Screening And Functional Mechanism Of Labor Onset Related LncRNAs On Mouse Myometrium

The uterus is the organ of female animals to give birth to fetus.The muscular tissue of uterus is mainly composed of uterine smooth muscle cells(MSMc),uterine endometrial cells(MEEc)and uterine stromal cells(MESc).Normally,the uterus is in a static state to maintain the stable environment needed for fetal growth.Meanwhile,the uterus grows to accommodate the fetus,amniotic fluid,and placenta.The normal labor onset in late pregnancy is a complex,multi-process,and multifactorial process which includes maternal-fetal interaction and immune response.The core of labor is uterine contraction,and the process of labor from start to success is also a process of gradually strengthening the ability of uterine contractions.Abnormal labor can be divided into preterm labor and delayed labor.There are many similarities between the molecular mechanism of preterm labor and term labor,including P4 functional reduction,inflammatory cytokine release,and the expression of contraction-related genes.The delayed labor is the result of various factors that causes contraction-related genes failing to express normally.It is significant to identify the specific mechanism of labor onset for the precise regulation of labor time in animal production,and the diagnosis of pregnancy status in human,even the prevention and treatment of pregnancy-related diseases.Lnc RNA is a kind of non-coding RNA.As an important regulatory element,it can regulate various bio-processes including cell differentiation,immune response and tumor migration.At present,the research of lnc RNA focuses on embryo implantation and uterine diseases.There are few functional reports between late pregnancy and labor onset on lnc RNAs.At first,differently expressed lnc RNAs from uteruses of mice in the periods of late pregnancy and labor onset were screened.And then,the function of these lnc RNAs were determined whether they influence labor onset or not.The study will provide data and theoretical support to the regulation of labor in animal production.It also will be benefit to identification of pregnancy stage,prevention and treatment of pregnancy diseases.1.Mouse uterine labor onset lnc RNA screeningPregnancy mice model was constructed.Differently expressed m RNAs and lncRNAs for sequencing were obtained from mouse uteruses on the 15.5th and the18.5th day of pregnancy.The sequencing results showed that 1654 m RNAs and 141 lnc RNAs were differently expressed in mouse uteruses on the 15.5th and the 18.5th day of pregnancy.The up-regulate lnc RNA-Gm44275 of the 18.5th day pregnancy,were selected through quantitative verification as candidate lnc RNAs for subsequent studies.The primary mouse uterine myocytes were isolated and identified,of which with extremely high purity were obtained.2.Effect of lnc RNA-Gm44275 on uterine function in mouseAlternative lnc RNAs by q RT-PCR validation.Under physiological condition,the concentration of P4 in mouse on the 18.5th day of pregnancy was lower than that of the 15.5th day.The expression level of lnc RNA-Gm44275 on the 18.5th day of pregnancy was higher than that of the 15.5th day.In vitro,the expression level of lnc RNA-Gm44275 in MSMc was higher than that in MESc and MEEc.But the expression level of lnc RNA-Gm44275 was significantly down-regulated in the mouse uterine myocytes treated with P4(P<0.05).Therefore,this lnc RNA was selected to study the effect on uterine function.The lnc RNA-Gm44275 interference sequence and over-expressed vector were constructed,and transfected into mouse uterine smooth muscle,to explore the effect of lnc RNA-Gm44275 on the function of labor onset.The expression of related genes and proteins was detected by q RT-PCR and WB.The q RT-PCR results showed that the expression levels of OXTR,IL6,PBP2 and EMP1 were significantly decreased after interfered lnc RNA-Gm44275(P<0.05).While only the expression level of IL6 was significantly increased after over-expressed lnc RNA-Gm44275(P<0.05).Protein expression showed that the expression level of OXTR was significantly decreased after interfered lnc RNA-Gm44275(P<0.05).The expression levels of IL6 and STAT3 were significantly increased after over-expression of lnc RNA-Gm44275(P<0.05).To further explore the effect of lnc RNA-Gm44275 on uterine function in vivo,ADVlnc RNA-Gm44275 and ADVNC adenovirus vectors were constructed and injected into the uterus of mouse on the 15.5th day of pregnancy(n=4/4).Parturition started at 33 h to 42 h after ADVlnc RNA-Gm44275 injected(n=4).While the ADVNC group did not deliver at 42 h after injected(n=3)and 48 h after injected(n=1).It showed that lnc RNA-Gm44275 could promote the secretion of inflammatory cytokines and activate the STAT3 signaling pathway in vitro.It also could promote the initiation of labor in vivo,resulting in preterm labor.3.Preliminary study on the mechanism of function of Lnc RNA-Gm44275The lnc RNA-Gm44275 was predicted to potentially target mi RNA,and a dual-luciferase vector was constructed for verification.The results showed that there was no targeting relationship between lnc RNA-Gm44275 and the screened mi RNA149-5p.The lnc RNA-Gm44275 was synthesized by in vitro transcription,labeled with biotin.Target proteins were pulled down and identified by mass spectrometry.The results showed that the potential binding proteins of lnc RNA-Gm44275 were mainly enriched in the regulation of actin cytoskeleton(mmu04810),the focal adhesion(mmu04510)and the proteoglycan in cancer(mmu05205).Through the above research,he following several rounds are obtained.1.There were 141 differently expressed lnc RNAs in the uteruses of mouse between the day of15.5 and 18.5 of pregnancy;2.The expression level of Lnc RNA-Gm44275 in MSMc was higher than that of MEEc and MESc;3.In vitro,lnc RNA-Gm44275 could affect the expression of OXTR,IL6 and STAT3;4.In vivo,lnc RNA-Gm44275 could promote labor onset;5.256 lnc RNA-Gm44275 binding proteins were found,of which FN1,A2 M and LTF were reported to be associated with labor or uterine contractility.