Screening And Identification Of Differential Circular RNA In Plasma Of Patients With Prostate Cancer

IntroductionProstate cancer（PCa）is the most common urinary system tumor in men and is the second most common malignant tumor in the male population worldwide.With the development of our society,the proportion of male malignant tumors in China is also increasing year by year.At present,the early clinical screening for prostate cancer mainly uses prostate specific antigen（PSA）combined with digital rectal examination.However,it is well known that the PSA examination has a high false positive rate and the digital rectal examination is too subjective.Therefore,a highly sensitive and highly accurate screening method is urgently needed in the clinic.Non-coding RNA（ncRNA）has been used as a diagnostic marker for the early diagnosis of a variety of tumors.Currently,research on non-coding RNA and prostate cancer is also in the process of development,The relationship between microRNA（miRNA）,lncRNA（long non-coding RNA,LncRNA）and prostate cancer has gradually been explored,but the relationship between circular RNA（circRNA）and prostate cancer has rarely been reported.Part Ⅰ Plasma differential circular RNA screening in patients with prostate cancerObjective:（1）High-throughput sequencing technology was used to obtain the expression profile of circulating RNA in patients with prostate cancer and benign prostatic hyperplasia.（2）Screening for differentially expressed circular RNA in prostate cancer plasma.（3）Initially determine the circular RNA of patients with prostate cancer and patients with benign prostatic hyperplasia.Methods:Four cases of clinically diagnosed prostate cancer and 4 cases of clinically diagnosed benign prostatic hyperplasia were collected.Plasma samples were collected and high-throughput sequencing of plasma samples was performed.The plasma of patients with benign prostatic hyperplasia and benign prostatic hyperplasia were screened according to the sequencing results.A circular RNA that is differentially expressed.The real-time Quantitative PCR Detecting System（qRT-PCR）technique was used to verify the differentially expressed circular RNAs in the preliminary screening to determine the circular RNA with significant difference.RESULTS:（1）A total of 7131 differentially expressed circular RNAs were identified in the plasma of prostate cancer,of which 3268 were up-regulated and 3863 were down-regulated.（2）Fold change>3,P<0.001 was set in the differentially expressed circular RNA,and 9 significantly up-regulated circular RNAs and 3 significantly down-regulated circular RNAs were initially selected.（3）Screening of small samples and screening for significant differences in expression of cyclic RNA showed that the expression of hsa_circ₀001296 in plasma of prostate cancer patients was significantly higher than that of the control group,and the expression of hsa_circ₀003258 in plasma of prostate cancer patients was significantly lower than that of the control group.Conclusion:（1）There is a lot of differentially expressed circular RNAs in the plasma of patients with prostate cancer and benign prostatic hyperplasia,and some of them have significant expression differences.（2）Small-scale verification of differentially expressed circular RNA showed that the expression of hsa_circ₀001296 in plasma of prostate cancer patients was significantly higher than that of the control group,and the expression of hsa_circ₃258 in plasma of prostate cancer patients was significantly lower than that of the control group.Part Ⅱ Expression verification and functional prediction of hsa_circ₀001296Objective:（1）To improve the stability of detection of circulating RNA in plasma of patients with prostate cancer.（2）Expand the experimental sample and verify the first part of the results by qRT-PCR detection of hsa_circ₀001296 in plasma of patients with prostate cancer and benign prostatic hyperplasia.（3）To explore the mechanism of hsa_circ₀001296 regulating the occurrence and development of prostate cancer.Methods:（1）qRT-PCR was used to verify the expression of hsa_circ₀001296 in prostate cancer cell lines,and the ring structure was verified by actinomycin assay.（2）Collection of 25 cases of clinical diagnosis of prostate cancer and 24 cases of clinical diagnosis of benign prostatic hyperplasia.Plasma samples were collected,and NADPH was used as an internal reference to increase the sample size to verify the significant difference in the expression of circular RNA（hsa_circ₀001296,up-regulated）by qRT-PCR.（3）Comprehensive analysis of bioinformatics methods to predict the biological function of hsa_circ₀001296.Results:（1）consistent with the chip,the overall expression level of hsa_circ₁296 in plasma of patients with prostate cancer was significantly higher than that of patients with benign prostatic hyperplasia.（2）hsa_circ₀001296 is expressed in prostate cancer cell lines pc3,C4-2,22RV1,LNCAP,DU145 and has a cyclic stable structure.（3）hsa_circ₀001296 may regulate the proliferation of prostate cancer by HMGB1 regulated by sponge adsorption of miR-582-5p.Conclusion:（1）hsa_circ₀001296 is significantly up-regulated in plasma of patients with prostate cancer,and it has certain diagnostic value for early diagnosis of prostate cancer.（2）Detection of circular RNA in human plasma is feasible and has obvious advantages for early diagnosis of prostate cancer.（4）hsa_circ₀001296 may regulate the proliferation of prostate cancer by HMGB1 regulated by sponge adsorption of miR-582-5p.