| Bovine mastitis is one of the most common diseases in the dairy industry.The disease is caused by the invasion and reproduction of pathogenic microorganisms in the breast.Mastitis can cause pathological changes in breast tissue.Circular RNA is a kind of molecule with the function of regulating the expression of gene.It is produced by a new type of alternative splicing.It can serve as a "sponge" of mi RNA.Circ RNA contains a large number of mi RNA response elements(MRE),which can serve as competing endogenous RNAs(ce RNA),combining mi RNA,inhibiting the function of m RNA,and regulating the expression of downstream genes.Based on the relevant characteristics of circular RNA,a new circular RNA(named circ0001186)was found by a high-throughput sequencing,which was significantly down-regulated in a heat-inactivated S.aureus-induced Mammary alveolar cells(MAC-T)model.Through the bioinformatic analysis,circ-0001186 affected the proliferation of MACT.Circ-0001186-overexpressing MAC-T cell line was built as the model to study the effects of the circ RNA on the biological function of MAC-T cells and the effects of the circ RNA serving as a mi RNA sponge were studied by RT-q PCR,cell counting kit(CCK-8),5-ethynyl-2’-deoxyuridine kit(EDU)and luciferase reporter assay.The experimental results were as follows:(1)Throughout the high-throughput sequencing in the previous study,the circ-0001186 sequence was obtained.Circ-0001186 is 874 bp in length and is formed by the cyclization of the second exon of ZNF609.RT-q PCR was used to detected the expression of circ-0001186 in breast tissue,heart,liver,spleen,lung,kidney,stomach and intestine.The results showd that the expression was lower in breast tissue than in other tissues.Circ-0001186-overexpressing MAC-T cell line was successfully constructed.RNase R enzyme was used to digest circ-0001186-overexpressing MAC-T cell,and the result showed that linear m RNA ZNF609 was significantly decreased,which proving the loop characteristics of circ-0001186.CCK-8 test was used to verify the viability of MAC-T cells after transfected with circ-0001186,the results showed that compared with the control group,the viability of MAC-T cell was significantly enhance in the circ-0001186-overexpressing group.Also,EDU test was used to verify the ability of proliferation of MAC-T,the result showed that overexpression of circ-0001186 promoted the proliferation of MAC-T cells.According to the results,circ-0001186 enhanced the viability and promote the proliferation of MAC-T cells.(2)Through the bioinformatic analysis,we found that mi R-3432 a may bind to circ-0001186,primers with wild-type and mutant-type were designed to amplify the sequence and combined with psi CHECK2 vector,co-transfect the plasmid with mi R-3432 a mimics and mimic NC into 293-T cells,and verified the combine of circ-0001186 and mi R-3432 a using the dual luciferase report assay.The results showed that the relative fluorescence intensity of the WT and mi RNA mimic group was lower than the other groups,which indicating that circ-0001186 combined with mi R-3432 a.Based on the above research results,circ-0001186 may affect the viability and proliferative capacity of MAC-T cells by combining with mi R-3432 a.CCK-8 test was used to verify the viability of circ-0001186-overexpressing MAC-T cells after transfect with mi R-3432 a mimic,mimic NC,mi R-3432 a inhibitor and inhibitor NC,the results showed that compared with the control group,cell viability was alleviated in the mi R-3432 a mimic group.Also,the results of EDU test showed that the proliferation of MAC-T cells was reduced in mi R-3432 a mimic group.Also,through the bioinformatic analysis,we found that m RNA Jun D may bind to mi R-3432 a.Primers with wild-type and mutant-type were designed respectively,and the dual luciferase report experiment was used to verify the binding.The results showed that relative fluorescence intensity of the WT and mi RNA mimic group was lower than the other groups,which indicating that Jun D can bind to mi R-3432 a.According to the results,circ-0001186 may regulate m RNA Jun D by combining with mi R-3432 a,and mi R-3432 a attenuated changes in cell function caused by overexpression of circ-0001186.This study investigated the biological function of circ-0001186 by constructing the circ-0001186-overexpressing MAC-T cell.The result showed that overexpression of circ-0001186 affected the proliferation capacity and cell viability of MAC-T cells.In addition,circ-0001186 can act as a "sponge" of mi R-3432 a,competitively binding to mi R-3432 a,and upregulating the expression of target m RNA Jun D.It also relief the enhancement of viability and proliferation in MAC-T caused by overexpression of circ-0001186,which reduced the viability of MAC-T cells and inhibited cell proliferation. |
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