Downregulation Of OPN Expression Inhibits The Differentiation Of White Preadipocyte To Brown Adipocyte

Background and objective:brown adipocyte?BA?plays an important role in body energy homeostasis and anti-obesity by"burning"fat,but the transcriptional regulation mechanism of BA differentiation has not been fully elucidated.Osteopontin?OPN?is a kind of cytokine which is involved in many physiological and pathological processes.Our previous study found that OPN is a positive regulatory factor that can induce BA differentiation.In this study,we constructed the adipocyte-specific adenovirus vector Ad-aP2-opn-shRNA and then used it to infect white pre-adipocyte?3T3-L1?and human umbilical vein endothelial cells?HUVEC?respectively.On the one hand,the infection specificity of Ad-aP2-opn-shRNA was observed;on the other hand,the effects of downregulating OPN expression on the differentiation from 3T3-L1 preadipocyte to mature brown adipocyte were investigated.Methods:?1?Screening of the optimal opn-shRNA coding sequence:the murine opn gene sequence?NM₀01204201?was obtained from GenBank and then the online design software of Ambion company was used to select three interference target site referring to small interfering RNA design principles:?1?GCAGACACTTTCACTCCAA;?2?GTCAGCTGGATGAACCAAG;?3?GGATGTGATCGATAGTCAA.Two complementary oligonucleotide sequences for each target site were designed,which is called as opn-shRNA-513,opn-shRNA-514,opn-shRNA-515.The three opn-shRNA expression sequences were cloned into the sticky ends of pU6-MCS-AMP plasmid to form 3 recombinant plasmids known as opn-shRNA-513,opn-shRNA-514,opn-shRNA-515 respectively.At the same time,a negative control plasmid was constructed.The plasmid sequences were identified by restriction enzyme and DNA sequencing.The silencing efficacy of opn-shRNA plasmid was estimated by Western blots.?2?Construction of the adipocyte-specific adenovirus vector carrying opn-shRNA?Ad-aP2-opn-shRNA?:the optimal opn-shRNA sequence and the plasmid carrying aP2-specific promoter were respectively digested by restriction endonuclease and linked to form a recombinant plasmid,i.e.,aP2-opn-shRNA,in a ligation system.Similarly,aP2-opn-shRNA and an adenovirus vector?Ad-MCS-SV40-EGFP?were digested by SacII/NheI and ligated to construct a recombinant Adenovirus Shuttle Plasmid?Ad-aP2-opn-shRNA?in a Cre/loxP recombinase system in HEK293.The sequence of Ad-aP2-opn-shRNA was confirmed by PCR and positive cloning sequencing.The concentration of adenovirus was tittered by useing the end point dilution method.?3?Verification of the adipocyte-specificity for Ad-aP2-opn-shRNA:the pre-adipocyte?3T3-L1?and human umbilical vein endothelial cells?HUVEC?were infected using the Ad-aP2-opn-shRNA,respectively.48 hour later,green fluorescent protein?EGFP?was visualized under a fluorescence microscope;?4?Effect of Ad-aP2-opn-shRNA on the differentiation from 3T3-L1 preadipocyte to BA:Ad-aP2-opn-shRNA was infected to 3T3-L1 preadipocytes and divided into 3 groups,?1?Control group,?2?negative control group?NC?,?3?adenovirus group?Ad-aP2-opn-shRNA?.On the one hand,lipid accumulation was observed using oil red O staining;on the other hand,the expression levels of the BA-specific molecular markers such as PRDM16 and UCP-1were detected using Western blots and sOPN levels in the supernatant were assayed using ELISA Kit.Results:?1?The recombinant plasmid was successfully constructed and screened the optimal silencing efficiency opn-shRNA,which is opn-shRNA-513;?2?an adipocyte-specific adenoviral vector?Ad-aP2-opn-shRNA?was successfully constructed and the adenoviral titer was up to 1×10¹⁰PFU/ml;?3?48 hours after infection of Ad-aP2-opn-shRNA,the intensity of green fluorescence in 3T3-L1 was significantly higher than that in HUVECS,sugesting the Ad-aP2-opn-shRNA is the adipocyte specific;?4?the results from the oil red O staining showed that lipid droplet in Ad-aP2-opn-shRNA group decreased than that in Contrl group and NC group;moreover,Western blot results showed that PRDM16 and UCP-1 expression levels in Ad-aP2-opn-shRNA group were significantly lower than Control group and NC group and there was significant difference?P<0.01?.Conclusion:?1?An adipocyte-specific adenoviral vector carrying opn-shRNA?Ad-aP2-opn-shRNA?was successfully constructed;?2?Downregulation of OPN expression inhibits the differentiation of white pre-adipocyte into brown adipocyte and the underlying mechanism needs further to be studied.