| Milk fat is the main nutrient in milk,which is favored by the food and beauty industries because it is rich in vitamins,trace elements and unique flavor.However,the quality of milk in our country is generally low and its competitiveness in international market is insufficient.Therefore,in addition to strictly strengthening the supervision of dairy production and the feeding management of dairy cows,it is urgent to improve the milk quality and milk yield of dairy cows’ mammary glands at the molecular level.Ln RNA is a class of non-coding RNA with a transcript of more than 200 nucleotides.It has a large number,complex types and divers e functions,and regulates the biological functions of the body in a variety of ways.Pseudogenes are derived from protein-coding genes,which are similar to their homologous sequences.Active pseudogenes are often considered as a subclass of lnc RNA and ar e often associated with many diseases.However,pseudogenes have not been reported to regulate DCMECs lactation,proliferation,apoptosis and ferroptosis.Research methods:In this study,Dairy cow mammary epithelial cells(DCMECs)were used as the researc h model.Firstly,RNA-seq technology was applied to find that there was a significant difference in the expression of miR-223 in the mammary tissue of dairy cow with high quality,low quality and dry milk stage.The results showed that miR-223 had an important regulatory function in lactation,and the ce RNA regulatory network of lnc RNA,miR-223 and m RNA was established by bioinformatics software.q PCR and dual luciferase reporter gene assay were used to verify the targeted binding ability of pseudogene RPS4XP1 to miR-223 and miR-223 to FOXO13’UTR region.Western blot was used to detect the expression of FOXO1 protein in lactation,dry milk,puberty and mastitis inflammatory tissues.The protein coding of the pseudogene RPS4XP1 was detected by p ET-28 a in vitro translation system.The miR-223 mimics/mimics NC,miR-223 inhibitor/inhibitor NC,si R-FOXO1,pseudogene RPS4XP1 eukaryotic overexpression vector and CRISPR-Cas9 knockout vector were transfected into DCMECs by liposome transfection technology.Immunofluorescence technique was used to identify the extracted primary DCMECs,and fluorescence microscopy was used to detect cell proliferation,apoptosis,and intracellular lipid droplet synthesis.Triglyceride secretion was detected by enzyme activity kit.Flow cytometry was used to detect cell apoptosis.The nuclear export and entry of FOXO1 protein were observed by ultrahigh resolution microscope.q RCR and western blot were used to detect the expression of genes and proteins related to lactation,proliferation and apoptosis,respectively.Co-transfection technology was used to co-transfect miR-223 inhibitor,si R-FOXO1,RPS4XP1 overexpression vectors and miR-223 mimics into DCMECs to verify the ce RNA regulatory network of pseudogenes RPS4XP1,miR-223 and FOXO1.To investigate the effect of miR-223 on the inflammatory response of DCMECs induced by lipopolysaccharide(LPS).The effects of pseudogenes RPS4XP1 and miR-223 on the expression of ferroptosis-related genes in DCMECs were investigated by PCR Array.The results showed that:(1)The wild type and mutant gene fragments of RPS4XP1 and FOXO1 3’UTR containing the predicted target binding site were ligated into the p Si CHECK2 gene reporter vector,and then co-transfected with miR-223 into Hela cells for detection of dual luciferase activity.The results showed that miR-223 specifically bound to RPS4XP1 and FOXO1 3’UTR,respectively.q PCR results showed that pseudogene RPS4XP1 negatively regulated the expression of miR-223 and positively regulated the expression of FOXO1.miR-223 negatively regulated FOXO1 gene expression.The results of super-resolution microscopy and western blot showed that miR-223 negatively regulated FOXO1 protein expression.(2)The p ET-28 a translation system was used to detect the protein coding of the pseudogene RPS4XP1.The results showed that the pseudogene RPS4XP1 had no obvious protein translation and was a non-coding RNA.Bioinformatics software predicted the subcellular localization of pseudogene RPS4XP1,and the results showed that the pseudogene RPS4XP1 was mainly located in the cytoplasm,followed by the nucleus.(3)The effect of miR-223 and pseudogene RPS4XP1 on milk fat synthesis of DCMECs was detected.The results showed that overexpression of miR-223 promoted milk fat synthesis,while inhibition of miR-223 expression had the reverse effect.Overexpression of pseudogene RPS4XP1 inhibited milk fat synthesis,however,the inhibitory effect of milk fat synthesis was cancelled by co-transfection of RPS4XP1 and miR-223 mimics.(4)The effect of miR-223 and pseudogene RPS4XP1 on the proliferation of DCMECs was detected by EDU.The results showed that overexpression of miR-223 promoted cell proliferation,while inhibition of miR-223 expression had the opposite effect.Overexpression of pseudogene RPS4XP1 inhibited cell proliferation,while co-transfection of RPS4XP1 and miR-223 mimics abrogated the inhibitory effect on cell proliferation.(5)Flow cytometry was used to detect the effect of miR-223 on apoptosis of DCMECs.The results showed that overexpression of miR-223 could inhibit cell apoptosis,on the contrary,inhibition of miR-223 expression could promote cell apoptosis.(6)Western blot was used to detect the expression of proteins related to lactation,proliferation and apoptosis.The protein expression levels of FASN,m TOR,p-m TOR,SREBP1,p-FOXO1,AKT,p-AKT,PPARγ,Cyclin D1 and Bcl-2 were significantly up-regulated,while the protein expression levels of Caspase3 and Bax were significantly down-regulated.When miR-223 was inhibited,the results were opposite.After FOXO1 knockdown,the protein expression of FASN,m TOR,p-m TOR,SREBP1,AKT,p-AKT,PPARγ,Cyclin D1 and Bcl-2 was significantly up-regulated,and the protein expression of Caspase3 and Bax was significantly down-regulated,except p-FOXO1 protein had no significant change.However,co-transfection of si R-FOXO1 and miR-223 inhibitor could cancel the above changes in protein expression.In addition,after overexpression of pseudogene RPS4XP1,the protein expression levels of FAS N,m TOR,p-m TOR,SREBP1,p-FOXO1,AKT,p-AKT,PPARγ,Cyclin D1 and Bcl-2 were significantly down-regulated,and the protein expression levels of Caspase3 and Bax were significantly up-regulated.However,when co-transfected with p CDH-RPS4XP1 and miR-223 mimics,the above changes in protein expression were offset.After knocking out the pseudogene RPS4XP1,the results were opposite to the overexpression group.(7)The results showed that LPS could promote the accumulation of FOXO1 protein in the nucleus and promote the expression of miR-223.Overexpression of miR-223 inhibited LPS-induced TNF-α protein expression and promoted FASN protein expression.Fluorescence microscopy showed that miR-223 could reduce the apoptosis rate induced by LPS and promote milk fat synthesis.(8)Ferroptosis PCR Array was used to detect the effect of pseudogenes RPS4XP1 and miR-223 on the expression profile of ferroptosis related genes in DCMECs.The results showed that pseudogene RPS4XP1 may regulate the expression of ferroptosis-related genes NOX3,CA9 and ALOX15 by targeting miR-223 to promote ferroptosis.In summary,the pseudogene RPS4XP1 is mainly localized in the cytoplasm,does not translate proteins,and has sequence conservation among human and bovine species.There was a ce RNA regulatory network between pseudogene RPS4XP1,miR-223 and FOXO1.Pseudogene RPS4XP1 can act as a molecular sponge of miR-223 and regulate the proliferation,apoptosis and milk fat synthesis of DCMECs by activating AKT/FOXO1 and AKT/m TOR signaling pathways.miR-223 can reduce the inflammatory response of DCMECs induced by LPS,i nhibit cell apoptosis,and promote milk fat synthesis.Knocking down the expression of pseudogene RPS4XP1 may inhibit ferroptosis of DCMECs by targeting miR-223. |
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