| FHL2(Four and a half LIM domains protein 2)as a transcriptional regulator,which is a member of LIM-Only protein family,FHL2 participate in many physiological regulatory processes such as gene expression and cell proliferation,differentiation,cell migration and apoptosis.Until now,most of study focused on human and mice.In our study,we analyze the data of transcriptome data of sheep ovary tissues with polytocous and monotocous group,which found that the expression of FHL2 was significantly different between the two groups.It was suggested that FHL2 plays an important role in follicular development of sheep.In order to prove this speculate,in this study,we foucus on gene expression,gene cloning,bioinformatics analysis and localization of FHL2 in ovine ovaries and granulosa cells.To investigate the effects of FHL2 on the growth of granulosa cells,steroid hormone secretion and related gene expression in sheep,we constructed overexpression and siRNA interference models in granulosa.Through construction of yeast two hybrid cDNA library,we screened and verified the interaction signal pathway related of FHL2 in sheep granulosa cells.It aims to reveal the molecular mechanism of which regulated follicular development in sheep.In this study,real-time PCR(RT-PCR)was used to detect the expression level of FHL2 gene in different tissues of sheep.The coding sequence of FHL2 gene was obtained by molecular cloning technology,and the sequence homology and evolution relationship among different species were compared by bioinformatics analysis.Immunohistochemistry was used to detect the expression and distribution of FHL2 protein in ovine ovary.The localization of FHL2 protein in ovarian granulosa cells was detected by cell immunofluorescence.The results showed that the expression level of FHL2 gene in ovary was higher than other tissues,and the CDS of FHL2 gene was 837 bp in ovary,which encoding 279 aa.The cDNA sequence of FHL2 was consistent with GenBank,and without amino acid mutation occurred.The homology of FHL2 amino acid sequence of sheep with human,cattle,pig,horse,chicken,dog and monkey was 86%,98.3%,91.7%,89.3%,76.5%,88.2%and 86.2%,respectively.FHL2 was mainly distributed in the preantral follicle and antral follicle.The results of cell immunofluorescence showed that FHL2 was located in the nucleus and cytoplasm of granulosa cells.pcDNA3.1-FHL2 was constructed by molecular cloning,the interference sequence of siRNA targeted siRNA was designed and synthesized.After transfection,the interference efficiency was explored and overexpression and interference of cell models were established in vitro.The results showed that:(1)the eukaryotic expression vector pcDNA3.1-FHL2 was successfully constructed and the expression of FHL2 was significantly increased at 24 h after transfection into sheep granulosa cells.(2)three siRNAs of FHL2 gene was successfully synthesized,and the optimal transfection time and interference effect were detected by RT-PCR and Western-blot,which indicating that siFHL2-2 could significantly reduce the expression of FHL2 at 72 h after transfection.AlamarBlueTM HS was used to detect cell proliferation,flow cytometry to detect cell apoptosis and cell cycle,ELISA to detect the changes of estrogen and progesterone secretion in granulosa cells.RT-PCR was used to detect the mRNA expression of apoptosis(Bcl-2,Bax,p53 and Caspase3),cell cycle(CyclinD1,CyclinB1 and p21)and hormone secretion(StAR,3?-HSD,CYP11,CYP19)related genes.The results showed that FHL2 overexpression model was used to confirm that 24 h after transfection,compared with the control group,FHL2 overexpression inhibited the ovarian granulosa cells growth(P<0.01);the proportion of normal cells was significantly reduced(P<0.05),the proportion of apoptosis was increased(P<0.05)in late period,the apoptosis gene of Bcl-2 expression levels was significantly down-regulated(P<0.05),the expression level of Bax and Caspase 3 genes significantly up-regulated(P<0.05),and the ratio of the Bcl-2/Bax significantly up-regulated(P<0.05),which indicated that overexpression of FHL2 promote granulosa cell apoptosis.The proportion of G0/G1 and S phase cells decreased(P<0.001),the expression level of Cyclin B1 was significantly down-regulated(P<0.05).The secretion levels of reproductive hormones and related gene expression levels were detected,the secretion levels of E2 and P in granulosa cells significantly decreased(P<0.05),the relative expression levels of CYP19A1 and 3?-HSD genes were down-regulated(P<0.05).Comfirmed by siRNA interference model,72 h after transfection,compared with control group,the interference of FHL2 expression promoted the growth of ovarian granulosa cells(P<0.01).The detection of apoptosis and apoptosis related gene expression level,the proportion of normal cells was significantly increased(P<0.05),the apoptosis rate in the late stage was significantly decreased(P<0.05),the expression level of Caspase3 gene was down-regulated(P<0.05),and the proportion of Bcl-2/Bax was up-regulated(P<0.05),and those of inhibit cell apoptosis.The proportion of cells in S phase and G2/M phase increased(P<0.05),The expression levels of cell cycle factors Cyclin D1 and p21 were up-regulated(P<0.05).The levels of reproductive hormone secretion and related gene expression were detected,the secretion of E2 was significantly increased(P<0.05),and the relative expression of CYP11A1,CYP19A1 and StAR genes were significantly up-regulated(P<0.05).The yeast two-hybrid library of sheep ovarian tissue cells was constructed by yeast two-hybrid technology,and yeast bait plasmid pGBKT7-FHL2 was constructed.The host proteins interacting with FHL2 protein were screened by yeast two-hybrid library.The results showed that the cDNA library of sheep ovarian tissue containing 3×108 recombinants was successfully constructed by yeast two-hybrid technique,and the bait vector pGBKT7-FHL2 was used to detect the non-toxicity and self-activation activity.Using pGBKT7-FHL2 bait plasmid,11 proteins(RPL31,RPS20,RPS12,COMMD1,AP-1,PLCD1,FOXO1,?-catenin,CREB,NF-?B,CYLD)interact with FHL2 were screened in yeast library,and Blast analysis showed that these proteins were mainly involved in protein synthesis,gene transcription,cell proliferation and differentiation,cell apoptosis,and signaling pathway regulation.The expression level of FHL2 in ovarian granulosa cells was studied by adding exogenous growth factor TGF-?1.Using TGF-?1 protein and several important signaling pathway inhibitors PI3K(Deguelin),MEK(Combimetinib),NF-?B(BAY11-7082),JNK(SP600125),MAPK(SB202190)were treated granulosa cells cultured in vitro,to carry out experimental verification of FHL2 signaling pathway.The results showed that exogenous addition of TGF-?1 protein caused the expression level of FHL2 to be up-regulated(P<0.05).On this basis,the above signaling pathway inhibitors were added,respectively.The results showed that the addition of PI3K and MARK signaling pathway inhibitors resulted in the down-regulation of FHL2 expression(P<0.05),which confirmed that PI3K and MAPK were signaling pathways of FHL2 regulating the expression of TGF-?1.In conclusion,the full length of FHL2 gene CDS sequence in sheep is 837 bp,encoding 279 amino acids,which is highly expressed in ovarian tissues and located in follicular granulosa cells.FHL2 affects granulosa cell proliferation,apoptosis,cycle,steroid hormone(E2 and P)secretion and related gene expression.11 proteins were screened by yeast two-hybrid,including RPL31,RPS20,RPS12,COMMD1,AP-1,PLCD1,FOXO1,?-catenin,CREB,NF-?B and CYLD,these proteins were involved in cell proliferation,apoptosis and other signaling pathways through interaction in ovine follicular development.PI3K and MAPK are signaling pathways that TGF-?1 regulates FHL2 expression. |
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