| BackgroundProstate cancer is the sixth most common malignancy among various cancer,and the second most common malignant tumor among men worldwide.In China,the incidence of prostate cancer is increasing every year,especially in developed cities,and prostate cancer has become a major problem for people health.To date,despite the surgical intervention and postoperative adjuvant chemotherapy for prostate cancer had made a big through,the 5-year survival rate is still at a low level for the reason that the lack of specific symptoms in the early stage and the effective screening methods.Therefore,it is of great important to explore the novel and more efficient biomarkers for prostate cancer.In recent years,with the rapid development of high-throughput sequencing technology,the non-coding RNAs(ncRNAs)in tumor microenvironment have attracted more and more attention,including circular RNAs(circRNAs),long non-coding RNAs(lncRNAs)and microRNAs(miRNAs)and etc.Among them,circRNA is a class of covalent cyclic structures that lack of the modification of 5’ and 3’terminus,making them resistant to the degradation of RNA enzymes and more stable than lncRNA.Furthermore,circRNA could also regular splicing,transcription and binding related proteins.Among all,circRNA＿100395 play as a specific role in breast and liver cancer,however,its function in prostate cancer is still unclear.In addition,numerous studies have shown that the microRNAs sponge adsorption of circRNA is the key mechanism for the regulation of tumor progression.In this study,the expression level of circRNA＿100395 in prostate cancer was analyzed by real-time quantitative polymerase chain reaction(RT-qPCR),and the relationship between its expression level and clinicopathological features and clinical prognosis of patients with prostate cancer was further analyzed to clarify the role of circRNA＿100395 in the development of prostate cancer.In addition,we found that microRNA-1228 and ORC6 may be the key downstream regulatory targets of circRNA＿100395 by means of a signal analysis,and further analyzed the expression level and correlation of circRNA＿1 00395 and microRNA-1228/ORC6 in tissue samples,and finally elucidated the possible role of circRNA＿100395/microRNA-1228/ORC6 axis in the development of prostate cancer.This study aimed to provide a novel and effective bio-marker for the diagnosis and treatment of prostate cancer.Part Ⅰ Expression pattern and clinical characteristics of circRNA＿100395 in prostate cancerObjectiveTo detect the expression level of circRNA＿100395 in prostate cancer and explore its clinical significance.Methods1.73 cases of Prostate cancer and paired non-tumor tissues were collected,which were further confirmed by pathological examination.2.The expression level of circRNA＿100395 were detected by real-time quantitative polymerase chain reaction(RT-qPCR)in prostate cancer and paired non-tumor tissues.Results1.The expression level of circRNA＿100395 in prostate cancer tissues were lower than that in normal tissues.2.The expression of circRNA＿100395 was closely association with the tumor size(P<0.001),Gleason score(P<0.001),lymphatic metastasis(P=0.0001),TNM stage(P=0.003).ConclusionsThe expression of circRNA＿100395 in prostate cancer tissue is significantly lower than that in normal tissue.Its expression level is closely associated with tumor size,Gleason score,lymph node metastasis and tumor TNM staging.Part Ⅱ The biological function of circRNA＿100395 in the regulation of cell proliferation and metastasis abilities in prostate cancerObjectiveTo detect the function of circRNA＿100395 in the regulation of cell proliferation,migration and invasion abilities of prostate cancer.Methods1.RT-qPCR assay was applied to detect the expression level of circRNA＿100395 in the normal RWPE cells and other prostate cancer cell lines(PC-3,DU 145,LNCAP,C4-2,22RV1 and VCAP cells).2.Lentivirus transfection assay was performed to construct the circRNA＿100395-upregulated PC-3 and DU 145 cell lines,and screened by puromycin.3.CCK-8 assay was used to detect the cell proliferation ability in PC-3 and DU145,after up-regulating circRNA＿100395 expression.4.Flow cytometry assay was applied to detect the distribution of cell cycle in PC-3 and DU 145,after up-regulating circRNA＿100395 expression.5.Transwell assay was applied to detect the ability of invasion and migration in PC-3 and DU 145 after up-regulating circRNA＿100395 expression.In addition,the western blotting assay was performed to evaluate the expression level of Epithelial-Mesenchymal Transition(EMT)signaling pathway related proteins.Results1.The results of RT-qPCR revealed that the expression level of circRNA＿100395 in prostate cancer cell were lower than that in normal cells,while PC-3 and DU 145 showed relatively lowest content(P<0.001).2.After lentivirus transfection,the circRNA＿100395 expression in PC-3 and DU 145 cells were up-regulated.(P<0.001).3.After circRNA＿100395 expression increased,the cell proliferation ability of PC-3 and DU145 cells were significantly inhibited,and numerous cells were arrested in G2/M stage.4.After circRNA＿100395 expression increased,the cell metastasis ability of PC-3 and DU 145 cells were dreamatically reduced.In addition,the results of western blotting revealed that the over-expression of circRNA＿100395 could upregulate E-cadherin protein expression,and inhibit N-cadherin,Vimentin and Slug proteins expressions.ConclusionsIn prostate cancer,up-regulating circRNA＿100395 expression could suppress cell proliferation ability through arresting numerous cells in G2/M stage.Meanwhile,the over-expression of circRNA＿100395 could inhibit cell invasion and migration abilities by inhibiting EMT related signaling pathway.Overall,circRNA＿100395 might be an important regulatory target for prostate cancer.Part Ⅲ Overexpression of microRNA-1228 could reverse the anti--tumor effect induced by up-regulated circRNA＿100395ObjectiveTo evaluate the cor-relationship between microRNA-1228 and circRNA＿100395 in prostate cancer.Methods1.Based on the bio-medical analysis technology,we predict the possible downstream target gene of circRNA＿100395,and further confirm their potential binding site.2.RT-qPCR assay was used to evaluate the expression level of microRNA-1228 in circRNA＿100395-overexpressed cell lines.3.The wild-type and mut-type 293T cell lines were constructed to analyze the relationship of circRNA＿100395 and microRNA-1228.4.The cell proliferation and metastasis abilities were detected in circRNA＿100395-overexpressed cells,after treated with microRNA-1228 mimics.Results1.Based on the results of bioinformatics analysis,the microRNA-1228 might be the target downstream of circRNA＿100395.2.The luciferase reporter assay results indicated that the luciferase activity was significantly lower in wild-type cells treated with microRNA-1228 mimics.3.RT-qPCR showed that microRNA-1228 was inhibited in circRNA＿100395 overexpressed cell lines.4.The cell proliferation and metastasis abilities were significantly promoted in circRNA＿100395-overexpressed cells,after treated with microRNA-1228 mimics.ConclusionsCircRNA＿100395 can directly bind microRNA-1228 in prostate cancer cells and directly down-regulate the expression level of downstream target gene microRNA-1228,thereby inhibiting the proliferation,metastasis and invasion ability of prostate cancer cells.Part Ⅳ CircRNA＿100395 regulate cell proliferation and metastasis abilities in prostate cancer via regulating microRNA＿1228/ORC6 axisObjectiveTo evaluate the cor-relationship between circRNA＿100395 and microRNA-1228/ORC6 axis in prostate cancer.Methods1.RT-qPCR assay was applied to detect the expression level of ORC6 in the normal RWPE cells and other prostate cancer cell lines(PC-3,DU145,LNCAP,C4-2,22RV1 and VCAP cells).2.The plasmid transfection assay were used to up-regulate the expression level of ORC6 in prostate cancer cells,and further detect their proliferation and metastasis abilities.Results1.The results of RT-qPCR revealed that the expression level of ORC6 in prostate cancer cell were lower than that in normal RWPE cells.2.The proliferation ability and invasion and metastasis abilities were inhibited after up-regulating the expression level of intracellular ORC6 in circRNA＿100395-overexpressed and microRNA-1228-overexpressed prostate cancer cell lines.ConclusionsIn summary,circRNA＿100395 could directly sponge microRNA-1228,followed by upregulate the expression level of ORC6,and finally inhibit cell proliferation and metastasis abilities in prostate cancer cells. |
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