway protein.Down-regulation of LINP1 reduced the expression of Wntl protein and β-catenin protein in the Wnt/β-catenin signaling pathway,and also reduced the expression of N-cadherin and Vimentin in the EMT pathway protein.Bladder cancer cell lines overexpress LINP1 and then add mir-148a-3p to inhibit Wntl and β-catenin protein expression;interfere with LINP1 and then inhibit mir-148a-3p,resulting in increased Wntl and β-catenin protein expression.Discussion1.The expression of LINP1 in bladder cancer tissue is higher than that in normal tissues adjacent to cancer;2.LINP1 promotes the proliferation,migration and invasion of bladder cancer cells in bladder cancer;3.There is an indirect relationship:LINP1 targets hsa-mir-148a-3p;4.LINP1,as a competitive endogenous RNA(ceRNA)of mir-148a-3p,indirectly regulates the expression of Wntl protein in the Wnt/β-catenin signaling pathway,and then regulates the EMT pathway to change the migration and invasion ability of bladder cancer cells.