Avian leukosis virus subgroup J (ALV-J) belongs to the family of retroviruses, a family member of the subfamily of lentiviruses, which can cause a variety of neoplastic diseases such as myeloid cell tumors, sarcomas, hemangiomas, renal tumors and erythroblastia. Symptoms, as well as avian myeloid leukemia, cause a high rate of death in infected chickens. The dispersed ALV-J strain is widely distributed, both horizontally and vertically, and often causes huge economic losses in the field of poultry farming. At present, our mechanism of tumorigenesis induced by ALV-J remains unclear, and the damage caused by eradication of ALV-J remains a major challenge.
Recently, the research team of Xie Qingmei from Huanan Agricultural University of Yunan, using the techniques of circular RNA sequencing , RIP and RNA pull down provided by Yunxu Biotechnology , revealed for the first time that the poultry chicken infected with retrovirus ALV-J was significantly up-regulated by circ. -Vav3 upregulates the expression of the target gene YAP1 by adsorption of gga-miR-375 by sponge, thereby promoting the occurrence of liver tumors. The study was published in RNA Biology ( IF: 5.216 ) under the heading " Circular RNA Vav3 sponges gga-miR-375 to promote epithelial-mesenchymal transition " . It is worth noting that the research team has previously published an article on the expression profile of circular RNA expression in ALV-J virus-resistant and virus-susceptible chicken liver tissues using the circular RNA sequencing service provided by Cloud Sequence Biotechnology ( Circular RNA alterations are involved in resistance to avian leucosis virus subgroup-J-induced tumor formation in chickens ), and 32 differentially expressed circular molecules were found, of which 12 were significantly increased in ALV-J resistant samples. Twenty of the expressions were higher in susceptible samples, and biosignal predictions indicated that these circular RNA molecules can regulate the signaling of immune pathways and thus change the immunity of organisms. This time, in-depth cooperation with our company, using RIP and RNA pull down technology to clarify its regulation through the adsorption of sponge to promote the formation of liver tumors. It has truly achieved a perfect progression from the study of circular RNA expression profiles to functional mechanisms, providing a good reference for the study of other species or disease types.
Previous work by the team revealed that gga-miR-375 was significantly down-regulated in chicken liver tumors infected with avian leukosis virus J subgroup (ALV-J). Combined with current research hotspots, gga-miR-375 may be associated with circRNA. In the present study, circular RNA sequencing results showed that circ-Vav3 was significantly up-regulated in chicken liver tumors infected with ALV-J. Further, it was confirmed by RIP , biotin RNA pull down and RNA-FISH experiments that circ-Vav3 is a sponge of gga-miR-375. In addition, the researchers confirmed that YAP1 is a target gene of gga-miR-375 by dual luciferase reporter gene assay, and further verified that circ-Vav3 releases the target gene YAP1 of gga-miR-375 through sponge adsorption, and then through circ- The Vav3/gga-miR-375/YAP1 axis affects the epithelial-mesenchymal transition (EMT) process to promote tumorigenesis and has been validated in clinical samples.
research content:
1. CircRNA expression profile and its binding site prediction
The researchers used ALV-J-infected chicken liver as a sequencing sample and detected differential expression of 2822 circRNAs by circular RNA sequencing (provided by Cloud Sequence Bios), of which 2808 circRNAs were significantly up-regulated. The cluster analysis, volcano map and scatter plot were used to analyze the expression of 25 circRNAs with up-regulated expression. It was found that circ-Vav3 was up-regulated most significantly. In addition, previous research by the team revealed that gga-miR-375 was most significantly down-regulated in the same sample. Next, TargetScan and miRanda software were used to predict the miRNAs that may be bound by 25 circRNAs. The results showed that circ-Vav3 and gga-miR-375 had the highest score.
2.circ-Vav3 sponge adsorption gga-miR-375
The nomenclature of circ-Vav3 is mainly based on the exon 8-11 of the VAV3 gene derived from the circular RNA; the predicted results show that there is a perfect targeting site between circ-Vav3 and gga-miR-375. Using the sponge mechanism to indicate the protein for AGO2 RIP experiments (provided by cloud-sequence organisms) , qPCR detection of captured AGO2 protein revealed significant enrichment of circ-Vav3 and gga-miR-375; and RNA pull down using circ-Vav3 probe The experiment (provided by Cloud Sequence Bio) detected the binding of gga-miR-375. RNA-FISH experiments confirmed that circ-Vav3 mainly exists in the cytoplasm, while gga-miR-375 is present in the cytoplasm and in the nucleus, revealing that circ-Vav3 and gga-miR-375 play a regulatory role through mutual binding.
3.gga-miR-373 negatively regulates YAP1 expression by binding to the 3'UTR region of YAP1, and overexpression of gga-miR-375 attenuates EMT
The biomarker revealed that YAP1 is a potential target gene of gga-miR-375, and further dual luciferase reporter gene confirmed that gga-miR-375 can inhibit the expression of YAP1 by binding to the 3'UTR region of YAP1; in vitro cell line DF Overexpression of gga-miR-375 in -1 significantly down-regulated the mRNA and protein levels of YAP1, revealing that gga-miR-375 inhibits the expression of YAP1 gene by directly binding to the 3'UTR region of YAP1. The EMT phenomenon is a precursor to tumor metastasis, which is accompanied by up-regulation of key genes (N-cadherin, Fibronectin, MMP2, ZEB1) and down-regulation (E-cadherin). Overexpression of gga-miR-375 revealed significant up-regulation of mRNA and protein levels of EMT-positive genes, down-regulation of mRNA and protein levels of negatively related genes, and demonstrated that EMT-related genes are not direct targets of gga-miR-375 . Therefore, gga-miR-375 inhibits the progression of EMT indirectly by inhibiting the expression of the YAP1 gene.
4.YAP1 overexpression promotes EMT and enhances cell migration ability
The researchers further demonstrated that overexpression of YAP1 significantly enhanced cell migration ability by Transwell experiments, and qPCR and WB also confirmed that the expression of EMT-related key genes was significantly changed in YAP1 overexpressing DF-1 cell line. This indicates that YAP1 can promote the occurrence of EMT and cell migration ability. 
5.circ-Vav3 sponge adsorbs gga-miR-375 up-regulates target gene YAP1 to promote EMT
After the accumulation of preliminary work, the researchers believe that circ-Vav3 adsorbs gga-miR-375 in the cytosol, thereby up-regulating the expression of YAP1 and EMT-related genes. Co-expression experiments showed that EMT positively related genes overexpressing gga-miR-375 and LCDH-circ-Vav3 (experimental group) relative to overexpressing gga-miR-375 and LCDH-circ-EV (empty group) The protein and mRNA levels of Fibronectin, N-cadherin, MMP2, ZEB1 and Vimentin were significantly up-regulated, while the protein and mRNA levels of the negatively related gene (E-cadherin) were significantly down-regulated; it was further confirmed that circ-Vav3 adsorbs gga-miR through sponge action. -375, upregulates the expression of its target gene YAP1, which in turn induces the occurrence of EMT.
6. Validation of ALV-J infected cell lines and clinical samples
After infection of DF-1 cell line by ALV-J virus strain, the expression of circ-Vav3 was significantly up-regulated and the expression of gga-miR-375 was down-regulated in cells by WB and qPCR. Meanwhile, YAP1 and EMT positive correlation genes were detected. Protein and mRNA levels were also significantly up-regulated, whereas protein and mRNA levels of EMT negative-related genes were significantly down-regulated. Liver samples from chickens infected with ALV-J obtained from chicken farms were subjected to the same test, and the same results were obtained, further consolidating the conclusions of the whole experiment.
to sum up:
"One stop in the end"! Cloud-sequence biological circular RNA research from spectrum ( cyclic RNA sequencing , whole transcriptome sequencing , circular RNA methylation sequencing ) to mechanismal research ( RNA pull down, RIP ), for your full coverage of differential circular RNA Molecular screening is an important research link in the verification of downstream molecular mechanisms. In addition, the circular RNA database circDB, which is established and maintained by Cloud Sequence , supports many customers to publish circular RNA results by virtue of wide species coverage, large data volume and many disease backgrounds. In the past, Yunxu Bio and experts, scholars and health care workers in the field of life sciences have progressed together to explore the mysteries of the unknown. Looking back on the past, from epigenetics to apparent transcriptomics, from genome to proteome, from intracellular to exosomes, cloud-sequence organisms rely on high-throughput sequencing technology for more and more clinical and basic biological research. Provide high quality research services. With our joint efforts, Cloud Preface customers publish more and more high-scoring articles.
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