Research Of Mov10 In The Regulation Of Mammary Epithelial Cell Proliferation In Dairy Goats Via MiR-21-5p/Smad7

The proliferative capacity of Goat mammary cells and the hormone levels in dairy goats are related to lactation levels in dairy goats.Previous studies found that Mov10(Moloney leukaemia virus 10)significantly promoted mammary epithelial cell proliferation.As an important RNA helicases,Mov10 is one of the important component proteins of the RNAinduced silencing complex(RISC)and an important ago cofactor for micro RNA mediated translational regulation.Mov10 protein was reported play important roles in inhibiting reverse transcriptase activity and 5′-3′ RNA helicase activity,also resolving RNA secondary structures through the RNA induced silencing complex ago,finally influence miRNAs expression and cell′s viability.Mov10 could regulate m RNA degradation and miRNA variable splicing.Previous study found that miRNA-21 plays an important role in the proliferation process of the Goat mammary epithelium.MiR-21-5p is an important member of the miRNA-21 family that has been shown to regulate proliferation in organs and tissues including the ovary,uterus,breast,heart,spleen,liver and thymus.At present,research of miR-21-5p is mainly focused on its role in the carcinogenesis and tumor proliferation,but less attention on its roles in regulating the development and lactation mechanism of the mammary gland.A previous study found that Mov10 significantly promoted the proliferation of breast cells,while a clear change in miR-21-5p with some association was found during this process,but the specific molecular mechanism was not clear.In this study,Goat mammary epithelial cells(GMECs)were used to systematically explore the role of Mov10 in regulating the production of miR-21-5p and to elucidate the mechanism of Mov10 regulates the proliferation of Goat mammary epithelial cells through miR-21-5p.The research results are as follows:(1)Mov10 affects epithelial proliferation and apoptosis of Goat mammary epithelial cellsIn previous study,cell bioactivity was changed accompany by the expression of Mov10 in Goat mammary epithelial cells.To explore the regulatory mechanism of this change,overexpression plasmid of Mov10 were cloned and constructed,and si-RNA were synthesized.Over-expression and knockdown efficiency plasmids and si-Mov10 were transfected into Goat mammary epithelial cells: the CCK-8 experiment results showed that the activity of Goat mammary epithelial cells significantly increased after overexpression of Mov10(P < 0.01).However,the results of q RT-PCR assay showed that the expression of proliferation marker genes CCNB1(P < 0.05)and CDC25C(P < 0.01)were up-regulated,which illustrated that Mov10 overexpression promoted cell proliferation.Results from both CCK-8 experiments and q RT-PCR experiments revealed that overexpression of Mov10 promotes proliferative activity in Goat mammary epithelial cells.Cell apoptosis was assessed using flow cytometry after knockdown of Mov10 in goat mammary epithelial cells,which showed that knockdown of Mov10 promoted apoptosis(P < 0.01).However,the expression levels of P53(P < 0.01),BAX(P < 0.05),and the BCL2/BAX ratio(P < 0.01),a marker of apoptosis,were determined by q RT-PCR,which showed that knockdown of Mov10 promoted the progression of apoptosis.Results from both flow cytometry and q RT-PCR experiments revealed that knockdown of Mov10 promoted apoptosis in Goat mammary epithelial cells.(2)Mov10 regulates miR-21-5p maturationThe study found that after overexpression of Mov10,the expression of Pre-mir-21(P <0.05)and miR-21-5p(P < 0.01)in cells was significantly decreased,The expression of Pre-mir-21(P < 0.01)and miR-21-5p(P < 0.01)in cells was significantly increased after knockdown of Mov10.The results of RNA binding protein immunoprecipitation assay(RNA Binding Protein Immunoprecipitation,RIP)experiments further demonstrated that Mov10 was an important factor in regulating miR-21-5p maturation,and Mov10 interacts with Pre-mir-21 and miR-21-5p.(3)MiR-21-5p targeted Smad7/TGF-β1 signal pathway regulates the proliferation of Goat mammary epithelial cellsIn this study,mimics and inhibitors of miR-21-5p were synthesized by chemical synthesis and transfected into Goat mammary epithelial cells,we used bioinformatics prediction software Target Scan and miRbase website screening to predict the target genes of miR-21-5p,and the screening found that the 3′UTR region of Smad7 contained the targeted binding site of miR-21-5p.We verified that miR-21-5p negatively regulates Smad7 gene m RNA expression by q RTPCR experiment(P < 0.05),and constructed wild-type(WT)and mutant(MUT)luciferase reporter vectors containing the Smad7 3′UTR region,and we verified,using a dual luciferase reporter assay,that miR-21-5p could decrease the luciferase activity of the wild-type vector containing Smad7(P < 0.05),whereas miR-21-5p had no significant effect on the luciferase activity of the mutant vector containing Smad7(P > 0.05).Above studies proved that Smad7 was a target gene of miR-21-5p,and miR-21-5p negatively regulated the expression of Smad7.In summary,in this study,we successfully cloned PLV-m Cherry-2A-Mov10 vector,demonstrated that Mov10 was involved in regulating the proliferation and apoptosis of GMECs,and demonstrated that Mov10 negatively regulates the expression of Pre-mir-21 and miR-21-5p.MiR-21-5p is able to target the Smad7 gene,and through Smad7/TGF-β1 signaling pathway regulates proliferation and apoptosis of Goat mammary epithelial cells.This study provides research materials for revealing the molecular mechanism of Mov10 participating in regulating the proliferation and apoptosis of Goat mammary epithelial cells.