Preliminary Screening And Functional Analysis Of CircRNA Differential Expression Profiles In Prostate Cancer And Benign Prostatic Hyperplasia

Background:Prostate cancer is the most common malignant neoplasm in male genitourinary system,especially in Western countries.It hsa high morbidity and mortality.In China,prostate cancer hsa become the fastest growing male malignant neoplasm in recent years.Prostate cancer hsa become one of the biggest diseases threatening men's health.Early detection and intervention is the key to effective treatment of prostate cancer,and also the key to prevent the death of patients with advanced prostate cancer who can not be cured.However,the commonly used serum prostate specific antigen?PSA?detection is not specific in the early diagnosis of prostate cancer,especially in elderly men with benign prostatic hyperplasia?BPH?which is interfering with PSA detection of prostate cancer.There is an urgent need for a more reliable biomarker to identify early prostate cancer and individuals at risk.Therefore,it is necessary to screen new markers to establish new early diagnostic markers of prostate cancer and explore their mechanisms in the pathogenesis and progression of prostate cancer.CircRNA is a covalently closed ring structure molecule formed by splicing of pre-mRNAs.It plays an important role in the regulation of proliferation,apoptosis and invasion of cancer cells.This indicates that circRNA is involved in the molecular mechanism of cancer progression.Many studies have shown that circRNA can be used as a biomarker for cancer diagnosis and prognosis.In recent years,circRNA hsa been extensively studied in various diseases,but there are few studies in prostate cancer.To solve these problems,the differential expression profiles of circRNA in prostate cancer were screened by gene chip technology compared with benign prostatic hyperplasia,which provided theoretical basis for screening new diagnostic markers of prostate cancer and exploring the role of circRNA in the pathogenesis and progression of prostate cancer.Objective:In this study,circRNA was screened from prostate cancer and benign prostatic hyperplasia by gene chip technology.The differentially expressed circRNA was randomly validated by RT-PCR.The differentially expressed circRNA was analyzed by bioinformatics in order to find potential markers for differentiating prostate cancer from benign prostatic hyperplasia,screening new diagnostic markers of prostate cancer,constructing ceRNA network of prostate cancer and lay a foundation in exploring the role of circRNA in the pathogenesis and progression of prostate cancer.Methods:Six prostatic tissue was elected as specimens,including 3 patients with prostate cancer and 3 patients with benign prostatic hyperplasia,were selected.Total RNA was extracted by Trizol method.The quality and concentration of total RNA were determined by NanoDropND-1000 spectrophotometer.The integrity of RNA was verified by agarose gel electrophoresis.Linear RNA was removed by RNase R ribonucleic acid exonuclease.CircRNA was amplified by primers and transcribed into fluorescent cRNA.The amplified and fluorescent labeled cRNA was hybridized with microarray probes,and the hybridized microarray was scanned by Agilent Scanner G2505C scanner.Use Agilent Feature Extraction software to extract data and normalize it.The fold change was used to screen differentially expressed circRNA,and bioinformatics prediction was carried out to find the associated microRNAs.Then KEGG and GO analysis were performed on the microRNAs that the most significant differential expressed circRNA associated.Finally,RT-PCR was used to randomly verify the expression of differential expressed circRNA in those prostate tissues.Results:1.Total of 13313 circRNA were detected.Compared with benign prostatic hyperplasia specimens,7287 were up-regulated and 6206 were down-regulated in prostate cancer specimens.There were 101 differentially expressed circRNA?p<0.05,FC value>2?,of which 26 were up-regulated,with hsa_circRNA₁02485 being the most differentially expressed and 75 down-regulated,and hsa_circRNA₀11333 being the most differentially expressed2.Five microRNAs were predicted for each differentially expressed circRNA by the target gene prediction software of microRNAs.It was found that each differentially expressed circRNA had five corresponding microRNAs and each microRNAs had more than one binding site,suggesting that differentially expressed circRNA could be used as a sponge of microRNAs to regulate gene expression through site adsorption.The results showed that there are binding sites between hsa circRNA 102485 and mir-103a-2-5p,mir-663a,mir-342-3p,mir-188-3p and mir-30b-3p,which has two binding sites with mir-103a-2-5p and mir-663a,and one binding site with mir-342-3p,mir-188-3p and mir-30b-3p.There are binding sites between hsa circRNA 011333 and mir-1277-5p,mir-3924,mir-5094,mir-4724-5p and mir-186-5p,which has five binding sites with microRNA-1277-5p,microRNA-3924 and microRNA-5094,and four binding sites with microRNA-4724-5p and microRNA-186-5p.3.Randomly selected differentially expressed circRNA for RT-PCR verification.Compared with the prostate hyperplasia specimens,the expression levels of hsa_circRNA₀04561,hsa_circRNA₁02101,hsa_circRNA₁00818 in prostate cancer specimens increased,while the expression levels of hsa_circRNA₁04195,hsa_circRNA₀18168 and hsa_circRNA₀21714 decreased,which were completely consistent with the results of gene chip.4.Bioinformatics analysis showed that hsa circRNA 102485 may be involved in ECM receptor interaction,fatty acid biosynthesis,polyadenine RNA binding,cell nitrogen metabolism,RNA binding,ion binding,biosynthesis and gene expression.Hsa circRNA 011333 may be involved in prion diseases,nucleic acid binding transcription factor activity,DNA template transcription,cell division cycle,RNA metabolism,cell protein metabolism,macromolecule complex assembly,cell death,and pressure response,protein binding transcription factor activity,ion binding,and cell protein regulation.Conclusions:1.Compared with benign prostatic hyperplasia,circRNA was differentially expressed in prostate cancer.2.There are more than one binding site between differentially expressed circRNAs and each corresponding microRNAs,which may be used as a microRNA sponge.The sponge interaction between hsa_circRNA₁02485 and microRNAs-663a,microRNAs-30b-3p,and between hsa circRNA 011333 and microRNAs-186-5p has important research value.3.Hsa circRNA 102485 may be related to ECM receptor interaction,fatty acid biosynthesis and metabolism,and hsa circRNA 011333 may be related to prion diseases,both of which may participate in basic cellular biological behavior.4.Hsa circRNA 011333/miR-186-5p/AKAP12 may be one of the ceRNA networks of prostate cancer.The correlation between hsa circRNA 102485 and miR-663a,miR-30b-3p may contribute to the construction of other prostate cancer ceRNA networks,all of which will provide a scientific basis for revealing the mechanism of progression between circRNA and prostate cancer.5.Hsa_circRNA₁02485 and hsa_circRNA₀11333 may be potential markers for differentiating prostate cancer from benign prostatic hyperplasia and new diagnostic markers for prostate cancer.