Research On The Function Of Microrna-29 Associated With Puberty Onset In Mice

Background: Micro RNAs(miRNAs)are a group of highly conserved endogenous non-coding small RNAs that are about 21-25 nucleotides.They can regulate biology process by target-binding and cleaving target m RNA or inhibiting m RNA translation.It is reported that all human miRNAs account for about 3% of the total number of genes,and may regulate about 30% of protein-coding genes in human cells.It shows that,the expression of miR-29b during mouse puberty onset changes significantly in the Gene microarray data in mouse brain tissue before our experiment.In addition,the expression of miR-29b was also detected in the hypothalamus tissues of mice with different growth cycle,and also appears significant differences,it shows that miR-29b expression may play an important role in the mammalian nervous system and regulate the growth and development of the individual.So miR-29b is inferred to a micro RNA that associated with mouse puberty onset.Objective: Constructed stably transfected miR-29b cells in mice cells GT1-7 sources hypothalamus and detected the effect of overexpression of miR-29b on target genes and puberty onset genes;Transfected micro RNA-29 mimics and explored the interaction between two micro RNAs from the same family;Constructed miR-29b overexpression mouse model by hypothalamus in situ injection and explored the relationship between miR-29b and sexual development by observing the differences of mice sexual development related phenotypes.Methods: Constructed miR-29a and miR-29b lentivirus expression vectors,packagedinto virus particles and transfected into GT1-7 cells.14 days later,screened stably transfected cells after working with bleomycin,and tested the expression of miR-29 with fluorescent quantitation PCR,to verify the stably transfected cells can be used as the miR-29 overexpression model.Transfected miR-29a mimic,miR-29b mimic into GT1-7 cells respectively and also into GT1-7cells,tested the interactions by fluorescent quantitation PCR,to study the miR-29 biological functions,through the sexual development related gene expression analysis.In the animal experiment,Injecting miR-29b lentiviral into C57/B6 female mice born 12 days,by the hypothalamus positioning injection.Observed and noted the sexual development phenotypes of miR-29b hypothalamus overexpressed mice,including weight gain,vaginal opening time,cage time,number of offspring and offspring mortality,to determine whether the miR-29 to participate in the control mice sexual development start or not.Results: The expression of miR-29b was significantly increased about 28 times in the GT1-7 cell model,and the expression levels of the three target genes were decreased in different degrees,DCX expression decreased by about 44%,Vdac1 expression decreased by about 16%,Pten expression decreased by about 17%.But LH-?,kiss-1,Inshulin,IGF-I,GPR54,Gn RH and leptin-R were not changed.Explored the interactions of two different micro RNA from the same family,after transfected miR-29a mimic,the expression of miR-29a increased about 91 times,but miR-29b has no significant changes,meanwhile,detected the other genes related to sexual development,only decreased 9.5%.Similarly,after transfected miR-29b mimics,the expression of miR-29b increased about 105 times,but miR-29a has no significant changes,meanwhile,detected the other genes related to sexual development,only Leptin-R decreased 10.2%.This experiment shows that,though miR-29a,miR-29b come from the same family,have the same sequences,the expression between both have no effects to each other.In animal experiments,the results showed that the expression level of miR-29b in the hypothalamus region of the injection site was significantly higher than that in the control group,indicating that the mouse model of miR-29b overexpression in hypothalamus was successfully constructed.In the detection of sex-related genes,kiss-1,Inshulin,IGF-I,GPR54,Gn RH,LH-? did not change in the three groups,the expression of Leptin-R was significantly decreased in lentivirus group.Compared with thecontrol group,the average weight of the mice injected with lentivirus was decreased about 2g,and the body weight was slow;vaginal opening time was 35.38 ± 0.9161(days),postponed for 2days;the time from birth to death was 25.33 ± 0.8165(day),delayed for 2 days;the number of born mice was 6.167 ± 0.167(only)than the control group will reduce one;the death rate of lentiviruses was 1.500 ± 0.224(only),and the number of dead mice per mouse was 1.5,compared with the control group.Conclusion: In the cell experiment,we successfully constructed the overexpressed miR-29b stably transfected cells with lentivirus,and tested three target genes decreased in different degrees in it but except Leptin-R the other genes related to sexual development have no significant changes.The miR-29 mimics transfection results showed that miR-29a and miR-29b did not interact with each other;In animal experiments,the mouse model of miR-29b overexpression in the hypothalamus was successfully constructed by injection of the lentivirus into the hypothalamic region of the mouse.The expression of miR-29b was increased about 4times.The results showed that kiss-1,Inshulin,IGF-I,GPR54 and Gn RH did not change in normal saline,empty vector FUGW and lentivirus.The expression of Leptin-R was significantly decreased in lentivirus group.Compared with the control mice,the model mice shows a series of phenotypic differences,such as: weight gain,vulva open,close cage time,number of offspring born and death.The results showed that the overexpression of miR-29b had an effect on the puberty development of mice,which provided a theoretical basis for further study of puberty development in mice.