| Circular RNAs?circRNAs?,formed by alternative splicing of pre-mRNA transcripts,are a stable and widespread form of non-coding RNA in human cells.recent study finding,circRNAs have many significant regulatory functions,for example,circRNAs can function as miRNAs sponge which can competitive binding intracellular miRNAs that blocking the inhibiting effects of their target genes,sequentially regulate miRNAs' targets.Besides,circRNAs may also play a role by binding to RBP?RNA-binding protein?or complementary base pairing with other RNAs.With the further researches,miRNAs can influence tumor occurrence and progress acquired more and more researchers' approval.At present,the role of circRNAs have been reported in gastric cancer,hepatic carcinoma,laryngocarcinoma,basal cell carcinoma,colorectal cancer and leucocythemia.However the role of circRNAs in prostate cancer has not been elucidated.Our preliminary experiments show that circ CDK13 expressed in prostate cancer is significantly higher than tissue adjacent to carcinoma and hyperplasia of prostate tissue,we suspect it may have effect on promoting cancer,so circCDK13 was selected as a candidate circRNA for the further researches.Part? The expression of circCDK13 and its function in prostate cancer.Objective: To investigate circCDK13 expression in prostate cancer and its impact on prostate cancer cell proliferation.Methods:1 CircRNA microarray analysis of prostate cancer and corresponding adjacent non tumor specimens,Select remarkably upregulated circRNAs,then verified by qRT-PCR.2 Design divergent primers that aim at back-splicing junction.The specificity of the RT-PCR was validated by 1% agarose gel electrophoresisand sanger sequencing.3 RNA in situ hybridization for circ-CDK13 in paired prostate cancer tissues and observe circCDK13 expression quantity and location.4 Loss and gain of circCDK13,qRT-PCR detecte the expression of circ-CDK13,Wound healing assay were performed to observe proliferation capability.5 Loss and gain of circCDK13,Western blot analysis Cycle related proteins.Results:1 circCDK13 expressed in prostate cancer is significantly higher than para-carcinoma tissue.CircRNA microarray analysis shows distinguishable circRNA expression profile in 3 samples of cancerous and adjacent tissues.A total 15 kinds of circRNAs were found dysregulated expression in prostate cancer tissue,including 8 circRNAs upregulated?hsacirc40578,hsacirc0001212,hsacirc0051239,hsacirc0002082,hsacirc0000517,hsacirc0001982,hsacirc0079929,hsacirc0054971?and 7 circRNAs downregulated?hsacirc0001296,hsacirc0000350,hsacirc0003748,hsacirc0001255,hsacirc0079385,hsacirc0001605,hsacirc0092360?.In this 15 kinds of dysregulated circRNAs,we select the most significantly upregulated hsacirc0079929 as the research object,due to it comes from CDK13 gene,we term this circular transcript circCDK13.Then q RT-PCR confirmed circCDK13 expressed in prostate cancer is significantly higher than para-carcinoma tissue.2 Identification of circCDK13 as a circ RNA.To verify circCDK13 is a circRNA,we design divergent primers that aim at back-splicing junction,cDNA and g DNA as template for RT-PCR,then RT-PCR product was validated by agarose gel electrophoresis.cDNA RT-PCR product include back-splice junction Segments of DNA nucleotides,but gDNA RT-PCR product didn't.Sanger sequencing verified the back-splice junction.RT-PCR amplification circCDK13,agarose gel electrophoresis toidentify the molecular weight is 660 bp.3 Verification that circRNA expression in clinical tissues.In order to further confirm circCDK13 expression quantity,HE staining was conducted to distinguish prostate cancer and adjacent tissues.RNA in situ hybridization for circ-CDK13?green?indicate that circ-CDK13 in prostate cancer is significantly higher than its para-carcinoma tissue.The result is consistent with microarray and qRT-PCR.4 Loss and gain of circCDK13 inhibits and promotes proliferation of prostate cancer cells,respectively.In order to verify circCDK13 biology function,we construct GFP-circ CDK13 plasmid and ordered si-circCDK13,Loss and gain of circCDK13,then q RT-PCR detectes the expression of circ-CDK13.We found that when transfect GFP-circCDK13,its expression increased;When transfect si-circ CDK13,its expression decreased.The reliability of si-circCDK13 and GFPcircCDK13 was confirmed.Since circCDK13 is upregulated in prostate cancer,it should promote the PC3 cells proliferation in vitro,To test this hypothesis,we carried out cell scratch experiment.The results showed that,overexpression of circCDK13 significantly increased PC3 cells proliferation and migrate ability;knockdown circCDK13 reveals opposite results.5 CircCDK13 affect tumor cell proliferation through regulating the cell cycle.In order to clarify the molecular mechanisms of circCDK13 promote prostate cancer cell proliferation,Western blot analysis was conduct for cell cycle-related protein.CDK4,CDK13 and CyclinD1 were significantly increased in PC3 cell with circCDK13 overexpressed.Knockdown circCDK13 reveals opposite results.These results indicate that prostate cancer cells with circCDK13 overexpressed promote tumor cell proliferation by regulating the cell cycle.Summary: CircCDK13 expressed in prostate cancer is significantly higher than para-carcinoma tissue,and circCDK13 is verified as a circRNA.Overexpression of circCDK13 significantly increased PC3 cells proliferation;Knockdown circCDK13 reveals opposite results.Circ CDK13 promote tumor cell proliferation by regulating the cell cycle.Part? CircCDK13 activate E2F5 signaling pathway promote prostate cancer cell proliferation by sponging miR-212-5p/449 a.Objective: To elucidate the mechanism of CircCDK13 activate E2F5 signaling pathway promote prostate cancer cell proliferation by sponging mi R-212-5p/449 a.Method:1 qRT-PCR was conducted to checkout the variation of miRNAs between Prostate cancer and para-carcinoma tissue.Bioinformatics methods predict the binding sites between circCDK13 and miR-212-5p/449 a.QRT–PCR analysis of mi R-212-5p/449 a level from the PC3 cell lysates after transfected with3?-end biotinylated circCDK13.Luciferase reporter assay detect whether circCDK13 binding mi RNAs.2 Hierarchical clustering analysis prostate cancer and adjacent non tumor specimens and screen out abnormal expression genes,then the result was verified by qRT-PCR.3 The possible binding sites between E2F5 and miR-212-5p/449 a were predicted by bioinformatics methods.E2F5 3?UTR and their mutant fragments were synthesized and connected to pmir-GLO double fluorescence report plasmid,then transfected PC3 cells and detection the fluorescence activity.RNA in situ hybridization for circ-CDK13 and Immunofluorescence staining of E2F5 in paired prostate cancer tissues to observe their expression quantity and location.4 Overexpression of E2F5 and q RT-PCR detected the variation of circular and liner CDK13;Overexpression of E2F5 then western blot check the variation of CDK13 and cell cycle related proteins.Luciferase reporter assay detect the interaction between E2F5 and circCDK13.5 Cell strain with circCDK13 stably expressed was established via transfected with AdGFP-circCDK13,then they were subcutaneous injection into the back of nude mice to observe the change of the tumor.Western blotanalysis for E2F5 and PCNA in tumor tissue.Immunofluorescence staining of tumor tissues were conducted to observe the expression of E2F5,P21 and PCNA.Results:1 circCDK13 is a ceRNA and reduces miR-212-5p/449 a in prostate cancer.In order to determine which miRNAs interaction with circCDK13,the expression of four miRNAs?miR-212-5p?miR-449a?mi R-375 and miR-578?were analyzed by q RT-PCR in prostate cancer and para-carcinoma tissue.The result showed that mi R-375 significantly increased but the other three rest have not found obviously difference.Subsequently,The binding sites between circCDK13 and miR-212-5p/449 a were predicted by bioinformatics methods.Two miR-212-5p binding sites and four miR-449 a binding sites were found,thus indicated that miR-212-5p/449 a interaction with circCDK13.In order to explore wether miR-212-5p/449 a combine with circCDK13,QRT–PCR analysis of miR-212-5p/449 a level from the oligo-pulldown PC3 cell lysates after transfection with 3?-end biotinylated circCDK13.We Found that the enrichment of miR-212-5p and miR-449 a obviously increased compared with the control group.Luciferase reporter assay detect whether circCDK13 binding miRNAs.In order to further validate the interactions between circCDK13 and miR-212-5p/449 a,pmirGLO-circCDK13 with luciferase activity was synthesized and transfected into PC3 cell with its control,relative luciferase activity were detected.MiR-NC,miR-212-5p,mi R-449 a and mi R-212-5p+miR-449 a were transfected into PC3 subsequently,measure the luciferase activity again.Luciferase reporter assay reveals that the fluorescence activity of pmirGLO-circCDK13 can be suppressed by mi R-212-5p and miR-449 a.The suppression was more distinct when mi R-212-5p and miR-449 a were co-transfected into PC3.2 The expression of E2F5 in prostate cancer is obvious higher than paracancer tissues.For the sake of determine the potential target gene of miR-212-5p/449 a,We use gene microarray analysis two groups of prostate cancer and corresponding adjacent non tumor specimens,nine genes were screened?E2F5,NAPB,HDGF,ELF3,FGF16,HINFP,UBTF,GDF11 and IGFBP3?.Subsequently,qRT-PCR verified that the expression levels of E2F5 in the prostate cancer group are significantly higher than those in corresponding nontumorous tissues.3 miR-212-5p/449a-mediated circCDK13 regulates E2F5 expression in prostate cancer.For the sake of explore wether mi R-212/449 a regulate E2F5,bioinformatics methods were used to predicted the binding sites between miR-212-5p/449 a and E2F5,found that Homo sapiens,Mus musculus,Bos taurus were highly homologous.For further verify the interaction of E2F5 with mi R-212-5p/449 a,the pmirGLO-E2F5 3?UTR with luciferase activities and their mutant fragments pmirGLO-E2F5 3?UTR mut were synthesized and transfected into PC3 cells respectively,relative luciferase activity were detected.MiR-NC,miR-212-5p,miR-449 a and miR-212-5p+miR-449 a were transfected into PC3 subsequently,measure the luciferase activity again.Luciferase reporter assay reveals that the fluorescence activity of pmirGLOE2F5 3?UTR can be suppressed by miR-212-5p or miR-449 a.The suppression was more distinct when miR-212-5p and miR-449 a were co-transfected into PC3.But when E2F5 3?UTR mutation the luciferase activity was almost not affected by mi R-212-5p and miR-449 a.The result suggest that E2F5 include target points of miR-212-5p and miR-449 a.RNA in situ hybridization for circ-CDK13 and Immunofluorescence staining of E2F5 were conducted to verify wether circCDK13 was related to E2F5.Found that circCDK13 with green and E2F5 with red were higher expression in prostate cancer than its paracancer tissue and locate in the same position.4 E2F5 promotes circCDK13 expression and form a feed-back loop regulation.To explore the relationship between CDK13 and E2F5,qRT-PCR detected the variation of circular and liner CDK13 after Overexpression E2F5 in PC3 cells.The expression of circular and liner CDK13 were significantly increased compared with the control groups.Overexpression of E2F5 then western blot check the variation of CDK4,CDK13,CyclinD1 and P21.The expression of cell cycle related proteins CDK4,CDK13 and CyclinD1 were high expression.But the expression of suppress cell cycle related proteins P21 was significantly decreased.Luciferase reporter assay was conducted to elucidate the interaction between E2F5 and CDK13.Fluorescent report carrier with CDK13 promoter was synthetized and transfected PC3 cells with Different quantity of pcDNA3.1-E2F5 together.Found that as the quantity of pcDNA3.1-E2F5 increased,pGL3-CDK13-Promoter luciferase activity is more and more strong.The above results show that the E2F5 expression not only can promote cell cycle increases CDK13 expression,but also can be a competitive combination of miR-212-5p and mi R-449 a,thus reducing the degradation of CDK13.5 circCDK13 promotes tumorigenesis of prostate cancer by activate E2F5.The above results show that the E2F5 can increase CDK13 expression in vitro,so whether CDK13 can regulate the expression of E2F5 in vivo? In order to prove this hypothesis,we have synthesized circCDK13 lentivirus?AdGFP-circCDK13?,tumor Cell strain with circCDK13 stably expression was established via transfected with AdGFP-circCDK13,After several generations of culture,cells were collected and resuspension into 1×PBS solution.The concentration was 1 × 106 cell/50 ?l PBS,and 50 ul resuspension solution were took subcutaneous injection into the back of nude mice,circCDK13 cells in right and its negative control group in left.Mice were sacrificed 21 days after tumor cells inoculation,and observe the change of the tumor.During breeding nude mice,measureed tumor size in 6,9,12,18,21 days respectively,tumor growth curve was drawed,also found that circCDK13 overexpressed tumor growth faster than the control group obviously.Western blot analysis for E2F5 and PCNA in tumor tissue,they obvious increased compared with their negative control group.The aboveresults suggest that circCDK13 can promote the expression of E2F5.To further verified the relationship between circCDK13 and E2F5,Immunofluorescence staining of tumor tissues were conducted to observe the expression of E2F5,P21 and PCNA.Found that compared with control group,the expression of E2F5 and PCNA was significant increased and the expression of P21 significant decreased in tumor tissue with circCDK13 overexpressed.Consistent with Western blot results.6 Proposed model for the mechanism of miR-212-5p/449 a mediates a feedback loop between circCDK13 and E2F5 regulates tumor cell growth in prostate cancer.Comprehensive the above results show that circCDK13 upregulated in prostate cancer and downregulated miR-212-5p/449 a by sponging them,the downregulated miR-212-5p/449 a removed the target suppress of E2F5,then E2F5 activate CDK13 and produce more circCDK13,eventually form a positive feedback loop of circCDK13/miR212-5p/449a/ E2F5 to promote prostate cancer progression.Summary:The expression of E2F5 in prostate cancer is obvious higher than paracancer tissue,but the expression of mi R-212-5p/449 a in prostate cancer is low,circCDK13 regulate E2F5 by sponging miR212-5p/449 a,then E2F5 regulate CDK13 in turn,form a positive feedback loop.Conclusion:1 CircCDK13 expressed in prostate cancer is significantly higher than para-carcinoma tissue.2 CircCDK13 affect tumor cell proliferation through regulating the cell cycle.3 The expression of E2F5 in prostate cancer is obvious higher than paracancer tissues.4 miR-212-5p/449a-mediated circCDK13 regulates E2F5 expression in prostate cancer.5 E2F5 promotes CDK13 expression and form a feed-back loop regu-lation. |
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