Screening,Identification And Functional Studies Of Long Non-Coding RNAs Differentially Expressed In Mammary Gland Of Dairy Cows

Mammary gland is an important organ for the synthesis and secretion of milk.The development cycles of mammary glands include pregnancy,lactation,and regression stages,which are affected or regulated by growth factors,hormones,coding genes and non-coding RNAs.In recent years,as a new regulator,long non-coding RNA?lnc RNA?has been attracted a growing attention and become the focus in life sciences.Studies have shown that lnc RNAs are involved in many biological processes,such as cell apoptosis,proliferation,differentiation and mammary gland development and lactation in human and mice.However,the role of lnc RNAs in dairy cow mammary gland is still unclear.In this study,Chinese Holstein mammary glands in the dry and 180-day lactation period were collected and sequenced by RNA-seq on an Illumina Hiseq3000 platform.The differentially expressed lnc RNAs and genes were screened based on the threshold of absolute value of fold change and P<0.05.Then,combining with their genomic location and potential interaction of differentially expressed lnc RNAs with mi R-221,four lnc RNAs were selected for subsequent functional studies,and the main results were as follows:1.Differentially expressed lnc RNAs and genes in dry and lactation periods?1?In dairy cow mammary glands in the dry and lactation periods,58,411,766 and 69,114,038 raw reads,and 54,805,136 and 65,069,892 clean reads were abtained,respectively.The percentage of clean reads was 93.83% and 94.15%.In addition,928 and 841 unannotated transcripts,and 64,316 and 61,791 known transcripts were obtained in the cow mammary glands in dry and lactation periods,in which 44,651 and 43,094 were m RNAs,respectively.?2?Totally 2,890 differentially expressed genes were found,in which 590 genes were significantly up-regulated and 2,300 down-regulated in lactation cow mammary gland.Gene ontology?GO?analysis showed that the significantly up-regulated genes were enriched in 69 GO terms,including negative regulation of cell growth,growth factor activity and sodium channel complex biological processes,etc.GO analysis showed that the significantly downregulated genes were enriched in 45 GO terms,including cell differentiation,cell adhesion and cell surface receptor biological processes,etc.In addition,KEGG analysis revealed that significantly up-and down-regulated genes in cow mammary gland in lactation were enriched in PPAR,PI3k-Akt,and TNF signaling pathways,etc.?3?Totally 3,746 differentially expressed lnc RNAs were found in cow mammary gland in dry and lactation periods,of which 1,014 were significantly up-and 2,732 down-regulated in lactation period.Combined with the genomic location of differentially expressed lnc RNA,target genes of 145 differentially expressed lnc RNA were predicted in cis and in trans.GO analysis of 47 potential target genes?in cis?showed that they were mainly enriched in the negative regulation of JAK-STAT,the cytokine-mediated,and positive regulation of phosphorylation?P<0.05?.GO analysis of 459 potential target genes?in trans?revealed that they were mainly involved in growth factor linkage,protein phosphorylation and positive regulation of cell differentiation biological processes.In addition,KEGG analysis showed that the potential target genes of 145 differentially expressed lnc RNAs were mainly enriched in 15 pathways,including NF-k B,MAPK,PI3K-Akt,and prolactin.2.Function study on the related lnc RNAsBecause mi R-221 could regulate the proliferation of bovine mammary gland epithelial cells?BMECs?,and four lnc RNAs(TCONS₀0000352,TCONS₀0040268,TCONS₀0087966 and TCONS₀0015196 potentially interacted with mi R-221,then the function of lnc RNAs was detected in BMECs transfected with their si RNAs and controls,respectively.MTT result showed that at 24 h after transfection,compared with their control groups,the viability of BMECs were dramatically higher in TCONS₀0000352 and TCONS₀0040268 interference groups?P<0.01?,while the interference of TCONS₀0015196 and TCONS₀0087966 significantly decreased the viability of BMECs?P<0.01?.However,at 48 h after transfection,there was no significant effect on the viability of BMECs in the four lnc RNA interference groups?P>0.05?.Ed U result further verified that the interfence of TCONS₀0040268 and TCONS₀0000352 significantly increased BMECs proliferation?P<0.05?,while the interference of TCONS₀0015196 and TCONS₀0087966 significantly decreased that after the transfection at 24 h?P<0.05?.Furtherly,the relative m RNA expressions of cell proliferation-related genes in BMECs were detected by q RT-PCR after inhibiting the related lnc RNAs.The results showed that the relative expression of CDKN1 A and CDKN1 B genes were significantly decreased after interfering TCONS₀0000352?P<0.01?,but the relative expression of CCND1 gene were dramatically increased compared with the control group?P<0.01?.After interfering TCONS₀0040268,the relative expression of CDKN1 A gene was very significantly decreased?P<0.01?,and the expression of CCND1 gene was significantly increased compared with the control?P<0.05?.As to TCONS₀0015196 and TCONS₀0087966,the relative expressions of CCND1 and CDK2 genes were dramatically decreased after interfering them,and the expression of CDKN1 A gene were significantly increased with the interfering with TCONS₀0015196?P<0.05?,while the relative expression of CCNE1 gene were significantly decreased with the inhibition of TCONS₀0087966?P<0.05?.3.The interaction between lncRNAs and miR-221The potential lnc RNAs interacted with mi R-221,which was related with mammary gland development and lactation,were predicted and found that TCONS₀0015196 and TCONS₀0087966 might interact with mi R-221 regulating lactation.Then dual luciferase reportor asssy was used to detect the effects of mi R-221 overexpression and inhibition on luciferase activities in HEK293 T cells.The results showed that the overexpression of mi R-221 significantly reduced the luciferase activity of TCONS₀0087966 target vector?P<0.01?,but the interference of mi R-221 expression had no significant effect?P>0.05?.In addition,the overexpression of mi R-221 significantly decreased TCONS₀0015196 luciferase activity?P<0.01?,while the interference of mi R-221 significantly increased its luciferase activity?P<0.01?.Taken together,the differentially expressed lnc RNAs and genes were screened and identified in dairy cow mammary glands in dry and lactation period,and the functions of related lnc RNAs interacted with mi R-221 were studied.This study would enrich the regulatory mechamisn of mammary gland development and lactation and provide a theoretical basic for the molecular breeding in dairy cattle.