| Min pig is a local pig breed formed by long-term breeding in Northeast China,and its cold resistance is far better than other pig breeds.Therefore,in-depth understanding and study of the proliferation and differentiation mechanism of Min pig adipocytes plays an important role in understanding the molecular mechanism of Min pig cold resistance.At the same time,it has long-term significance for promoting the protection of germplasm resources,improving the cold resistance of other pig breeds by using their cold resistance characteristics,and cultivating new cold-resistant varieties.miR-155 is a multifunctional small molecule RNA that plays an important regulatory role in cell proliferation and differentiation,but its regulatory effect on porcine adipocyte proliferation and differentiation is rarely reported.In this study,the target gene of miR-155 was identified by dual luciferase method,and the expression pattern and role of its target gene CEBPB in regulating proliferation and adipogenesis were detected by overexpression and interference of miR-155 during the differentiation of Min pig preadipocytes.To determine the regulatory effect of miR-155 on CEBPB and its effect on adipogenesis.The results of this study will provide experimental basis for further revealing the mechanism of porcine adipocyte formation and regulation.The main results are as follows :1.The expression of miR-155 and CEBPB in different tissues of Min pigs under normal temperature and cold stimulation was detected by qPCR.Under normal temperature conditions,the expression of miR-155 was the lowest in the kidney,followed by the heart,and the expression levels in the back muscle,spleen,liver,back fat,and lung increased in turn.The expression level of CEBPB in the heart was the lowest,followed by that in the kidney,and the expression levels in the back muscle,liver,spleen,back fat and lung increased in turn.The expression of miR-155 was up-regulated in inguinal fat and chest fat,and down-regulated in buttock fat and back fat.The expression of CEBPB gene was up-regulated in back fat,inguinal fat and chest fat,and down-regulated in buttock fat.The results showed that miR-155 and CEBPB genes had obvious tissue expression specificity in Min pig adipose tissue,and cold stimulation could significantly change their expression patterns.2.The wild-type and mutant vectors of CEBPB gene were successfully constructed.The luciferase activity of the transfected cells was detected by double luciferase activity assay.The results showed that the luciferase activity was significantly inhibited after co-transfection of wild-type plasmid and miR-155 mimics.The results showed that CEBPB was a target gene of miR-155,and miR-155 had a negative regulatory effect on CEBPB.3.Overexpression of miR-155 in the proliferation stage of preadipocytes resulted in a decrease in the expression of proliferation marker genes CDK4 and PCNA.CCK8 results showed that the cell activity of the experimental group was lower than that of the control group.The positive rate of EdU staining cells was lower than that of the control group.Cell cycle detection showed that the cell proliferation index was lower than that of the control group.On the contrary,the expression of proliferation marker genes increased after interference with miR-155.CCK8 results showed that the cell activity of the experimental group was higher than that of the control group.The positive rate of EdU staining was higher than that of the control group.Cell cycle detection showed that the cell proliferation index was higher than that of the control group.In addition,overexpression of CEBPB also increased the expression of proliferation marker genes.CCK8 results showed that the cell activity of the experimental group was higher than that of the control group.The positive rate of EdU staining in the experimental group was higher than that in the control group.Cell cycle detection showed that the cell proliferation index was higher than that of the control group.The results showed that overexpression of miR-155 inhibited the proliferation of preadipocytes,and interference with miR-155 and overexpression of CEBPB promoted cell proliferation.4.Interference with miR-155 increased the expression of genes related to fat markers and those promoting fat generation during induction of differentiation,while the expression of inhibitory genes decreased.During the cell proliferation stage,interference with miR-155 increased the expression of proliferation markers,EdU staining,and cell proliferation index.These results suggest that interfering with miR-155 can promote the differentiation and proliferation of Min pig preadipocytes.4.In the process of inducing preadipocyte differentiation,overexpression of miR-155 led to a decrease in the expression of fat marker genes PPARG,FASN,FABP4 and adipogenic genes CEBPB,FADS1,FGF2,PROX1.At the same time,the expression of genes HSL and SOCS1 that inhibit adipogenesis increased.On the contrary,interference with miR-155 decreased the expression of fat marker genes and adipogenic factors,while increased the expression of adipogenic genes.In addition,overexpression of CEBPB increased the expression of fat marker genes and adipogenic genes,while reducing the expression of adipogenic genes.The results showed that overexpression of miR-155 could inhibit the differentiation of preadipocytes,while interference with miR-155 and overexpression of CEBPB could promote the differentiation of preadipocytes.In summary,miR-155 can inhibit cell proliferation and differentiation,and its mechanism may play a role by inhibiting the expression of CEBPB.The results of this study are helpful to understand the molecular mechanism of cold resistance in Min pig,and provide experimental basis for further study of the molecular mechanism of cold resistance in Min pig. |
