Expression Profiles Of MRNA/lncRNA And Their Related Functions In The Ovaries Of Polycystic Ovary Syndrome Rat Model

Background: Polycystic ovary syndrome(PCOS)is one of the most common heterogeneous reproductive and metabolic diseases,affecting approximately 5-10% of women in reproductive age worldwide.The clinical manifestations of the syndrome are highly variable,including androgen excess,irregular menses,and polycystic ovarian morphology.People with PCOS often indure infertility,obese and accompany with hirsutism,insulin resistance(IR),and dyslipidemia.In addition,they have an increased risk of endometrial cancer,type 2 diabetes(T2DM),and cardiovascular disease.However,the exact pathophysiology of PCOS remains unclear.At present,the main purpose of PCOS is to adjust the menstrual cycle,promote ovulation,improve living habits,and prevent long-term complications with conservative treatment.Therefore,establishing an ideal PCOS animal model is particularly important for the study of PCOS.There are many methods for the existing PCOS animal models,including DHEA,E2,letrozole and so on.It was found that the use of letrozole 200?g/d to construct PCOS rat model can simultaneously show ovarian morphology,endocrine and metabolic conditions similar to human PCOS,and therefore is suitable for the study of the clinical mechanism of PCOS.With advances in gene sequencing technology,it has been found that a large part of the genome is transcribed as long non-coding RNAs(lncRNA),which are RNA molecules with more than 200 nucleotides in transcripts that do not have protein-coding capabilities.Lnc RNA has become key regulators in many biological processes,such as genomic imprinting regulation,DNA metabolism,X chromosome inactivation,transcriptional activation,and chromatin modification.Specific lncRNAs are involved in a variety of human diseases such as cancer and cardiovascular,neurological and reproductive system diseases.The study found that the expression of lncRNA in peripheral blood,cumulus cells and granulosa cells of PCOS and normal patients was different,but the specific mechanism was unclear.Therefore,more research will help us to improve our understanding of lncRNAs and may help discover new PCOS diagnostic markers and therapeutic agents.Objective: To establish a PCOS animal model with letrozole,obtain the mRNA/lncRNA expression profile in the ovaries of PCOS animal models through deep sequencing,verify the deep sequencing results by RT-PCR,and perform bioinformatics analysis.Specific mRNA/lncRNA was selected for expression analysis and the effect of certain lncRNA on the function of rat ovarian granulosa cells was investigated to explore possible pathogenic mechanisms of lncRNA for PCOS.Method: 1.Establishment of PCOS rat model with letrozole(1)Letrozole was administered to the PCOS model continuously for 23 days;(2)The estrus cycle was measured by vaginal smear,and the sex hormones in the peripheral blood were measured by chemiluminescence and the morphology of the ovaries was observed by HE staining.2.The study of mRNA expression profile of ovary in PCOS rat model(1)mRNA expression profiles in ovaries of PCOS animal model was obtained by deep sequencing;(2)Bioinformatics analysis of differentially expressed mRNA;(3)Verification of the accuracy of deep sequencing by RT-PCR.3.Expression and analysis of Plet1 in rat reproductive system and PCOS model(1)RT-PCR was used to detect the expression of Plet1 in the reproductive system of normal rats in different oestrous cycle;(2)RT-PCR and Western blot were used to detect the expression of Plet1 in reproductive system of normal rats;(3)Immunohistochemical method was used to detect the expression and location of Plet1 in normal rat reproductive system;(4)RT-PCR,Western blot was used to detect the expression of Plet1 in the ovary and uterus of PCOS model.4.The study of lncRNA expression profile in ovaries of PCOS animal model(1)The lncRNA expression profile of ovaries of PCOS rat model was obtained by deep sequencing;(2)Bioinformatics analysis of differentially expressed lncRNAs;(3)Verification of the accuracy of deep sequencing by RT-PCR.5.Expression and function analysis of lncRNA CD36-005 in PCOS model(1)The expression of lncRNA CD36-005 and CD36 in the ovaries of PCOS model was detected by RT-PCR;(2)RT-PCR was used to detect the expression of lncRNA CD36-005 in the ovaries and uterus during different estrous cycles in normal rats;(3)Construction of recombinant adenovirus carrying lncRNA CD36-005 of rat;(4)Primary cultured rat ovarian granulosa cells and FSHR immunofluorescence identification of rat ovary granulosa cells;(5)RT-PCR detection of the expression of lncRNA CD36-005 and CD36;(6)The effect of lncRNA CD36-005 on cell viability detected by CCK8;(7)Influence of lncRNA CD36-005 on cell cycle was detected by flow cytometry;(8)The effect of lncRNA CD36-005 on apoptosis was detected by flow cytometry.Result: 1.Establishment of PCOS rat model with letrozole The PCOS rat model was successfully constructed and characterized as: The body weight of PCOS rats significantly exceeded the normal group,lost normal estrus cycle,appeared sex hormone disorder,and ovarian polycystic morphology.2.The study of mRNA expression profile of the ovaries in PCOS rat model(1)There were differences in mRNA expression levels between the PCOS model and the normal control group determined the difference in pathological conditions of PCOS;(2)Bioinformatic analysis showed these differential mRNAs may be involved in multiple biological processes such as sex hormone metabolism,lipid metabolism,cell fusion and embryo adhesion,and are closely related to the pathogenesis of PCOS;(3)The results of RT-PCR validation of differentially expressed mRNAs were in consistent with deep sequencing results,suggesting that the deep sequencing results have high reliability and accuracy.3.Expression of Plet1 in rat reproductive system and PCOS rat model(1)Plet1 is expressed in the ovary and uterus of rats and is mainly expressed in ovarian granulosa cells,luteal cells,and endometrial stromal cells;(2)The expression of Plet1 in the uterus and ovary of normal rats was not related to the estrus cycle,and the expression of Plet1 in the uterus was significantly higher than that in the ovary;(3)The expression of Plet1 in ovary and uterus of PCOS rat model was lower than that of normal control rats;(4)Plet1 may participate in the pathogenesis of PCOS by affecting follicular development and endometrial receptivity.It is necessary to further explore whether Plet1 can be used as a marker of adverse pregnancy outcome in PCOS.4.The study of lncRNA expression profile in ovarian tissue of PCOS rat model(1)PCOS rat and the control groups differed in the expression level of lncRNA,and determined the difference in PCOS pathology;(2)Bioinformatic analysis showed these differences lncRNA may participate in many biological processes such as reproductive metabolism,insulin metabolism,cell fusion,and is closely related to the pathogenesis of PCOS;(3)The RT-PCR validation results of differentially expressed lncRNAs were in agreement with the deep sequencing results;(4)Analysis of ce RNA network showed the lncRNA-mi RNA-mRNA expression network may be involved in the regulation of PCOS pathogenesis.5.The expression and its potential functions of lncRNA CD36-005 in PCOS(1)The expression of lncRNA CD36-005 in ovary and uterus of normal rats was highest in the estrus stage;(2)The expression of lncRNA CD36-005 was higher in the ovary than in the uterus;(3)Lnc RNA CD36-005 has a positive co-expression relationship with CD36,and lncRNA CD36-005 may be involved in the regulation of CD36 expression;(4)Lnc RNA CD36-005 can reduce the viability of ovarian granulosa cells and decrease the cell cycle S phase,which may be involved in the pathogenesis of PCOS by inhibiting cell proliferation.Conclusion: 1.Letrozole successfully constructed a PCOS rat model that can be used for PCOS study;2.There were differences in mRNA/lncRNA expression profiles between PCOS rat ovaries and normal rats;3.Bioinformatic analysis showed these differential mRNA/lncRNA may be involved in multiple biological processes such as sex hormone metabolism,insulin metabolism,cell fusion and embryo adhesion,and are closely related to the pathogenesis of PCOS;4.RT-PCR validation results are in consistent with the deep sequencing results,suggesting the deep sequencing results have high reliability and accuracy;5.The ce RNA network analysis showed lncRNA-mi RNA-mRNA expression network may be involved in the regulation of PCOS pathogenesis;6.Plet1 is mainly expressed in ovarian granulosa cells,luteal cells,and endometrial stromal cells;The expression of Plet1 is different in the ovary and uterus of PCOS rat model;Plet1 may participate in the pathogenesis of PCOS by affecting follicular development and endometrial receptivity.Whether Plet1 can be used as a marker of adverse pregnancy outcome in PCOS needs further investigation;7.The lncRNA CD36-005 and CD36 were highly expressed in the ovary of the PCOS model and had a positive co-expression relationship.The lncRNA CD36-005 may be involved in the regulation of CD36 expression;8.Lnc RNA CD36-005 reduced the viability of ovarian granulosa cells and decreased the cell cycle S phase,which may be involved in the pathogenesis of PCOS by inhibiting cell proliferation.