Expression Profiles And Preliminary Applied Research Of Circular RNAs In Prostate Cancer

Part Ⅰ Expression Profiles of Circular RNAs in Prostate Cancer Cell Lines and TissuesObjective:To obtain expression profiles of circular RNAs（circRNAs）in prostate cancer-related cell lines and tissues using RNA-Seq.To screen circRNAs with differential expressions in those cell lines and tissues.To perform functional prediction and analysis of those circRNAs.Methods:Three prostate cancer-related cell lines（normal prostatic epithelial cell line RWPE-1,prostate cancer primary tumor cell line 22RV1,prostate cancer bone metastasis cell line PC-3）and 65 pairs of prostate cancer and paracancerous tissues were selected for RNA-Seq and bioinformatics analysis.Results:A total of 9,545circRNAs were identified in the three prostate cancer-related cell lines,of which 845 were expressed in all three cell lines,and a large number of circRNAs with differential expressions were also found.GO functional analysis and KEGG pathway analysis suggested that the host genes of those circRNAs may have important biological functions and play key roles in many important molecular signaling pathways.By algorithms of DCC,find_circ,and circRNA_finder,a total of 277,122 circRNAs were identified in 65pairs of prostate cancer and paracancerous tissues,of which 82,797 were identified by all the three algorithms.Unsupervised clustering and PCA analysis revealed significant differences in circRNAs expression profiles between prostate cancer and paracancerous tissues.9 circRNAs were significantly up-regulated,while 86 circRNAs were significantly down-regulated in prostate cancer tissues.The functional predictions of the 95 circRNAs with differential expressions suggested multiple binding sites for the corresponding miRNAs.Conclusion:circRNAs are widely expressed in prostate cancer-related cell lines and tissues,and there are a large number of circRNAs with differential expressions.Bioinformatics analyses showed that those circRNAs in prostate cancer may exert biological function by competitively adsorbing miRNAs,or participate in progression of prostate cancer together with their host genes,and may have potential diagnostic values.Part Ⅱ Validation and Functional Research of Circular RNAs with Differential Expressions in Prostate CancerObjective:To verify circRNAs sequencing results in prostate cancer-related cell lines and tissues.To analyze the significance of different expressions of circular RNA-circFUT8in prostate cancer-related cell lines.To explore the role and its significance of circular RNA-circAMACR in the diagnosis and progression of prostate cancer.Methods:The circRNAs with differential expressions were selected and verified by qRT-PCR in normal prostatic epithelial cell line and prostate cancer cell lines,prostate cancer and paracancerous tissues.circFUT8（low abundance expression in normal prostatic epithelial cell line RWPE-1,with progressively higher expression levels in prostate cancer primary tumor cell line 22RV1 and prostate cancer bone metastasis cell line PC-3）was selected as the study object,and the difference in expression levels between the prostate cancer cell lines and normal prostatic epithelial cell line was verified by qRT-PCR.Considering the function of its host gene,the role of circFUT8 in metastasis and androgen-independent transformation of prostate cancer was analyzed.circAMACR(with the most significant up-regulated expression in prostate cancer tissues,fold change=6.19,P=1×10^-13)was selected as the study subject,and the different expression levels between prostate cancer tissues and paracancerous tissues,prostate cancer cell lines and normal prostatic epithelial cell line were verified by qRT-PCR.siRNA was used to interfere with the expression of circAMACR in prostate cancer cells and the effect of circAMACR on the biological behavior of tumor cells was observed to explore its role and significance in the diagnosis and progression of prostate cancer.Results:The qRT-PCR verification results of circRNAs with differential expressions in prostate cancer-related cell lines and tissues were basically consistent with the results of RNA-Seq.The expression of circFUT8 in prostate cancer cell lines was significantly higher than that of normal prostatic epithelial cell line.The expression of circFUT8 in prostate cancer metastasis cell lines（PC-3,DU145）was significantly higher than that of prostate cancer primary tumor cell line（22RV1）.The expression of circFUT8 in androgen-independent prostate cancer cell lines（PC-3,DU145）was also significantly higher than that of androgen-dependent prostate cancer cell line（LNCap）.The expression of circAMACR in prostate cancer tissues was significantly higher than that in paracancerous tissues.The expression of circAMACR in prostate cancer cell lines was also significantly higher than that in normal prostatic epithelial cell line.circAMACR is mainly expressed in cytoplasm.Interfering with the expression of circAMACR in prostate cancer cells inhibited the proliferation and migration of tumor cells.Conclusion:The expression profiles of circRNAs in prostate cancer-related cell lines and tissues obtained by RNA-Seq are reliable.circFUT8 has a unique differential expression pattern in prostate cancer-related cell lines and may be involved in the metastasis processes and androgen-independent transformation of prostate cancer.circAMACR is tumor-specific expressed in prostate cancer and has certain tumor biological effects during the progression of prostate cancer.Part Ⅲ Preliminary Study on the Application of Plasmatic Circular RNAs in Detection of Prostate CancerObjective:To improve the stability of circRNAs detection in the plasma of prostate cancer patients.To determine the optimal number and combination strategy of internal control genes for plasma qRT-PCR detection in patients with prostate cancer and benign prostatic hyperplasia.To explore the role and its significance of plasmatic circRNAs in detection of prostate cancer.Methods:The method of extracting total RNA from plasma samples was improved,and the qRT-PCR reaction conditions for detecting plasmatic circRNAs were optimized.qRT-PCR detection of 8 candidate internal control genes includingβ-Actin,GAPDH,18s rRNA,β₂M,GUSB,HPRT1,PPIA,and SDHA was performed in the plasma samples of each 10 patients with prostate cancer or benign prostatic hyperplasia,respectively.NormFinder and geNorm software were used to analyze experimental data.qRT-PCR detection of 29 circRNAs with differential expressions（9up-regulated and 20 down-regulated）was performed in the plasma samples of 45 patients with prostate cancer and 30 patients with benign prostatic hyperplasia.Results:Two internal control genes were optimal for plasma qRT-PCR detection in patients with prostate cancer or benign prostatic hyperplasia.The optimal internal control gene combinations were GAPDH and PPIA.When qRT-PCR was performed in plasma,there were no amplification curves for 9 circRNAs,and there were no target bands for 14 circRNAs during electrophoresis.There were 3 circRNAs with obvious interference bands during electrophoresis,and only 3 circRNAs（circAPLF,circLPAR1,and circSLC45A4）were stably expressed in the plasma of patients with prostate cancer or benign prostatic hyperplasia.The overall expression levels of circAPLF and circSLC45A4 in the plasma of prostate cancer patients were significantly lower than those in benign prostatic hyperplasia.There was no significant difference in the expression levels of plasmatic circLPAR1between patients with prostate cancer or benign prostatic hyperplasia.Conclusion:Detection of plasmatic circRNAs in prostate cancer patients is feasible.However,it should be optimized according to the specific experimental system.The internal control gene combination of GAPDH and PPIA can be used as a standardized factor for the qRT-PCR detection of plasma samples in patients with prostate cancer or benign prostatic hyperplasia.circAPLF and circSLC45A4 were stably expressed in plasma of patients with prostate cancer,and the overall expression levels were significantly lower than those in benign prostatic hyperplasia,which may have certain application value in the diagnosis of prostate cancer.