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Prostate cancer is a human-specific disease. It is one of the most common malignant tumors in men and women in Europe and America. It has the highest incidence of prostate cancer in the United States, and its mortality rate is second only to lung cancer. Researchers at the University of Toronto in Canada first selected 144 prostate cancer samples with detailed clinical information for whole transcriptome sequencing (RNA-seq), and identified more than 7,000 circRNA molecules, which were revealed by bioinformatics analysis. The expression characteristics of circRNA were closely related to the invasion and metastasis characteristics of prostate cancer. Subsequently, large-scale shRNA library technology was used for knockout verification. It was found that about 11.3% of circRNAs are closely related to the proliferation characteristics of prostate cancer cells, and the linear transcripts of these circRNA-derived genes are not. Affects cell proliferation, indicating a special function of circRNA. Finally, the researchers focused on the significant correlation of circCSNK1G3 in prostate cancer, revealing that circCSNK1G3 can interact with miR-181 to regulate cell proliferation, which is different from the more recent circRNA sponge mechanism, which stabilizes miR-181 in prostate cancer. The discovery of this activity provides a new research idea for circRNA. 
1. Analysis of the expression profile of localized prostate cancer 
To sum up: there are sufficient funds, there are enough samples, using the new molecular circRNA as a molecular marker, combined with the mechanism, the summit CNS is no longer far away.
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Https://sci-hub.tw/10.1016/j.cell.2019.01.025
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On February 7th, Prof. Paul C. Boutros and Professor Houshen Hansen He from the University of Toronto, Canada conducted a complete transcriptome sequencing of 144 prostate cancer samples, combined with subsequent functional mechanisms to reveal new regulatory mechanisms for prostate cancer circRNA! The research was published in the famous academic journal Cell ( IF 31.4 ) under the title " Widespread and Functional RNA Circularization in Localized Prostate Cancer ". Next, let's look at how researchers use high-throughput sequencing technology RNA-seq and large-scale shRNA library technology to reveal the expression and biological function of circRNA in localized prostate cancer.
Article introduction:
research content:
The authors performed transcriptome analysis of 144 prostate cancer specimens by whole transcriptome sequencing (the cloud sequencing organism provides this service ) technology, displaying total RNAs (n=38,359) and high abundance techniques in the form of pictures, and 144 cases of prostate. The transcriptome analysis of the cancer specimens shows the distribution of total RNAs (n=38,359) and high-abundance RNAs (n=18,152) in each sample, and cluster analysis of high-abundance RNA in each sample. To show the difference between the samples. Through fusion gene analysis, a large number of fusion gene transcripts were identified, and more than 10% of the SU2C metastatic transcriptome found recurrence and fusion, revealing that the fusion gene is involved in invasive localized diseases.
2. Localized prostate cancer circular RNA expression profile
The authors identified a total of 76,311 circRNA molecules using CIRCexplorer, including 62 fusion gene-derived circRNA molecules, and validated high-reliability circular RNA molecules by RNase R and expanded sample size. Find the circular RNAs specifically expressed by tissue cells and their expression patterns in different tissue types; analyze the prostate disease by circular RNA of circBase database, and analyze the linear transcripts of parental genes corresponding to circular RNA To investigate the correlation between circRNA and linear RNA.
3. Differential expression of circular RNA is associated with prostate cancer progression <br> The researchers clustered 144 prostate cancers, compared to linear RNA, the strongest pattern of circular RNAs and their presence in each sample. The overall abundance is consistent. In addition, the prognosis of patients with extreme circRNA distribution was significantly worse than that of patients with stable circRNA distribution, and the extreme circRNA index group was also associated with a higher incidence of metastasis. This correlation excludes the effects of linear transcripts from the parental source, and there are indications that this specifically expressed circRNA is closely related to the development of prostate cancer.
To assess the functional importance of these specific circRNAs, the most abundant 2000 circular RNAs in prostate cancer cell lines were selected for shRNA functional deletion screening, targeting sites for trans-cleavage sequences, and then knocking out these circular RNAs. The cell line was subjected to whole transcriptome sequencing , and the linear RNA was depleted at the same time to ensure the effectiveness of the screening. The results showed that these specific circRNAs significantly promoted the proliferation of cells, rather than linear RNA.
4. Functional characteristics of circCSNK1G3 <br> The circular RNA molecule circCSNK1G3 is very specific, and its circular and linear forms have similar expression abundance. It has been verified that the circular RNA form is essential in four prostate cancer cell lines. The corresponding linear transcript is not required. To investigate the mechanism by which circCSNK1G3 promotes cell proliferation, the researchers performed full-transcriptome sequencing (RNA-seq) analysis by knocking out and over-expressing circCSNK1G3 in the PC-3 cell line. GSEA enrichment analysis revealed that many of the genes up-regulated in over-expressed samples were down-regulated in knockout samples and vice versa. A similar pattern was observed for the other two circular RNAs, circSTAU2 and circGOLPH3, indicating that the phenotype observed in knockout and overexpression experiments was not caused by the extra-target effect. At the same time, this phenomenon did not occur after knocking out its linear transcript CSNK1G3. It was finally confirmed that the promotion of circCSNK1G3 on prostate cancer cell proliferation was not related to its corresponding linear transcript.
5. circCSNK1G3 synergistically stabilizes miR-181b/d activity to promote prostate cancer <br> Cyclic RNA usually interacts with miRNA to play a regulatory role. Firstly, 10 candidate miRNAs interacting with circCSNK1G3 were found by prediction, and circCSNK1G3 and miRNA were further analyzed. The correlation between the five miRNAs that were significantly associated with circCSNK1G3 expression was determined to be miR-181a/b/c/d and miR-196b, respectively. By circCSNK1G3 pull down (cloud biological order for this service) was found only miR-181b / d significantly enriched knockout circCSNK1G3 reduce miR- 181b / d abundance, overexpression increased miR- 181b / d abundance. A similar phenomenon was observed for ciRS-7, and knocking out ciRS-7 reduced its interacting miR-7. Overexpression of miR-181b/d in PC-3 cell line significantly reduced CBX7 abundance. CBX7 is a well-characterized miR-181b target and a tumor suppressor of prostate cancer. Consistently down-regulated with miR-181b/d after ctCSNK1G3 knockdown, CBX7 abundance increased significantly. Similarly, in the miR-181b/d overexpressing cell line, cell cycle genes such as CDK1, CDC25A are upregulated and downregulated in the circCSNK1G3 knockout cell line. Consistent with cell cycle gene up-regulation, miR-181b/d overexpression promotes PC-3 cell proliferation. More importantly, overexpression of miR-181b/d significantly attenuated the knockdown-induced cell proliferation arrest of circCSNK1G3. In summary, circCSNK1G3 promotes the proliferation of prostate cancer cells through interaction with miR-181b/d, and this new regulation is different from the sponge mechanism.
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