Study On The Regulation Of CircRNA-006258/miR-574-5p/EVI5L On Mammary Epithelial Cells Proliferation And Milk Synthesis In Dairy Goats

The function of lactation is an important economic trait in the industry of dairy goat.The mammary gland is a main organ with economic value for the dairy goat.They are the simultaneous processes of the development of the breast and lactation and the grouth of MECs.And its activity and number are closely related.More and more literatures proved that non-coding RNA are widely involved in breast development and physiological activities of lactation,including: miRNA regulate the 3 ‘untranslated region of m RNA,and circRNA affect miRNA by regulation of the "sponge" effect and so on.Basing on pre-sequencing of our laboratory revealed that miR-574-5p was differentially expressed during peak lactation and lactation in dairy goats,and the molecular mechanism of miR-574-5p's regulatory effect in GMECs is unclear.In this experiment,the epithelial cells of Guanzhong dairy goat were used as the research object.Based on the sequencing and software prediction crossover,we got the database of miR-574-5p target gene and circRNA,these helped us to selecte the circRNA and m RNA for this study.In this test,epithelial cell culture system was established in vitro,and the du-luciferase reporter system was used to detect the targeting relationship between miR-574-5p and EVI5 L,circRNA-006258.Using RT-q PCR,Western blot,CCK-8 Ed U,apoptosis,etc.detected the regulatory effects of circRNA-006258/miR-574-5p/EVI5 L on the proliferation and milk synthesis of GMECs.The main research results of this experiment are as follows:1.EVI5 L was a target gene of miR-574-5pEVI5L dual-luciferase wild-type and mutant reporter vectors were constructed,and double-luciferase activity was detected.EVI5 L wild-type vector significantly down-regulated dual-luciferase activity,while the mutant vector has no obvious changes,and it is concluded that EVI5 L is a target gene of miR-574-5p;it is known from RT-q PCR and WB tests that miR-574-5p mimic can reduce the expression of EVI5 L at m RNA and protein levels,And miR-574-5p inhibitor significantly increased the expression of EVI5 L at both m RNA and protein levels.It was concluded that EVI5 L is a target gene of miR-574-5p and is inhibited by miR-574-5p.2.Circ RNA-006258 relieves EVI5 L inhibition by miR-574-5p through spong-adsorptionThe constructed circRNA-006258 wild type and mutant CHECK2 vector and miR-574-5p mimics,inhibitor and NC,inhibitor NC were transferred to GMECs respectively,and the double luciferase activity was obtained.The double luciferase of the wild type vector was obtained.The activity was significantly reduced,but the mutant vector had no significant change.By synthesizing si-circRNA-006258,si-circRNA-006258 and NC were transfected into GMECs,respectively,by RT-q PCR,WB test detected the m RNA and protein expression of EVI5L after treatment,and found that the m RNA and protein levels of EVI5L in the si-circRNA-006258 group were significantly lower than those in the NC group.It was concluded that circRNA-006258 released the inhibitory effect of miR-574-5p on EVI5L through sponge adsorption.3.Circ RNA-006258/miR-574-5p/EVI5 L axis regulates mammary epithelial cell proliferation and milk synthesis signal pathwayConstruct an overexpression vector of EVI5 L,synthesize the interfering RNA of EVI5 L and circRNA-006258,and transfect them into GMECs,and explore the signals of circRNA-006258/miR-574-5p/EVI5 L axis to regulate mammary epithelial cell proliferation and milk synthesis by WB test path.EVI5 L is known to be a Rab23 homologous GTPase activating protein.The results of this test show that the circRNA-006258/miR-574-5p/EVI5 L axis regulated the cell cycle of GMECs through Rab23/ITGB1/TIAM1/Rac1-TGF-?/Smad pathway promoted S phase;it promoted the protein expression of Bcl2 and reduce the protein expression of Bax,thereby promoting the proliferation of GMECs,increasing its cell viability and inhibiting apoptosis;activating the PI3K/AKT-m TOR signaling pathway to promote the production of triglycerides and ?-casein in GMECs was also investigated.In summary,this experiment initially verified that circRNA-006258 acted as a molecular sponge for miR-574-5p to adsorb it,thereby releasing the inhibitory effect of miR-574-5p on EVI5 L.Circ RNA-006258/ miR-574-5p/EVI5 L axis was constructed to comprehensively elaborate the molecular mechanism that affects proliferation and milk synthesis of dairy goat mammary epithelial cells,providing a scientific basis for lactation and precision breeding of dairy goats.