The Presence And Characteristic Analysis Of Testis Derived Circular RNA In Human Seminal Plasma

Part Ⅰ Circ RNA deep sequencing and identifying in human testicular tissue Background and Objective: As major executors of epigenetic regulation,non-coding RNAs(nc RNAs) are important elements in transcription and post-transcription regulation. Circular RNAs(circ RNAs), the newly discovered class of nc RNAs, has become the star molecule in transcription study. Gene expression and its epigenetic regulation play vital roles in spermatogenesis, and the abnormality of which may result in infertility and illness of offspring. In this part, we did some preliminary work in exploring this new class of nc RNAs in human testis. And we hope to lay the foundation for further studies of their potential roles in spermatogenesis. Methods: Total RNA was isolated from human testis tissue using TRIzol. After ribosomal RNAs depletion and linear RNA digestion by RNase R,c DNA library were generated for deep sequencing on Illumina Hi Seq 3000 platform in PE150 sequencing mode. Then we randomly choose 50 circ RNAs of different expression levels and lengths for q RT-PCR validation and alternative circularization analysis. NCBI/PrimerBLAST website was used to design the divergent primers for q RT-PCR according to the initiation site and their circular form. QRT-PCR was performed to analyze the expression of circ RNAs and m RNAs before and after RNase R digestion. Results: In human testis tissue, 15996 circ RNAs(read counts ≥ 1) were identified. Among them 10792 were not included by circ Base before, therefore are first reported. And we found that 94.6% of their host genes transcribe 6 or less circ RNAs. In these host genes, 1017 were discovered to form circ RNAs for the first time, and they are found to be closely related to spermatogenesis, sperm structure and functions in Gene Ontology analysis.Further, we observed that most circ RNAs(70.6%) are derived from protein coding exonic regions.Among the 55 randomly chosen circ RNAs, 22 were already included in circ Base and 33 were first predicted. Thirty out of the 55 can be specifically amplified, with 19 in circ Base and 11 first discovered. As in the 30 successfully validated circ RNAs, 11 were generally expressed and 16 were specifically expressed in human testis. Nine(3 in circ Base and 6 firstly discovered) of the 55 circ RNAs can be detected but were accompanied with non-specific amplification.We chose three genes(STK31 、 RNF17 and SAE1) to design primers for their corresponding genes. By which we successfully validated their alternative circularization characteristic. After RNase R treatment, m RNAs of 9 host genes degraded significantly while their circular counterparts remained intact. On the contrary, the expression of circ RNAs from several genes(HIST1H2BA、WDR26 and TBL1XR1) was up-regulated to 4 times fold, revealing the high RNase R resistence property of circ RNAs. Conclusions: The present study predicted and identified the presence of circ RNAs in human testis tissue. More than tens of thousands of new circ RNAs and more than thousands of circ RNA generating host genes were found by us.These testis derived circ RNAs are possessed with alternative circularization and RNase R endurance property. And these circ RNAs may serve as novel epigenetic research hotspot for human spermatogenesis.Part Ⅱ The expression and characterization of human testis-derived circ RNAs in seminal plasma Background and Objective: There are abundant free m RNAs and mi RNAs in seminal plasma. With their high stability, easy accessibility and comprehensive representation of different reproductive organs(testis, epididymis, seminal vehicles and prostate glands), they may serve as novel non-invasive biomarkers for male reproductive diseases. Also, they are of significant value in reproductive epigenetic research. In this research we take the circ RNAs that were validated in the first part as target, analyze their expression and characterization in seminal plasma, and thus explore their potentials in serving as novel biomarkers and research target for reproductive diseases. Methods: Semen samples of males with normal seminal parameters were collected. Seminal plasma was isolated and RNAs were extracted. QRT-PCR was performed to analyze the expression of circ RNAs which were validated in the first part. Seminal plasma was placed in room temperature for 0h, 12 h and 24 h. The expression of each time point was examined to explore the stability of circ RNAs in seminal plasma. And filter membrane with 0.1um pore and 30 k Da ultrafiltration spin were used to analyze the circ RNA expression of untreated seminal plasma, filtrate and eluate from the filter membrane, filtrate and eluate from the ultrafiltration spin(after filtration by filter membrane). And by which we may get to know the possible existence form of circ RNAs in seminal plasma. Results: 20 testis-derived circ RNAs which are validated in the first part can be detected in seminal plasma, no matter they are generally or testis specifically expressed. There was no difference in each time point(0h,12 h, 24h) when circ RNAs were housed in room temperature, indicating the high stability of testis-derived circ RNAs in seminal plasma. In the 0.1um pore filter membrane and 30 k Da ultrafiltration spin experiment, circ RNAs mainly exist in the filtrate from the filter. And after we subject the filtrate to ultrafiltration, most circ RNAs were retained in the concentration tube. These findings suggest that seminal free circ RNAs are present in the seminal plasma in form of protein complex. And the two-step filtration method may result in the concentration and enrichment of the circ RNA-protein complex. Conclusions: Testis-derived circ RNAs, including those testis specially expressed circ RNAs, can be stably detected in seminal plasma. The stability of them may come from their protein-RNA complex form, which result in escape from RNase degradation. Seminal plasma circ RNAs may serve as novel biomarkers in male productive physiology and pathology research.