Mechanism Of CircMTHFD2L On The Development And Progression Of Canine Mammary Tumors

Canine mammary gland tumor is a common disease of canine tumor diseases,accounting for about half of tumor diseases,and malignant canine breast tumors account for about 50%.currently,the mechanism of occurrence and development of canine breast tumors is still unclear and cannot provide scientific guidance for clinical diagnose and treatment.In order to study the role of circ MTHFD2 L in canine mammary tumors on the development of canine mammary tumors,this study explored the existence of micro RNA(mi RNA)sponge mechanism of circ MTHFD2 L in canine breast tumors.The laboratory-preserved original canine breast tumor cell(CHMP)was used as an experimental material,and the RNA binding protein immunoprecipitation(RIP)experiment was used to combine the mi RNAs and circ RNAs bound to the Ago2 protein(argonaute 2,Ago2).RNA Pull down and found that circ MTHFD2 L binds to the Ago2 protein and is significantly different from the negative probe,with the basis of Ago2-mediated mi RNA sponge.In order to verify and detect the mi RNAs adsorbed by circ MTHFD2 L,RNA pull down experiment was used to precipitate mi RNAs and proteins bound with circ MTHFD2 L,and a total of 101 differentially expressed mi RNAs and 44 differentially expressed proteins were pulled down.At the same time,the circ MTHFD2 L sequence was used to predict the mi RNAs bound to it from the bioinformatics website(Target Scan Human7.2),and then combined with the 101 differentially expressed mi RNAs detected from the RNA pull down,a total of 2 mi RNAs were screened.The mi RNAs involved in the development of breast tumors are cfa-mi R-34 a and cfa-mi R-449 a,respectively.In order to further verify that circ MTHFD2 L acts as a sponge to adsorb cfa-mi R-34 a and cfa-mi R-449 a,a dual luciferase reporter gene experiment was designed.The results showed that the binding rate of betweencfa-mi R-34 a,cfa-mi R-449 a and circ MTHFD2 L is not high,indicating that circ MTHFD2 L not be a sponge adsorbing cfa-mi R-34 a or cfa-mi R-449 a.On the other hand,the previous validation results showed that circ MTHFD2 L was significantly expressed in canine mammary tumor tissues,suggesting that it may play a tumor suppressor role in breast tumors,and according to biological information,circ MTHFD2 L may be involved in the mechanism of apoptosis.Circ MTHFD2 L was overexpressed in CHMP,and the expression levels of three apoptosis-related genes of Bcl-2,Bax and p53 were detected by real-time PCR.The results showed that overexpression of circ MTHFD2 L had no significant effect on apoptosis,and there was no significant change in the expression of apoptotic factors,indicating that the effect of circ MTHFD2 L on cell apoptosis was not significant,it may be function in other ways.In summary,this study explored the role of circ MTHFD2 L in mi RNA sponge mechanism and the effect of circ MTHFD2 L overexpression on apoptosis,for the follow-up study,circ MTHFD2 L provided data support for the development of canine mammary tumors.