| Prostate cancer(PCa)is the most common malignancy of the male reproductive system,and the incidence increases with age.In China,there is an ascending trend of the incidence in some developed areas in recent years especially in Shanghai,where PCa has ranked into the first common male reproductive system cancer.Therefore,it is becoming more and more imperative to provide the theoretical basis of molecular mechanism for the treatment of PCa.Circular RNA(circular RNA,circRNA)is a kind of non-coding RNA formed by covalently linking the 5 'and 3' ends of linear RNA precursors by reverse splicing mechanism,which is also related to transcription and post-transcriptional regulation of genes.Nowadays,with the continuous development of molecular biology,bioinformatics and high-throughput sequencing technology,circRNA has been found to exist not only in bacteria,fungi,plants,but also in mammals.As a competitive endogenous RNA(CeRNA),circRNA,miRNA,lncRNA and protein have an interaction relationship with each other.With in-deep researches,circRNA has been proved to involve in a variety of processes of diseases and tumors.CircRNA is potentially expected to provide a new theoretical mechanism to related diseases and tumors for its rich expression,unique and stable structure,and tissue expression specificity.However,the mechanism and function of circRNA in the development and progression of PCa is still unclear.To address the role of circRNA plays in the pathogenesis and progression of PCa,normal prostate epithelial cells RWPE-1,prostate carcinoma epithelial cell 22RV1 and PCa bone metastatic cell PC3 were selected to be detected via high-throughput circRNA sequencing,representing normal tissue,tumor and metastatic tissue respectively.The results showed that the expression of circRNA-CDR1 as was significantly higher nearly 200 times in PC3 cell lines than that in normal prostate epithelial cell lines,suggesting that CDR1 as may be a crucial molecular in the progression of PCa.CDR1 as was famous for its "miRNA sponge" function for the sake of dense distribution of a variety of miRNA binding sites on its sequence.And there has been a former deep study about CDR1 as so that we choose it as our target in this study.Firstly,we verified the sequencing results by qPCR in several common prostate cancer cell lines and a consistent result was obtained.Then we detected the characteristics of CDR1 as as a circRNA by qPCR and agarose gel electrophoresis.PC3 and C4-2,in which CDR1 as highly expressed,were selected for cell functional verification.CCK8 assay,Edu proliferation assay,flow cytometry cycle and apoptosis assay,transwell migration and invasion experiments were detected in PC3 and C4-2 after siRNA interference.The results showed that CDR1 as had an effect on PCa cell proliferation,cycle,migration and invation.The results of nucleoplasm separation essay showed that CDR1 as mainly existed in the cytoplasm and miRIP essay indicated that CDR1 as may modulate the expression of downstream genes through binding with miR-23a/b.The high-throughput sequencing results of mRNA based on the cell samples dealt with siRNAs of CDR1 as demonstrated that CCND1 and CXCL5 were significantly downregulated and which was validated by qPCR and western blot experiments.The predictive results by multiple databases illustrated that there were binding sites of miR-23a/b on the sequence of CCND1 and CXCL5,corresponding study reported that CCND1 and CXCL5 played an important role in the cycle and metastasis of cancer.In summary,previous studies on circRNA have focused on elucidating the mechanism of their formation.At present,more studies are contributed to their functions in diseases.With the advantages of stability,high conservation and strong expression specificity,circRNA has an important research value in the study of disease pathogenesis or being a diagnostic biomarker.Our results confirmed that CDR1 as performed as a ceRNA to up-regulate the expression of CCND1 and CXCL5 by inhibiting miR-23a/b,thereby promoting the progression of PCa.As we know,it is the first study to clarify the mechanism of CDR1 as in PCa progression as a ceRNA,which may serve as a potential target for the treatment of PCa or a biomarker for the diagnosis.Part? Profiling and bioinformatics analyses of circular RNA expression in prostate cancer cells.Objective To explore the profiling of circular RNA expression in PCa cells.Methods(1)The circRNAs were enriched by the specific sequencing of circRNA.(2)The expression of circRNA in normal prostate epithelial cell RWPE-1,prostate carcinoma cell 22RV1 and prostate cancer bone metastatic cell PC3 was detected by high-throughput sequencing.(3)Bioinformatics analyses were applied in sequencing results.(4)Sequencing results were validated by RT-qPCR.Results(1)A total of 9545 kinds of circRNAs were identified in the three cell lines.(2)There were hundreds of differentially expressed circRNAs in the three cell lines.(3)GO analyses and KEGG pathway analyses showed that host genes of differential circRNAs were closely related to progression of PCa.(4)Compared with RWPE-1,CDR1 as was upregulated nearly 200 times in PC3.(5)CDR1as had multiple miRNA binding sites,such as miR-7-5p and so on.Conclusion The expression of circRNA in prostate cancer cell lines is very abundant,and there are many differentially expressed circRNAs among these cell lines,which may be closely related to the development of PCa.CDR1 as highly expressed in PC3 and has association with many important miRNAs,which may play an important role in proliferation and metastasis.Part ? The expression validation of CDR1 as in prostate cancer cell lines and tissuesObjective To detect and validate the expression results of CDR1 as in PCa cell lines and tissues.Methods(1)We obtained the sequence of CDR1 as through the high-throughput sequencing and which was verified in the Circnet database,the accuracy of our sequencing results was confirmed.(2)Sequencing results of CDR1 as expression were verified by qPCR in three cell lines.(3)The amplified fragments were verified by sanger sequencing.(4)Three PCa cell lines(LNCaP,DU 145 and C4-2),kidney cancer cell line 786 O and bladder cancer cell line T24 were added to demonstrate the expression specificity of CDR1 as.(5)The characteristics of circRNA were verified by agarose gel electrophoresis of the convergent and divergent primer amplifications from the cDNA and gDNA of PC3.(6)RNase R digestion was used to verify the stability characterastics of CDR1 as.(7)The distribution of the CDR1 as in the sub-localization of cells was carried out by nuclear separation experiment.(8)We analysed the expression of CDR1 as in 65 pairs of tissues.Results(1)CDR1as was an antisense strand of circRNA from X chromosome containing 1485 bases with the circRNA ID of has-circ-0001946 chrX: 139865339-139866824.(2)The expression of CDR1 as in PC3 was more than 1000 times that of RWPE-1 and nearly 30 times that of 22RV1.(3)The sequence of sanger sequencing was matched to the correct sequence.(4)CDR1as specifically expressed in prostate cancer cell lines,also highly expressed in C4-2 about 200 times that of RWPE-1.(5)The convergent primer amplification products were observed in both cDNA and gDNA but the divergent primer amplification product was only observed in cDNA,indicating that there was no identical circular DNA molecule in the genome.(6)The expression of CDR1 as was slightly affected by RNase R digestion,but the expression of line transcript ?-actin was significantly decreased.(7)Nuclear-cytoplasm separation experiment showed that CDR1 as was mainly distributed in the cytoplasm.(8)The expression of CDR1 as is positively related to PSA level,r = 0.30.Conclusion CDR1 as is an antisense strand circRNA derived from X chromosome,which specifically expresses in PCa cell lines PC3 and C4-2,and was mainly distributed in the cytoplasm.Part ? Functional identification of CDR1asObjective To explore the function of CDR1 as in PCa cells.Methods(1)Cells were transfected with siRNAs to interfere CDR1 as expression.(2)The proliferation of PC3 and C4-2 cells processed with siRNAs was detected by CCK8 assay.(3)Cell cycle and apoptosis of PC3 and C4-2 cells processed with siRNAs was detected by flow cytometry.(4)The ability of migration and invasion of PC3 and C4-2 cells processed with siRNAs was examined using transwell chambers.(5)Stable CDR1 as knock-down cell line was constructed by virus transfection.(6)Tumor-bearing mice model was constructed using CDR1 as knockdown stable cell line,and then its impact on tumorigenicity was explored.Results(1)The expression of CDR1 as was down-regulated by more than 80% after transfection with siRNAs efficently.(2)The ability of proliferation was inhibited after being processed with siRNAs.(3)Cell cycle arrested in G1 phase after being processed with siRNAs,no effect was detected on apoptosis.(4)The ability of migration and invasion of cells processed with siRNAs was weakened.(5)The knockdown efficiency of stable cell line was more than 90%.(6)The ability of tumorigenicity in stable cell with CDR1 as knockdown was decreased.Conclusion CDR1 as can affect the proliferation of prostate cancer cells through interfering cell cycle without affecting the apoptosis.CDR1 as can promote the invasion and metastasis of PCa cells.The knockdown of CDR1 as can reduce the tumorigenicity of cancer cells.Part ? The mechanism of CDR1 as in prostate cancer cellsObjective To explore the mechanism of CDR1 as performed in PCa cells.Methods(1)The miRIP method was used to explore the miRNAs binding with CDR1 as.(2)Sequencing of the mRNAs of cell lines processed with siRNAs was used to find out the downstream targets.(3)RT-qPCR was used to verify sequencing results.(4)Multiple databases were used to predict the target binding sites between mRNAs and miRNAs.(5)Dual luciferase reporter detection was used to verify the binding of miRNAs and CDR1 as.(6)To further explore the relationship among CDR1 as,miRNA and mRNA we used miRNA mimics transfection method and miRNA inhibitors transfection rescue experiment.(7)Western blot was used to detect the expression of CCND1 and CXCL5 and EMT markers.Results(1)CDR1as could sponge miRNAs such as miR-183,miR-23 a and miR-23 b and so on.(2)CCND1 and CXCL5 gene expression was downregulated after CDR1 as was down-regulated.(3)CCND1 had binding sites with miR-183,miR-23a/b and CXCL5 had binding sites with miR-183,miR-23a/b,miR-200b/c.(4)Dual luciferase reporter assay showed that there was interaction between CDR1 as and miR23a/b.(5)The expression of CCND1 and CXCL5 was down-regulated after miR23a/b overexpressed,and the expression of CCND1 and CXCL5 was restored with transfection of miRNA23a/b inhibitors in cells processed with CDR1 as siRNAs.(6)The results of Western blot showed that the expression of CCND1 and CXCL5 was down-regulated after being processed with CDR1 as siRNAs,while the expression of Ncadherin and Snail was down-regulated in PC3 knockdown groups,Ecadherin was up-regulated and Snail was downregulated in C4-2 knockdown groups.Conclusion CDR1 as regulates the expression of CCND1 through miR-23a/23 b and further promotes the cell cycle progression of PCa cells.At the same time,miR-23a/b regulates the expression of CXCL5 and changes the expression of EMT marker to promote the migration and invation of PCa cells. |
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