Regulatory Mechanism Of LncRNA MPNCR On The Proliferation Of Bovine Mammary Epithelial Cells

The development of mammary gland undergoes periodic changes such as growth,differentiation and degradation along with the animal's reproductive change and lactation cycle.The proliferation and apoptosis of mammary epithelial cells throughout the entire process of lactation cycle.The mammary gland epithelial cells could synthesize and secrete milk protein,so the number of mammary epithelial cells and lactating capacity of single cell are very important in milk production.It was found that long-noncoding RNAs(lnc RNAs)and micro RNAs(mi RNAs)participated in mammary gland development and lactation cycle by regulating genes expression.The previous RNA-seq result of mammary gland tissues during the lactation and dry period of dairy cow showed that the expression of TCONS-71659(named lnc RNA MPNCR,mammary proliferation-associated long noncoding RNA)was significantly higher during lactation than that during dry period,so we speculated that lnc RNA MPNCR participated in bovine lactation regulation.Therefore,we conducted MTT assay and Ed U assay to explore the effect of lnc RNA MPNCR on bovine mammary epithelial cell line(BMECs)viability and proliferation.Then we performed RT-q PCR and Western Blot assay to study its regulatory mechanism as a competitive endogenous RNA(ce RNA)to the proliferation of bovine mammary epithelial cells.The main results were as follows:1.In order to explore the function of lnc RNA MPNCR on bovine mammary epithelial cells,the pc DNA3.1(+)-lnc RNA MPNCR and pc DNA3.1(+)vectors were transfected into BMECs.MTT assay result showed that overexpression of lnc RNA MPNCR extremely reduced cell viability(P <0.01)compared with the control group.Ed U assay result showed that the overexpression of lnc RNA MPNCR obviously inhibited the proliferation of BMECs(P <0.01).2.To explore the regulatory mechanism of lnc RNA MPNCR on the proliferation of BMECs,its subcellular localization was performed.The results showed that MPNCR expressed in the nucleus and cytoplasm.Bioinformatics analysis showed that there were binding sites between mi R-31 and lnc RNA MPNCR.The interaction between them was explored by dual-luciferase test and RT-q PCR.Dual-luciferase assay result showed that the fluorescence activity ratio of lnc RNA MPNCR in BEMCs(renilla enzyme activity/firefly enzyme activity,R/F)was significantly reduced with the transfection of mi R-31 mimic(P<0.05).RT-q PCR result showed that the expression of mi R-31 was dramatically reduced after transfected pc DNA3.1(+)-lnc RNA MPNCR vector in BMECs(P<0.01).RT-q PCR result also showed that the overexpression of mi R-31 in BMECs obviously inhibited the expression level of lnc RNA MPNCR(P<0.01),while the expression of lnc RNA MPNCR were significantly up-regulated after the inhibition of mi R-31(P<0.05),suggesting that lnc RNA MPNCR regulated the expression of mi R-31.3.To explore the function and target genes of mi R-31,mi R-31 mimic(or mimic NC)and mi R-31 inhibitor(or inhibitor-NC)were transfected into BMECs,respectively.Compared with their control groups,MTT assay showed that the overexpression of mi R-31 extremely promoted the cell viability(P<0.01)and the inhibition of mi R-31 significantly reduced the cell viability of BMECs(P<0.05).Ed U assay further showed that the proliferation of BMECs was promoted extremely transfected with mi R-31 mimic(P<0.01).The result of dual-luciferase assay showed that,compared with the control group,mi R-31 dramatically decreased the dual-luciferase activity ratio(renillase anzyme activity/ firefly enzyme activity,R/F)of CAMK2 D 3'UTR(P<0.01).RT-q PCR and Western Blot showed that mi R-31 dramaticaly inhibited the expression of CAMK2D(P<0.01),meanwhile the expression of CAMK2 D was up-regulated with the inhibition of mi R-31(P<0.05).The above results indicated that CAMK2 D was a target gene of mi R-31.4.To investigate the regulatory effect of lnc RNA MPNCR on CAMK2 D,the pc DNA3.1(+)-lnc RNA MPNCR and its control vector pc DNA3.1(+)were transfected into BMECs,respectively.The results of RT-q PCR and Western Blot showed that the overexpression of lnc RNA MPNCR dramatically up-regulated the expression of mi R-31 and CAMK2 D,which was the target of mi R-31,simultaneously at m RNA and protein level(P<0.01).The rescue experiment result further revealed that lnc RNA MPNCR inhibited BMECs proliferation by targeting mi R-31(P<0.05)after transfected with pc DNA3.1(+)-lnc RNA MPNCR vector & mi R-31 mimic,pc DNA 3.1(+)-lnc RNA MPNCR & mi R-31 mimic and pc DNA 3.1(+)-lnc RNA MPNCR & mi R-31 mimic-NC into BMECs.Taken together,lnc RNA MPNCR sponged mi R-31 and up-regulated the expression of proliferation-related gene CAMK2 D,a downstream target gene of mi R-31,inhibited the proliferation of BMECs.In this study,we revealed the influence and regulatory mechanism of lnc RNA MPNCR in BMECs.And it would provide a theory basis for further exploration of lnc RNA molecular mechanism in BMECs.