| 1.Backgroud:Bladder cancer is one of the most common genitourinary system malignancies in China,among which bladder urothelial carcinoma is the most common.In China,the incidence of bladder cancer ranks 13th among systemic malignant tumors,among which the incidence of male patients with bladder cancer ranks seventh,and the incidence of female patients with bladder cancer ranks 17th.In systemic malignant tumors,the mortality rate of bladder cancer ranks 13 th,among which males rank 11th,and females rank 16th.According to the degree of tumor cells infiltrating the muscle layer of the bladder,bladder cancer can be divided into two categories:non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC).NMIBC is the most common type of bladder cancer,accounting for about 70%-75%of bladder cancer patients.The remaining 25%-30%of bladder cancer patients are MIBC,and even distant metastases.The main treatment for NMIBC is transurethral resection.When there are high risk factors for recurrence and progression,intravesical infusion of immune preparations or chemotherapy drugs can be performed after surgery to prevent tumor recurrence and progression.Transurethral resection combined with intravesical infusion of BCG is one of the most common treatment strategies for NMIBC,but more than 50%of patients will relapse,and 10%-30%of NMIBC will develop into MIBC or even metastasize.The prognosis of MIBC is poor,and its 5-year survival rate is only 25%.At present,the pathogenesis of bladder cancer has not yet been clarified,and further research is needed to find new targets to provide a theoretical basis for the treatment of bladder cancer.Epithelial-mesenchymal transition(EMT)is a multi-step process in which epithelial cells lose their epithelial properties and acquire mesenchymal properties,thereby giving cancer cells the properties of migration and invasion.A large number of studies have shown that EMT is the initial event of invasion and metastasis of various malignant tumors including bladder cancer.The initiation and development of EMT involve different signal pathways and signal crosstalk,which are regulated at the transcription,post-transcription,translation and post-translational levels.TGF-?activates Smad2 and Smad3 through a tetrameric complex composed of type I and type II receptors,and then binds to Smad4.The trimeric Smad complex translocates into the nucleus,and cooperates with transcription regulators to inhibit or activate EMT transcription factors.Studies have found that TGF-? mediates the expression of genes such as Snail,E-cadherin,Zebl and Twist through the Smad3/Smad4 complex.Long non-coding RNA(lncRNA)is a non-coding RNA with a length greater than 200 nucleotides,and lncRNA is one of the emerging regulators involved in a variety of biological processes.Studies have found that lncRNA participates in a wide range of biological processes through a variety of pathways,including proliferation,adhesion,invasion,metastasis,stemness,apoptosis,genomic instability,and EMT.More and more evidences show that multiple lncRNAs are involved in EMT of bladder cancer through different pathways.Our previous studies found that PlncRNA-1(prostate lncRNA-1)is highly expressed in prostate cancer tissues and prostate cancer cells.PlncRNA-1,as an endogenous competitive RNA,regulates androgen receptor expression through miR-297-3p and miR-34c-5p to promote the occurrence and development of prostate cancer.In addition,PlncRNA-1 can also regulate the proliferation and EMT of prostate cancer through TGF-?1.However,there is currently a lack of research on the expression level,biological function and mechanism of PlncRNA-1 in bladder cancer.In this study,we aim to explore the expression level of PlncRNA-1 in bladder cancer and its biological function,further explain the molecular mechanism of its regulation,and provide potential molecular markers for the clinical diagnosis and prognosis of bladder cancer.At the same time,it laid the experimental and theoretical foundation for the targeted therapy and drug development of bladder cancer.2.Objective1)To explore the correlation between the expression level of PlncRNA-1 and the clinical data such as age,sex,pathological grade and staging of bladder cancer patients.2)Functional experimental studies clarify that PlncRNA-1 regulates bladder cancer cell proliferation and EMT.3)To explore the expression level of PlncRNA-1 is regulated by the methylation of its gene promoter region.4)Exploring that PlncRNA-1 promotes the progress of bladder cancer by regulating the miRNA-136/smad3 axis,providing new strategies and new targets for the diagnosis and treatment of bladder cancer.3.Methods3.1 The expression of PlncRNA-1 in bladder cancer and its predictive value1)Twenty eight pairs of bladder cancer tissues and adjacent tissues were collected and cryopreserved in liquid nitrogen or fixed with formalin.At the same time,collect various clinical information of patients.2)Total mRNA was extracted from frozen tissues by Trizol and reverse transcripted into cDNA.The expression of PlncRNA-1 in bladder cancer and adjacent tissues was analyzed by real-time quantitative PCR(RT-qPCR).3)R software was used for statistical analysis.T test was used to analyze the differential expression of PlncRNA-1 in each subgroup.Chi-square test was used to analyze the differential distribution of PlncRNA-1 high expression group and low expression group in different subgroups.3.2 PlncRNA-1 regulates the proliferation and EMT of bladder cancer1)Total mRNA was extracted from several bladder cancer cell lines(5637,T24,J82 and RT4)by Trizol,and then reverse transcripted into cDNA.The expression of PlncRNA-1 in several bladder cancer cell lines was detected by RT-qPCR.2)Design and synthesize PlncRNA-1 small interfering RNA(siPlncRNA-1),use RT-qPCR to verify the interference efficiency of siPlncRNA-1.3)The expression of PlncRNA-1 was knocked down in bladder cancer cells.CCK-8 assay was used to detect the regulation of PlncRNA-1 on the proliferation of bladder cancer cells.The clone formation assay was used to detect the regulation of PlncRNA-1 on the cloning ability of bladder cancer cells.The wound healing assay was used to detect the regulation of PlncRNA-1 on the migration of bladder cancer cells.Transwell assay was used to detect the regulation of PlncRNA-1 on the migration and invasion of bladder cancer cells.4)The expression of PlncRNA-1 was knocked down in bladder cancer cells.Western blot was used to detect the expression level of EMT markers in bladder cancer cells5)The regulation of PlncRNA-1 on the proliferation of bladder cancer cells was detected in vivo.Nude mice were used for subcutaneous tumor implantation.The volume and growth curve of implanted tumor were measured.The expression of Ki-67 in implanted tumor was detected by immunohistochemistry(IHC).3.3 Hypomethylation of the promoter region of PlncRNA-1 can promote its expression1)Bioinformatics analysis:the methylation level of the promoter region of PlncRNA-1 and the correlation between methylation level and expression level were analyzed by Wanderer and Mexpress.2)Bisulfite amplicon sequencing(BSAS)was performed on two cells(T24 and 5637)with different expression of PlncRNA-1 to explore the methylation modification sites in the promoter region of PlncRNA-1 gene.3)Bladder cancer cells were treated with different concentrations of 5-Aza-2'-deoxycytidine(AZA)and DNA was extracted from the cells.Cytosine was transformed into uracil by bisulfite transformation kit,and then the first-generation sequencing was performed to detect the methylation level at position 131 in the gene promoter region of PlncRNA-1.At the same time,the total mRNA was extracted and reverse transcripted into cDNA.RT-qPCR was used to detect the regulatory effect of AZA on the expression of PlncRNA-1.4)Six pairs of bladder cancer tissues were randomly selected to extract DNA.Cytosine was transformed into uracil by bisulfite transformation kit.Bisulfite sequencing(BSP)combined with first generation sequencing was used to detect the methylation level at position 131 in the promoter region of PlncRNA gene.At the same time,the total mRNA of the 6 pairs of bladder cancer tissues selected above was extracted and reverse transcribed into cDNA.RT-qPCR was used to detect the expression level of PlncRNA-1 in bladder cancer tissues.3.4 PlncRNA-1 promotes the progression of BC by miRNA-136/Smad3 axis1)Total mRNA was extracted from 28 pairs of frozen tissues using Trizol and reverse transcribed to cDNA.The expression levels of Smad3 mRNA in bladder cancer tissues and adjacent noncancerous tissues were analyzed by RT-qPCR.Pearson correlation coefficient was used to test the correlation between the expression levels of Smad3 mRNA and PlncRNA-1 in bladder cancer tissues.At the same time,the expression levels of Smad3 protein in bladder cancer tissues and adjacent noncancerous tissues were analyzed by IHC.2)Localization of PlncRNA-1 in the subcellular structure of bladder cancer cells was used by FISH3)After knockdown of PlncRNA-1 expression in bladder cancer cells,RT-qPCR was applied to detect the expression levels of Smad3 mRNA,while Western blot was applied to detect the expression levels of Smad3 protein and phosphorylated-Smad3 protein.At the same time,miRNA-seq was used to detect the differential changes of miRNA after knocking down the expression of PlncRNA-1,and GO enrichment analysis and KEGG enrichment analysis was performed for miRNA predicted targets.4)After extracting miRNA from 28 pairs of frozen tissue with Trizol and reverse transcribing to cDNA.RT-qPCR was used to analyze the expression levels of miRNA-136 in bladder cancer tissues and adjacent noncancerous tissues5)After knocking down the expression of PlncRNA-1 in bladder cancer cells,RT-qPCR was used to detect the expression level of miRNA-136.6)Knock down or overexpress miRNA-136 in bladder cancer cells,RT-qPCR was used to verify the efficiency of knockdown or overexpression.The expression levels of PlncRNA-1 and Smad3 mRNA were determined by RT-qPCR,while the the expression levels of Smad3 protein and phosphorylated-Smad3 protein were determined by Western blot.7)Plasmids of PlncRNA-1 wild-type and mutant were constructed,followed by double transfection of plasmids and miRNA-136 mimics in 293T cells.Dual luciferase assay was applied to verify that PlncRNA-1 could act as a competing endogenous RNA for miRNA-136.8)Rescue experiment:after knockdown of PlncRNA-1 in bladder cancer cells,miRNA-136 was knocked down again.The expression levels of Smad3,phosphorylated-smad3 and EMT markers(Vimentin,E-cadherin and N-cadherin)protein were determined by Western blot.4.Results4.1 The expression of PlncRNA-1 in bladder cancer and its predictive value1)PlncRNA-1 was found to be significantly highly expressed in 71.43%(20/28)of bladder cancer tissues compared with paired paracancerous tissues.2)Subgroup analysis showed that PlncRNA-1 was highly expressed in ?65 years of age group(p=0.048),single tumor group(p=0.049),MIBC group(p=0.0075)and T3-T4 stage group(p=0.019).3)In bladder cancer tissues,bladder cancer patients were divided into PlncRNA-1 high expression group and low expression group according to the median of PlncRNA-1 expression level.In PlncRNA-1 low expression group,T1-T2 stage accounted for 100%(14/14),while MIBC stage accounted for 42.86%(6/14).In PlncRNA-1 high expression group,T1-T2 stage accounted for 57.14%(8/14),while MIBC stage accounted for 85.71%(12/14).Based on the expression level of PlncRNA-1,we can predict the degree of tumor invasion and T stage in patients with bladder cancer.4.2 PlncRNA-1 regulates the proliferation and EMT of bladder cancer1)There are differences in the expression level of PlncRNA-1 in different bladder cancer cell lines(T24,J82,RT4 and 5637),with the highest expression level in 5637 cells and the lowest expression level in T24 cells.2)Design and synthesize the interfering RNA of PlncRNA-1.Compared with the control group,siPlncRNA-1 significantly reduced the expression level of PlncRNA-1 in T24 and 5637 cell lines.3)PlncRNA-1 can inhibit the proliferation and cloning ability of bladder cancer cells:CCK-8 experiments proved that knockdown of PlncRNA-1 in bladder cancer cells could obviously inhibit the proliferation ability of cells.The clone formation experiments further showed that knockdown of PlncRNA-1 could obviously inhibit the cloning ability of cells.4)PlncRNA-1 can inhibit the migration and invasion ability of bladder cancer cells:The wound healing experiment proves that knockdown of PlncRNA-1 in bladder cancer cells obviously inhibits the migration ability of cells.Transwell experiments proved that knockdown of PlncRNA-1 in bladder cancer cells significantly inhibit the migration and invasion of cells.5)PlncRNA-1 could regulate the expression levels of EMT related proteins:compared with the control group,knockdown of PlncRNA-1 obviously decreased the expression of N-cadherin protein and Vimentin protein,and upregulated E-cadherin protein expression.6)In vivo study showed that knockdown of PlncRNA-1 could obviously inhibit the growth of tumors,reduce the weight of tumors,and reduce the volume of tumors.The immunostaining analysis of seeded tumors showed that knockdown of PlncRNA-1 significantly decreased Ki-67 protein expression.4.3 Hypomethylation of the promoter region of PlncRNA-1 can promote its expression1)Application of the Wanderer and Mexpress online tool found that the methylation level of PlncRNA-1 gene promoter region was decreased in bladder cancer tissues and the methylation level of PlncRNA-1 promoter region was negatively correlated with the expression level of PlncRNA-1.2)BS AS was used to analyze the methylation level of the transcription start site(TSS,2000 bp upstream and 1000 bp downstream)of the PlncRNA-1 gene in bladder cancer T24 and 5637.The study found that the 131 position of the PlncRNA-1 promoter(located at position 36157603 on chromosome 21)has a high methylation level,and there is a significant difference in the methylation level of the two cell lines.3)BSP combined with first-generation sequencing showed that different concentrations of AZA can significantly inhibit the methylation of PlncRNA-1 promoter region after treating bladder cancer cells.At the same time,10 clones were randomly selected from the products of BSP amplification and sequenced for the first generation.It was found that with the increase of AZA concentration,the number of methylated clones decreased.Therefore,AZA can effectively inhibit the methylation level of PlncRNA-1 promoter.4)After using different AZA concentrations to treat bladder cancer cells,the study found that as the concentration of AZA increases,the expression level of PlncRNA-1 gradually increases.5)Randomly selected 6 pairs of bladder cancer tissues and adjacent tissues for BSP and first-generation sequencing.It was found that the methylation level of PlncRNA-1 promoter at position 131 in bladder cancer tissues was significantly lower than that of adjacent tissue.6)RT-qPCR was performed on total mRNA extracted from bladder cancer tissues and adjacent tissues using 6 selected pairs as above.It was found that the expression level of PlncRNA-1 was obviously higher in bladder cancer tissues than in adjacent tissues.4.4 PlncRNA-1 promotes the progression of BC by miRNA-136/Smad3 axis1)Compared with adjacent tissues,Smad3 is significantly up-regulated in 75.00%(21/28)of bladder cancer tissues.Correlation analysis showed that the expression levels of PlncRNA-1 and Smad3 mRNA in bladder cancer tissues were positively correlated(R=0.82,p=9.7e-08).2)Use FISH to confirm that PlncRNA-1 is mainly distributed in the nucleus of bladder cancer cells and partly in the cytoplasm.3)Knockdown of PlncRNA-1 in bladder cancer cells can significantly down-regulate the expression levels of Smad3 mRNA,Smad3 protein and phosphorylated-Smad3 protein.4)After knocking down PlncRNA-1 in bladder cancer cells,miRNA sequencing was performed,and it was found that 36 miRNAs were differentially expressed,of which has-miRNA-136-5p was the miRNA with the largest fold change.GO analysis and KEGG analysis of miRNA target genes found that the biological processes include transcription,classical Wnt signal transduction and intracellular signal transduction.The miRNA target genes are mainly located in the nucleus and cytoplasm.The main molecular functions of miRNA target genes include protein-DNA binding and transcriptional activity.The main pathway processes of miRNA target genes include multiple cancer pathways,focal adhesions,regulation of actin cytoskeleton,mTOR signaling pathway,Wnt signaling pathway,and transcriptional activity.5)Compared with adjacent tissues,the expression of miRNA-136 in bladder cancer tissues was significantly down-regulated(p=0.0078).6)Knockdown of PlncRNA-1 in bladder cancer cells can significantly up-regulate the expression of miRNA-13 6.7)Knockdown or overexpression of miRNA-136 in bladder cancer cells and RT-qPCR validated its effectiveness.At the same time,knockdown or overexpression of miRNA-136 expression in bladder cancer cells can significantly regulate the expression levels of PlncRNA-1,Smad3 mRNA,Smad3 protein and phosphorylated-Smad3 protein.8)Dual luciferase reporter gene detection found that miRNA-136 can directly bind to PlncRNA-1.9)Rescue experiment:after knocking down PlncRNA-1 in bladder cancer cells,knocking down miRNA-136 again.Rescue experiments found that knocking down miRNA-136 can rescue the changes in the expression of Smad3 protein,phosphorylated-Smad3 protein and EMT marker protein caused by the down-regulation of PlncRNA-1.5.Conclusion1)PlncRNA-1,as an oncogene,is overexpressed in bladder cancer tissues,and its expression level is related to tumor invasion,T stage,age and tumor number.The expression level of PlncRNA-1 can be used as a predictor of tumor invasion and T stage in patients with bladder cancer to a certain extent.2)Functional studies confirm that PlncRNA-1 can regulate the proliferation and EMT of bladder cancer cells.3)In bladder cancer,there is hypomethylation at 131(36157603 on chromosome 21)of PlncRNA-1 promoter,and the hypomethylation of PlncRNA-1 promoter can promote the up-regulation of PlncRNA-14)Mechanism studies found that PlncRNA-1 promotes the progress of bladder cancer by regulating miRNA-136/Smad3 axis,which provides new strategies and new targets for the diagnosis and treatment of bladder cancer. |
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