| Heat stress has important effects on lactation of pigs.Especially in the south of China,heat stress has a great impact on pig production.As an important endocrine organ,the pituitary gland can participate in the regulation of lactation through growth hormone and prolactin secreted by itself.As a new type of regulatory molecule,non-coding RNA can participate in the regulation of lactation at the transcriptional or post-transcriptional level.However,most researches have mainly focused on the breast tissue of dairy cows,while it is still blank in other animals or tissues.It has been reported that heat stress affects lactation,endocrine status and non-coding RNA expression in animals,but the relationship and mechanism are not clear.The effects of heat stress on the expression of non-coding RNA in the pituitary of sows have not been reported.In order to analyze the role of non-coding RNA in heat stress affecting sow lactation,this study explored the differentially expressed noncoding RNAs and their interactions in pituitary gland of sows under heat stress.This study found the changes of key non-coding RNAs related to lactation in the pituitary gland and its mechanism,expounding the molecular mechanism of pituitary regulation of lactation under heat stress from the perspective of non-coding RNAs.The main research contents and results are as follows:1.To study the effects of heat stress on lactation performance and related hormones.Four large-scale breeding pig farms were selected in the Pearl River Delta,East,West and North regions of Guangdong.During the winter(December,January,non-heat stress period)and summer months(July,August,heat stress period)of 2017-2018,recorded the production indicators of the Landrace(3 to 5 parity)in each unit of four farms,such as live litter size,the litter birth weight and the weaning weight of piglets.In Shanwei pig farm,the body surface temperature and rectal temperature of lactating sows in winter and summer were measured.In Guangzhou pig farm,six healthy sows(Landrace)that had finished lactation were selected respectively in winter and summer,and the blood and pituitary tissue were collected after euthanizing the sows.An automatic biochemical analyzer was used to analyze the biochemical indicators in the serum.The levels of HSP70,SOD,COR,and PRL in the serum were detected by ELISA kit analysis.The m RNA and protein expressions of PRL in pituitary tissue were detected by real-time fluorescence quantitative PCR and Western Blot.The results were shown as follows:(1)Compared with non-heat stress,heat stress significantly increased(P<0.01)the sow body surface temperature(39.6℃ vs 36.8℃,39.6℃ vs 36.5℃,40.4℃ vs 36.8℃)and rectal temperature(38.9 ℃ vs 38.5 ℃).(2)Under heat stress,the live litter size of sows and the number of alive weaning piglets were significantly reduced(P<0.05)or had a tendency to decrease.In addition,heat stress significantly reduced(P<0.05)the weaning litter weight and the 21 days total milk production of sows(124.4 kg vs 150.0 kg,167.1 kg vs 192.1 kg,178.1 kg vs 205.4 kg,182.2kg vs 234.6 kg).(3)Compared with non-heat stress,the total protein and globulin of sow serum were significantly increased under heat stress(P<0.05);the glucose concentration was extremely reduced(P<0.01);LDH and GOT concentrations were significantly increased(P<0.01);and there were no significant changes in concentrations of urea,albumin,total cholesterol,triglycerides,and GPT(P>0.05).Heat stress significantly increased(P<0.05)serum HSP70 content,significantly reduced(P<0.05)the activity of SOD in serum,as well as significantly reduced(P<0.05)the levels of COR and PRL in serum.(4)Heat stress significantly inhibited(P<0.01)PRL protein expression in pituitary of sows.2.To investigate the effects of heat stress on the expression of non-coding RNA in the pituitary of sows.Three samples were selected from sow pituitaries collected in winter and summer respectively,and RNA was extracted and the whole transcriptome sequencing was performed to obtain pituitary m RNAs,lnc RNAs,circ RNAs and micro RNA(mi RNAs)expression profiles under heat stress and non-heat stress.First,the characteristics of the detected m RNAs and non-coding RNAs were analyzed,including length,type,and chromosomal location.Then,differential expression analysises of m RNAs and non-coding RNAs were performed,the sequencing results were verified using q RT-PCR detection.GO and KEGG analysises were performed on target genes of differentially expressed non-coding RNAs.The functions of differentially expressed circ RNAs and lnc RNAs,as well as their potential interactions with pituitary mi RNAs were predicted.Finally,the interaction analysis of differentially expressed m RNAs and non-coding RNAs was performed,and a ce RNA network that may be related to lactation regulation was constructed.The results were shown as follows:(1)There were 96 m RNAs differentially expressed in the pituitary of heat-stressed sows,of which 51 were up-regulated and 45 were down-regulated.There were 462 lnc RNAs differentially expressed,of which 232 were up-regulated and 230 were down-regulated.There were 59 circ RNAs differentially expressed,of which 42 were up-regulated and 17 were down-regulated.There were 78 mi RNAs differentially expressed,of which 35 were upregulated and 43 were down-regulated.(2)Differentially expressed mi RNAs may target genes that related to pituitary hormone synthesis and secretion;differentially expressed lnc RNAs and circ RNAs may regulate the synthesis and secretion of pituitary FSH,LH,GH and PRL through interactions with their potential mi RNAs.(3)Multiple lnc RNAs/circ RNAs-mi R-130a-TGFBR2 interactions may be involved in PRL regulation.3.To investigate the effects of mi R-130a-3p on PRL expression and its regulatory mechanism.From the analysis of the effects of heat stress on the non-coding RNA of sows pituitary,it was found that mi R-130a-3p expression changes and may be involved in PRL regulation.Rat GH3 cells were selected as the cell model,after mi R-130a-3p mimic and inhibitor were synthesized and transfected into GH3 cells,the expression of PRL was detected at the m RNA and protein levels.The target genes of mi R-130a-3p were predicted by Target Scan and RNAhybrid,and the target relationship between mi R-130a-3p and its target gene was verified by dual luciferase reporter vector,q RT-PCR and Western Blot.Then,the inhibitor and the overexpression vector of the target gene were used to verify the target gene’s regulatory function on PRL and the recovery function on mi R-130a-3p’s action.Finally,heat stress tests were performed on mice and GH3 cells to detect the effects of heat stress on PRL,mi R-130a-3p and its target gene.The results were shown as follows:(1)q RT-PCR,Western Blot,and cell immunofluorescence detection revealed that mi R-130a-3p mimic significantly inhibited(P<0.05)the m RNA and protein expressions of PRL;(2)The dual luciferase reporter gene detection system confirmed that mi R-130a-3p directly targets ERα,and q RT-PCR,Western Blot,and cell immunofluorescence detection revealed that mi R-130a-3p mimic significantly inhibited(P<0.05))the m RNA and protein expressions of ERα;(3)ERα inhibitor inhibited the expressions of ERα and PRL proteins.ERα eukaryotic overexpression vector restored the inhibitory effect of mi R-130a-3p on PRL,and mi R-130a-3p inhibited PRL expression by targeting ERα;(4)The 2 h daily heat stimulation for 7 consecutive days significantly increased(P<0.05)mi R-130a-3p level,and significantly reduced(P<0.05)ERα and PRL expressions in mouse pituitary.41°C significantly increased(P<0.05)mi R-130a-3p expression level,and significantly reduced(P<0.05)ERα and PRL expressions in GH3 cells.Mi R-130a-3p may be involved in regulating the reduction of PRL expression induced by heat stress.In summary,this study found that heat stress could increase sow body surface temperature and rectal temperature,reduce sow lactation performance,serum PRL concentration,and pituitary PRL protein expression;heat stress could also change expressions of pituitary m RNAs,lnc RNAs,circ RNAs and mi RNAs.The differently expressed lnc RNAs and circ RNAs may regulate the synthesis and secretion of pituitary FSH,LH,GH and PRL through interactions with their potential mi RNAs;mi R-130a-3p inhibited PRL expression by targeting ERα and may be involved in regulating the reduction of PRL expression induced by heat stress.This study firstly excavated the differentially expressed non-coding RNAs in the pituitary of sows under heat stress,confirming that mi R-130a-3p may be involved in the process of heat stress affecting lactation,and revealed a new approach of heat stress affecting lactation. |
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