The Molecular Mechanism Of MiR-133b Regulating Follicle Development

The data from China National Health and Family Planning Commission report that the incidence of infertility is about 7%-10% in 2013. Anovulatory infertility is a common type of female sterility in many pathogenesis of infertility. The morbidity of anovulatory infertility is up to 25%-35% in infertility and mainly results from follicular maturation disorder. The molecular mechanism of follicle maturation disorder is a hot project in the field of human reproduction.Follicular development is an extremely complex process, including the follicular construction and function changes at various phase of ooctye, which is regulated by endocrine regulation, local factors, genes and micro RNA. Though some interesting progress was found in the mechanism controling ovarian follicle development in human, there are many questions to still be involved in the field.In this project, we plan to screen the differential expression mi RNA at various phase of human ooctye under the human ovaries simulative conditions by micro RNA microarray. We want to select the specific high-expression of mi RNA as a research target and identify its potential binding sites to the target gene by a dual luciferase reporter assay. We will comfirm the target gene of the mi RNA by immunofluorescence, Western blot and Realtime-PCR. We prefer to clarify the molecular mechanism in the development and maturation of oocyte regulated by mi RNA and search for the drug target to regulate oocyte maturation and method for prevention and treatment of infertility.Part I The different expression of mi RNAs at the various phase of human ooctyeObjective To screen the different expression of mi RNAs at various phase of human ooctye. To select the specific high-expression of mi RNA targeting at some phase of human ooctye and predict the potential target of the specific high-expression of mi RNAMethods The oocytes at GV and MI phase were collected and pre-treated with IGF-1 to imitate the internal environment of human ovaries.The different expression of mi RNAs in human ooctye at GV and MI phase was screened by microarray and speculated the targets of specific higher-expressing mi RNA by bioinformatical resource.Result The results from mi RNA microarray indicated that 183 mi RNAs were up-regulated and 117 mi RNAs were down-regulated in GV oocytes and 145 mi RNAs were up-regulated and 200 mi RNAs were down-regulated in MI oocytes in the analogous internal environment of human ovaries. Our results showed that the expression of mi R-133 b was 32 fold in IGF-1 treated in MI oocytes than without IGF-1 treated oocytes. However thare was no change in mi R-133 b expression at GV oocytes with or without pre-treated by IGF-1. The data from bioinformatics suggested that TAGLN2 might be a potential target of mi R-133 b binding at No.215 to 250 nucleotide sequences of TAGLN2 3’ UTR.Summary 183 mi RNAs were up-regulated and 117 mi RNAs were down-regulated at GV oocytes. 145 mi RNAs were up-regulated and 200 mi RNAs were down-regulated at MI oocytes in the analogous internal environment of human ovaries. A specific high-expression of mi RNA at MI oocytes is mi R-133 b targeting at TAGLN2 3’ UTR.Part II Interaction between mi R-133 b and TAGLN2Objective To definite that TAGLN2 is a tegart of mi R-133 b by analyzing the interaction between TAGLN2 and mi R-133 b.Methods TAGLN2-3’UTR and TAGLN2-3’UTR mutant expression vector was constructed by gene recombination(result in gene named as psi CHECK-TAGLN2-3’UTR and psi CHECK-TAGLN2-3’UTR-m, respectively). mi R-133 b with psi CHECK or psi CHECK-TAGLN2-3’UTR or psi CHECK-TAGLN2-3’UTR-m repectively co-transfected into 293 T cells. The binding reaction between TAGLN2 3’ UTR and mi R-133 b was analyzed by Dual-Luciferase report gene system. The effect of mi R-133 b on TAGLN2 expresion was tested by Realtime-PCR and Western blot. Expression and subcellular localization of TAGLN2 were detected in MI phase oocytes treated with or without IGF-1 by immunofluorescence.Results Both expression vectors of psi CHECK-TAGLN2-3’UTR and psi CHECK-TAGLN2-3’UTR mutant were successfully constructed. The expression of luciferase report gene was significant decreased in 293 T cells cotransfected by mi R-133 b and psi CHECK-TAGLN2-3’UTR and was no change in 293 T cells cotransfected by mi R-133 b and psi CHECK or psi CHECK-TAGLN2-3’UTR-m. The binding site of mi R-133 b to TAGLN2 was the sequence from 215 to 250 nucleotide of TAGLN2-3’UTR. mi R-133 b mimic down-regulated TAGLN2 expression, but mi R-133 b inhibitor up-regulated TAGLN2 expression. TAGLN2 was located in the cytoplasm, but lilttle in neuleus of the oocyte at MI phase.Summary The tegart gene of mi R-133 b is TAGLN2 locateing in the MI oocyte cytoplasm. Mi R-133 b binds to the sequences from 215 to 250 nucleotide of TAGLN2-3’UTR and down-regulates the expression of both TAGLN2 m RNA and protein.Part III The mechanism of the oocyte maturation regulated by mi R-133 b targeting TAGLN2Objective To investigate the regulation of mi R-133b/TAGLN2 signal pathway on folicular development.Methods First, The expression level of TAGLN2 in ovaries on 4 weeks oldand 8 weeks old female ICR mice was detected by Western blot. Second, we randomly divided the 4 weeks old mouse into 4 groups, each group included six mice. The expression level of TAGLN2 in preantral follicle, antral follicle, preovulatory follicle and corpus luteum were measured by immunohistochemistry. Third, we randomly divided the 8 weeks old mouse into 3 groups, each group included 10 mice. The expression level of TAGLN2 in GV, GVBD, MII oocyte were measured by immunofluorescence. Fourth, we randomly divided the 8 weeks old mouse into 3 groups, each group included 8 mice. The oocyte diameter and zona pellucida thickness were measured after injecting TAGLN2 si RNA into ovary. Fifth, we randomly divided the 8 weeks old mouse into 5 groups, each group included 8 mice. Count the number of eggs from each mouse.The expression of TAGLN2 on ovary was analyzed by Western blot and Realtime-PCR after injecting mi R-133 b minic, mi R-133 b minic negative control, mi R-133 b inhibitor, mi R-133 b inhibitor negative control and solvent control into ovary. Sixth, we divided all the GV oocyte into 5 group, observe the mature rate of oocyte after injecting mi R-133 b minic, mi R-133 b minic negative control, mi R-133 b inhibitor, mi R-133 b inhibitor negative control and solvent control into eggs by microinjection. Analyze the effect of mi R-133 b on oocyte maturation.Result The results from western blot showed that TAGLN2 was expressed both in the ovaries of 4 or 8-week old mice and that the expression level of TAGLN2 was dramatically higher in the ovaries of 8-week old mice than those of 4-week old ones(p=0.032<0.05). The results from immunohistochemistry indicated that TAGLN2 was presented in preantral follicles, antral follicles, preovulatory follicles, luteal, and that TAGLN2 was mainly located in granulosa cell cytoplasm and envelope, but little in nucleus. Accompanied with the development of follicles, the expression level of TAGLN2 in granulosa cells was gradually reduced, but dramatically increased in the luteal phase(p=0.006<0.01). The results from immunofluorescence staining showed that TAGLN2 expressed in the mice oocyte cytoplasm and envelope. The expression level of TAGLN2 in GVBD was lower than GV oocytes(p=0.00<0.01) and lowest in MII oocytes(p=0.02<0.05). TAGLN2-si RNA significantly increased the oocyte diameter(p=0.01<0.05), but had no effect on the diameter of zona pellucida-free oocyte(ZP-free OD)(p>0.05). Mi R-133 b mimic increased the number of oocyte at both GV and MI phase(p>0.05) and mi R-133 b inhibitor significantly decreased the number of oocyte at both GV and MI phase(p<0.05) after ovary injected with mi R-133 b mimic、mi R-133 b mimic NC、mi R-133 b inhibitor and mi R-133 b inhibitor NC. The results from ovary microinjection data suggested that mi R-133 b mimic induced the maturation of oocyte(p<0.05) and that mi R-133 b inhibitor lowered the maturation of oocyte(p<0.05). Mi R-133 b mimic down-regulated(p<0.05), but mi R-133 b inhibitor up-regulated TAGLN2 expression.Summary TAGLN2 expression is negatively related to the oocyte maturation and located at the membrane and cytoplasm of granulosa cell. Mi R-133 b inhibites the TAGLN2 expression and promotes the oocyte maturation in mouse.