Effect And Mechanism Of LncRNA Meg3 On The Puberty In Female Rat

Objective:This study investigated the expression patterns of maternally expressed gene 3（Meg3）at different developmental stages on the reproductive axis of female rats,and the effects of Meg3 knockdown on the initiation of puberty,the transcription level of puberty-related genes,ovarian morphology and serum gonadal hormone concentration in female rats,which provides scientific theoretical basis for regulating the puberty of female animals from the perspective of reproduction.Methods:Firstly,female rats in the infancy（10 d）,prepubertal（25 d）,pubertal （32.50±1.22 d）,and adult（63.33±1.63 d）life stages were used to collect hypothalamus,pituitary gland,ovary,respectively.RT-q PCR was adopted to analyze the m RNA transcription levels of lncRNA Meg3 gene at different developmental stages.Then the sh RNA sequence was designed,recombinant plasmid was constructed,and 293T cells were transfected to package lentivirus.The hypothalamic cells of female rats were isolated and cultured.On the fifth day of cell culture,gonadotropin-releasing hormone neurons were identified by immunohistochemical staining and followed infected with lentivirus.After infection of 72 h,the expression of Gnrh,Kiss1 m RNA and other puberty-related genes were detected by RT-q PCR and the expression of Wnt,β-catenin protein was evaluated by Western blot.Prepubertal female rats at PND 25 were randomly divided into the following three groups:lentivirus group（lncRNA Meg3-sh RNA1,n=8）,negative control group（empty vector,n=8）,and blank control group（dd H₂O,n=8）.Following intracerebroventricular injection,the time of vaginal opening was recorded.Hypothalamus,pituitary gland,ovary and blood samples were taken at the day of VO or the lncRNA Meg3-sh RNA1 group was matched the same age when the control group animals’vulva was opened.RT-q PCR was used to analyze the transcription level of puberty-related genes.The right ovary was weighed,the size was measured,histological sections were prepared,and histological structure were observed.Serum estradiol（E₂）and progesterone（P₄）were detected by ELISA.Results:（1）The transcription level of lncRNA Meg3 in the hypothalamus of female rats was significantly lower in prepuberty than in infancy（P<0.05）,and no significant difference was found among infancy,puberty,and adulthood（P>0.05）.In the pituitary,the level of lncRNA Meg3 was the highest at the adult stage,which was significantly higher than that at infancy,prepuberty,and puberty（P<0.05）;there was no significant difference in the expression of lncRNA Meg3 among infancy,prepuberty,and puberty（P>0.05）.In the ovary,the lncRNA Meg3 level was significantly lower in infancy,puberty,and adulthood than in prepuberty（P<0.01）and reached the lowest level in adulthood;the lncRNA Meg3 level in infancy and adulthood was significantly lower than that at puberty（P<0.05）.No differences were found between infancy and adulthood（P>0.05）.（2）The results showed that the cells of the hypothalamus were mainly Gn RH neurons.After lentivirus infection of hypothalamic neurons for 72 h,compared with blank control and negative control group,the transcription levels of Gnrh and Kiss1m RNA in lncRNA Meg3-sh RNA1 group were decreased（P<0.05）,but there was no significant difference in the transcription level of Rfrp3 m RNA（P>0.05）.The Wnt（P<0.01）andβ-catenin proteins were increased（P<0.05）.（3）After intracerebroventricular injection in prepubertal female rats,the average day of VO in the lncRNA Meg3-sh RNA1 group was significantly delayed compared with the control groups（P<0.05）.When the vulva was opened in rats,the hypothalamic transcription level of lncRNA Meg3,Gnrh m RNA were decreased（P<0.05）,but the transcription level of Rfrp3 m RNA was increased（P<0.05）,while there was no significant difference in the transcription levels of Kiss1 and Tgfβm RNA（P>0.05）.There was no significant difference in the transcription levels of lncRNA Meg3,Gnrh R,Fshβ,Lhβm RNA in the pituitary gland and lncRNA Meg3,Fsh R,Lh R,Cyp19a1,17β-HSD,Star and Cyp11a1 m RNA in the ovary（P>0.05）.The longitudinal diameter and longitudinal perimeter of the ovary in the lncRNA Meg3-sh RNA1 group were significantly higher than those of in the control groups（P<0.05）,but the transverse diameter and transverse perimeter of the ovary and ovary index were not different（P>0.05）.There was no difference in the number of primary follicles,secondary follicles and corpora lutea（P>0.05）.The E₂ and P₄ levels in lncRNA Meg3-sh RNA1 group were significantly lower than those in the control groups（P<0.05）.（4）After intracerebroventricular injection in prepubertal female rats,when the lncRNA Meg3-sh RNA1 group was matched the same age which the vulva was opened in control animals,the hypothalamic transcription levels of lncRNA Meg3,Gnrh,Kiss1 and Tgfβm RNA were significantly decreased（P<0.05）,but the levels of Rfrp3 m RNA were not significantly different between groups（P>0.05）.The transcription level of Lhβm RNA in the pituitary was higher than in control groups（P<0.05）,but there were no significant differences in lncRNA Meg3,Gnrh R and Fshβm RNA（P>0.05）.In the ovary,the transcription levels of Lh R and 17β-HSD m RNA were significantly decreased（P<0.05）,and there was no significant change in the levels of lncRNA Meg3,Fsh R,Cyp19a1,Star and Cyp11a1 m RNA（P>0.05）.No significant differences were found in transverse diameter,transverse perimeter,longitudinal diameter,longitudinal perimeter and ovary index in lncRNA Meg3-sh RNA1 group（P>0.05）,while the number of corpora lutea in lncRNA Meg3-sh RNA1 group was significantly reduced（P<0.05）.The levels E₂ and P₄ were also significantly decreased（P<0.05）.Conclusion:（1）LncRNA Meg3 is expressed on the reproductive axis of female rats at different developmental stages,and changes with the developmental stages;（2）LncRNA Meg3 gene knockdown affects the hypothalamus–pituitary–ovarian axis puberty-related genes and serum gonadal hormone levels in female rats,and delays the onset of puberty;（3）knockdown of lncRNA Meg3 down-regulated Gnrh and Kiss1m RNA transcription levels in hypothalamic neurons and promotes Wnt andβ-catenin expression,suggesting that lncRNA Meg3 may affect rat puberty through the Wnt/β-catenin pathway.