The Temporal And Spatial Expression Pattern Of Ubiquitin-conjugating Enzyme 2C(UBE2C) In Zebrafish Embryo Development

Objective:Detected the expression microarray through a preliminary study of lnc RNA and m RNA of liver at different points of time of fetal and adult mice,finding that multiple coding genes and noncoding genes were located in the core areas of coexpression network,which the highest coexpression relationship index is UBE2 C molecular,a member of ubiquitin conjugating enzyme E2 family.The chip results showed that it had the highest expression in liver of fetal mice of 14.5 days,which the fluorescence value was 31643,while adult mice liver expression value was only 152,a difference of 200 times.Is the phenomenon of UBE2 C expression highly in the liver of fetal mice suggests that it plays an important regulatory role in the process of embryonic development?Is it related with the growth of tissues and organs? We have great interest in these problems.In eukaryotic cells,Ubiquitin-proteasome system(UPS) involves in regulating life process such as cell cycle,cell apoptosis,gene transcription, chromatin stability,DNA damage repair,antigen presentation and so on through mediating the ubiquitination and degradation in target protein of cytoplasm and nucleus.The ubiquitin molecule binds to substrate proteins covalently under the catalysis of Ubiquitin-activating enzyme(E1), Ubiquitin- conjugating enzyme(E2),Ubiquitin-protein ligase(E3),making the target protein identified by 26 S proteasome and degraded finally.As a member of ubiquitination conjugation enzyme family which regulates mitosis,UBE2 C is necessary in cell cycle progression and degradation of cyclins which can discern anaphase-promoting complex(APC/C),then transfers the activated ubiquitin to substrate protein,making substrate polyubiquitylation,helping substrate hydrolysis by the proteasome.During mitotic metaphase, APC/CCDC20(a member of)mediates degradation of securin and cyclin B,through which the UBE2 C can promote mitotic anaphase.Before late phase of M,chromosomal protein complex- cohesion binds sister chromatid closely together,while separase can crack cysteine of cohesion resulting in separating of sister chromatid, at the same time the activity of separase can be depressed by securing.In consequence,the degradation of securin and activating of separase mediated by UBE2C-APC/CCDC20 are essential in starting cells entering anaphase of mitosis.While the degradation of cyclin B mediated by UBE2C-APC/CCDC20 is the first step in termination of M phase.In the late M phase,cyclin dependent kinases-1(CDK1)reacts into complex with cyclin B,at this time the Cdk1 is active,but with the degradation of cyclin B,activity of Cdk1 decreases,causing Cdh1 dephosphorylation. Cdh1 combines with APC instead of CDC20.Then APC/CCdh1 forms,managing the ubiquitination of regulatory factors after M phase ends.In recent years,UBE2 C arouses general interests in the field of oncology research.In vivo and vitro experiments,overexpressed UBE2 C can stimulate cell proliferation and anchorage independent growth.UBE2 C expresses so little that it could not be detected in normal tissue,however,it expresses aberrantly high in carcinoma tissues such as nasopharyngeal cancer,breast cancer,lung cancer,liver cancer,prostate cancer and so on and relates to prognosis.It is reported that UBE2 C transgenic mice appear precocious degradation of cyclin B,extra centrioles,chromosome lagging and aneuploidy phenomenons,thus extensive spontaneous tumors emerge.But UBE2 C promoting mechanism expressing in carcinoma is not clear yet.With the development of develop molecular biology,molecular biology and tumor immunology,more and more researches indicate that growth of early embryonic tissues and organs is obviously overlapped with tumorigenesis in gene, protein and signaling pathway.Therefore researching embryonic development is necessary to find out the mechanism in tumor developing. However,we can hardly find the related report in whether UBE2 C participates in regulating embryonic development and how to regulate embryonic development in each tissue.In this article, we are willing to use the mode creature-zebrafish to detect UBE2 C expression in embryos at different times with the help of Real-Time PCR and whole embryo in situ hybridization,in order to further study the function and offer new thoughts of tumor research.Methods:Choosing Zebrafish embryos at 2 hours post fertilization(hpf), 4hpf,8hpf,10 hpf,12hpf,24 hpf,48hpf,72 hpf and 7days post fertilization(dpf) to extract RNA,after the transcription reversed,tested the expression in different times of UBE2 C using Real-Time PCR;marked the UBE2 C gene RNA probe by the composed digoxigenin,fixed the embryos at different stages for in situ hybridization.Observed the temporal and spatial expression pattern of UBE2 C in zebrafish during embryonic development.Results: From the Real-Time PCR results,we could see that UBE2 C expressed early in the 0hpf(1cell),reaching the highest in the 4hpf(the blastocyst stage),after the 48hpf(the incubation period),its expressing decreased to a lower level.In situ hybridization showed that UBE2 C expressed early in the 0hpf and concentrated at the blastoderm,from the cleavage stage(0.75-2.25hpf) until the 12 hpf,UBE2C distributed extensively;from 24 hpf, the expression signal showed some tissue specificity, after 48 hpf, the expression signal mainly concentrated in some organs such as statocyst,liver,senital system and so on.Conclusions: The expression of zebrafish ube2 c had obvious maternal expression pattern,suggesting that it regulated embryonic development possiblly through participation in ubiquitination and degradation of maternal protein.Its specific expression in the otic vesicle,liver and reproductive system also gave research direction for the function of UBE2 C in embryonic development, and provided new ideas for using in tumor research.