| IntroductionBladder cancer is the most common malignant disease of urinary problems.It has ranked the ninth in the world.About 430000 people are diagnosed with bladder cancer every year.Although the diagnosis and comprehensive treatment strategies of bladder cancer have been improved significantly,and the factors of poor prognosis of bladder cancer patients are related to malignant biological behaviors such as rapid proliferation,lymphatic/hematogenous metastasis and direct invasion,the molecular mechanism of malignant biological behaviors such as occurrence,development,recurrence and metastasis of bladder cancer is still unclear.To explore the molecular mechanism in the development of bladder cancer and seek potential therapeutic targets and pathways can provide a new direction for the diagnosis and treatment of bladder cancer.For the new RNA,circular RNAs were once considered as "by-products" of gene splicing after transcription.cricRNAs are newly defined non coding RNAs,which can regulate gene expression at both transcriptional and post transcriptional levels.With the birth of a new generation of sequencing technology and the development of bioinformatics analysis technology,many studies have found that cricRNAs are widely expressed in mammalian cells,and corresponding specificity,cell,growth stage,disease occurrence and development.Moreover,many reports have confirmed that some circular RNAs play an important role in different tumors.For example,cricRNAs interact with miRNAs to regulate transcription factors and classical and non classical signaling pathways to promote tumor development.A number of studies have shown that cricRNAs is related to the pathological characteristics,prognosis and chemosensitivity of bladder cancer.Basic research has further confirmed that the expression of cricRNAs in bladder cancer is related to a variety of tumor biological functions.We analyzed the data set of cricRNAs related to bladder cancer(GSE97239)and found that circFOXO3 was one of the most differentially expressed cricRNAs in bladder cancer tissues and normal bladder tissues.FoxO3 is one of the important members of FoxO subfamily.FoxO3 gene can simultaneously encode circular FOXO3(circFOXO3)and linear FOXO3(FOXO3 mRNA),which are closely related to apoptosis,autophagy,cell cycle,oxidative stress,cell differentiation,metabolism and immune response.CircFOXO3 is derived from the second exon of FOXO3 and has 2571 nucleotide sequences.Up to now,the specific mechanism of cricFOXO3 regulating tumor progression is not clear.It can be expressed as tumor suppressor and tumor promoter in different tumors.Previous studies have shown that this mechanism seems to be diversified.At present,we have recognized the role of non coding cricRNAs as a potential miRNA "sponge".Compared with most linear RNAs,cricRNAs has no 3 'terminal,so it is more resistant to the degradation of exonuclease.cricRNAs is characterized by high spongy capacity(sequences that can contain multiple miRNA binding sites)and relatively high expression level.These characteristics indicate that cricRNAs is more effective than linear non coding RNA.The sponge effect of cricRNAs is miRNA and tissue-specific:some cricRNAs only affect miRNAs of specific families in specific tissues.However,the exact circFOXO3 sponge miRNA remains controversial.The classical pattern of gene regulation by cricRNAs-miRNA-mRNA,so we will explore the possible miRNA and target genes of circFOXO3 in this study.We selected circFOXO3 as our research object and confirmed that circFOXO3 was low expressed in bladder cancer tissues and cell lines by qRT-PCR.Objective to further explore the effect of circFOXO3 on the biological function of bladder cancer through in vitro experiments;to further explore the molecular mechanism of circFOXO3 involved in the biological regulation of bladder cancer cells through in vitro molecular biology experimental technology;to explore its potential clinical significance through the correlation between its expression and clinical indicators.Methods:1.The expression level and biological significance of circFOXO3 in clinical tissue and cell line level1.1.49 cases were selected for pathological type of bladder urothelial carcinoma and 2cm normal bladder tissue beside cancer.RNA was extracted respectively,and the expression level of circFOXO3 in bladder cancer tissues and adjacent normal tissues was detected by qRT-PCR.The clinical data of patients with bladder cancer were collected and the clinical characteristics of patients with bladder cancer were analyzed by tabulation.The patients were divided into high expression group and low expression group according to the expression level of circFOXO3.Kaplan Meier survival analysis was used to test whether there was significant difference in endpoint(survival time or survival rate)between the two groups.1.2 qRT-PCR was used to detect the expression of circFOXO3 in human bladder cancer cell line 5637,human bladder cancer cell line EJ,human lung metastatic bladder cancer cell T24,human bladder transitional cell carcinoma cell UM-UC-3 and bladder epithelial immortalized cell SV-HUC-1.In vitro,circFOXO3 was overexpressed in bladder cancer cell lines EJ and T24,and the biological changes of bladder cancer cells were observed.CCK-8(cell counting kit-8)was used to detect the proliferation of bladder cancer cells,and and use Transwell to determine specific migration and invasion mechanisms.2.circFOXO3/miR-9-5p/TGFBR2 regulatory axis exists in bladder cancer cells.2.1 Bioinformatics analysis database and software TargetScan,miRanda and Circbase are used to predict miRNA and target genes that may be adsorbed by CircFOXO3.2.2 Overexpression of circFOX03 vector was transfected into EJ and T24 cells,and the expression level of mir-9-5p was detected by qRT-PCR2.3 EJ and T24 cells were transfected with synthetic mir-9-5p mimics,and the expression level of circFOXO3 was detected by qRT-PCR;Luciferase reporter gene experiment detected the luciferase activity of circFOX03wt reporter plasmid or mut reporter plasmid to verify the interaction of mir-9-5p with circFOXO3 and TGFBR2.2.4 In order to verify the regulatory effect of circFOXO3 on TGFBR2 and interfere with circFOXO3 and siTGFBR2,qRT-PCR and other methods were used to determine the specific expression of protein and TGFBR2 mRNA.3.circFOXO3 inhibited the proliferation,migration and invasion of bladder cancer cells through circFOXO3/miR-9-5p/TGFBR2 regulatory axis.3.1 The expression level of TGFBR2 in bladder cancer tissues and adjacent normal tissues was detected by qRT-PCR.3.2 Transfection of circFOXO3 Ctrl vector,circFOXO3 overexpression vector,circFOXO3 over expression vector and siTGFBR2 in bladder cancer cells EJ and T24,CCK8 test the proliferation and proliferation of bladder cancer cells in each group.Results:1.The expression level and biological significance of circFOXO3 in bladder cancer tissues and cell lines.1.1 qRT-PCR results showed that the expression level of circFOXO3 in bladder cancer tissues was lower than that in adjacent normal bladder tissues,and the relative expression levels of circFOXO3 were 1.36+0.53 and 3.81+0.62,respectively.Kaplan Meier test survival analysis showed that patients with lower expression level of circFOXO3 had shorter overall survival(OS)than those with higher expression level;The median survival time in the low expression group of circFOXO3 was 27.8 months.The overall survival time of bladder cancer patients was negatively correlated with the expression of circFOXO3.The expression level of circFOXO3 was not significantly different from that of the patient's age and gender there was a close positive correlation with T stage and lymphatic metastasis1.2 The relative expression of circFOXO3 in human bladder cancer cell line 5637,human bladder cancer cell line EJ,human lung metastatic bladder cancer cell T24,human bladder transitional cell carcinoma cell UM-UC-3,bladder epithelial immortalized cell SV-HUC-1 were 1.03+0.17,0.69+0.08,0.65+0.93,0.33 0.03,0.27+0.27,respectively.The expression level of circFOXO3 was low and the expression level of EJ and T24 was high.We selected it as the cell line for follow-up study;CCK8 results showed that overexpression of circFOXO3 could inhibit the proliferation of T24 cells and EJ Cells.Transwell experiment showed that up regulating the expression of circFOXO3 could inhibit the migration and invasion of T24 cells and EJ Cells.2.circFOXO3/miR-9-5p/TGFBR2 regulatory axis exists in bladder cancer cells.2.1 Bioinformatics analysis database Cirbase,TargetScan and miRanda are used to predict miRNA and target genes that circFOXO3 may be spongy.It was found that mir-9-5p may be a potential candidate for further research.Then,we found a potential binding site that may be regulated by mir-9-5p on TGFBR2,a downstream gene of mir9-5p.2.2 qRT-PCR showed that the expression level of mir-9-5p decreased after up regulating the expression of circFOXO3.2.3 Double luciferase gene level detection report shows that BC cells with circFOXO3 WT and mir-9-5p overexpression vector have lower luciferase level than those with circFOXO3 mutant and mir-9-5p overexpression vector,and BC cells with TGFBR2 WT and mir-9-5p expression vector have lower luciferase level than those with TGFBR2 mutant and mir-9-5p expression vector,It was confirmed that mir-9-5p could interact with circFOXO3 and TGFBR2.2.4 In RNA interference experiment,qRT-PCR results showed that in EJ and T24 cell lines,the expression level of TGFBR2 mRNA in circFOXO3 overexpression and siTGFBR2 co transfection group was lower than that in circFOXO3 overexpression group alone,while the expression level of TGFBR2 mRNA in circFOX03 overexpression,siTGFBR2 and TGFBR2 overexpression co transfection group was higher than that in circFOXO3 overexpression and siTGFBR2 co transfection group,but lower than that in circFOXO3 overexpression group.Western blot showed that in EJ and T24 cell lines,the protein expression level of TGFBR2 in the co transfection group with overexpression of circFOXO3 and siTGFBR2 was lower than that in the single circFOXO3 overexpression group,while the protein expression level of TGFBR2 in the co transfection group with overexpression of circFOXO3,siTGFBR2 and TGFBR2 was higher than that in the co transfection group with overexpression of circFOXO3 and siTGFBR2,but lower than that in the circFOXO3 overexpression group.Therefore,qRT-PCR and Western blot showed that circFOXO3 could regulate the transcription and translation expression level of TGFBR2.3.CircFOXO3 inhibited the proliferation,migration and invasion of bladder cancer cells by circFOXO3/miR-9-5p/TGFBR2 regulatory axis.3.1 The results of qRT-PCR showed that the expression of TGFBR2 in bladder cancer tissues was significantly lower than that in normal tissues.3.2 CCK8 analysis showed that up regulating the expression of circFOXO3 could inhibit the proliferation of EJ and T24 cells,while TGFBR2sirna weakened this inhibitory effect.3.3 Transwell analysis showed that circFOXO3 overexpression inhibited the migration and invasion of EJ and T24 cells.Compared with the circFOXO3 expression vector transfection group,the circFOXO3 overexpression vector and TGFBR2 siRNA co transfected bladder cancer cells had greater migration and invasion ability.Conclusion:1.the expression of circFOXO3 in bladder cancer and bladder cancer cells was lower than that in normal urothelial tissues.The low expression of circFOXO3 in bladder cancer patients is associated with late clinical stage,higher pathological grade,distant metastasis and poorer prognosis..CircFOXO3 has cancer suppressive properties and may have a significant effect on bladder cancer.It provides a new important theoretical basis for searching for the plasma target of bladder cancer.2.circFOXO3 regulates the proliferation,migration and invasion of bladder cancer cells through miR-9-5p/TGFBR2 pathway,and provides a theoretical basis for new therapeutic targets of bladder cancer. |
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