Spliceosome Loc66273 Variation Before 2 To 3 T3 - L1 Fat Cells To Differentiate Into Fat Effect Of Proliferation And Apoptosis

Part I The bioinformatics analysis and cellular localization of LI-2geneObjective:To understand the structure, function and subcellular localization of IL-2gene and study the distribution of LI-2gene and protein in various tissues of mice, for providing a basis for further research.Methods:The structure and function of LI-2gene and protein were analyzed by bioinformatics analysis, and its subcellular localization was observed under a fluorescence microscope.The mRNA levels of LI-2in various tissues of mice were examined by Semi-Quantitative RT-PCR,while its protein levels were examined by Western-blot. Regulatory genes related to CREB were screened by High-throughput screening technology of CREB luciferase enzymatic trans-reporting system..Result:.(1) The LI-2gene located on chromosome7E2in mouse,and the translatability framework encoding122amino acids is789bp, whose predicted molecular weight is13.2kDa and isoelectric point is7.77; Phylogenetic analysis showed that LI-2gene was high conservative in a variety of species, but had no significant homology with any known genes and proteins. Conservative domain analysis showed there was a prediction Mth938domain (amino acid residues5-120) in LI-2;(2) LI-2gene was expressed in fat, skeletal muscle, small intestine, lung and kidney tissue,while was higher in fat and skeletal muscle than others; Subcellular localization of LI-2suggested that the EGFP-LI2fusion protein diffusely distributed in the cytoplasm, having a high concentration around the nucleus;(3)CREB luciferase reporter gene assay system results suggested that the over-expression of LI-2increased the activity of CREB to2.5times than that of the blank control group.Conclusions:LI-2gene is highly conserved in a variety of species in vivo, suggesting that it has important functions. LI-2is highly expressed in white adipose tissue.High-throughput functional screening platform based on the quantitative expression of CREB luciferase trans-reporting system show that LI-2gene is one of the new genes which has linked to the transcriptional activity of CREB. This information prompted the LI-2gene may be related to the adipogenic differentiation. Part Ⅱ Expression profile of LI-2in the MDI induced differentiation of3T3-L1preadipocytesObjective:To study the expression changes of LI-2in MDI induced adipogenic differentiation of3T3-L1preadipocytes.Methods:This part of the experiment we use oil red staining,Western-Bloting and Semi-Quantitative RT-PCR to observe the MDI induced changed cell shapes, fat contents of3T3-L1preadipocytes and detected mRNA and protein levels of LI-2in3T3-L1preadipocytes on0,2,4,8days.Result:(1) Two days after MDI-induced3T3-L1preadipocytes, cells turned to be round and light gradually, and fusiform preadipocytes gradually became oval.Small amounts of lipid droplets in the cytoplasm were stained by Oil Red O to bright red.Six days after induction, larger lipid droplets appeared around the nuclei; Eight days after induction we can see more than90%of cells with the phenotype of mature fat cells.<2)Semi-Quantitative RT-PCR results showed that4days after MDI induction, the mRNA levels of LI-2in3T3-L1preadipocytes were significantly increased (P<0.05), and continued increasing during the differentiation process; Western blot results showed that the protein levels of IL-2in3T3-L1gradually increased with the differentiation induced,and by the4th day after induction there were significant differences (P<0.05).Conclusions:Classic cocktail MDI induction can successfully build the model of adipogenic differentiation of3T3-L1preadipocytes, to lay the foundation for the subsequent experiments.With the differentiation of3T3-L1preadipocytes, we found that the LI-2mRNA and protein levels were significantly increased compared with those of early differentiation, which further confirmed the relationship between LI-2and adipogenic differentiation. Part Ⅲ Effects of LI-2overexpression on the proliferation and differentiation of3T3-L1preadipocytesObjective:To study the effect of LI-2on3T3-L1adipogenic differentiation and cell proliferation and apoptosis in the case of cell overexpression of LI-2.Methods:(1)The cell shapes of pcDNA3.1-LI-2plasmid transfected3T3-L1preadipocytes, pcDNA.3.1empty vector transfected3T3-L1preadipocytes group and MDI induced3T3-L1preadipocytes were observed using an inverted microscope respectively. Oil red staining was used to observe the lipid droplet deposition in cells of each group;(2) Cell proliferation activity detection(MTT assay) was to detect the proliferation status of the three group cells cultured for eight days;(3)The apoptosis rates of cells in three groups were analysed by flow cytometry;(4) Cell cycle analysis of the three group cells were performed by flow cytometry;(5)Real-time fluorescence quantitative PCR and Western blotting were to detect mRNA levels and protein levels of LI-2and adipogenic genes in the differentiation of3T3-L1preadipocytes.Result:.(1)LI-2overexpression in3T3-L1preadipocytes in the absence of MDI induction lead that the cells gradually turned to round and light,and shaped gradually from the fusiform to oval, as the same with MDI-induced3T3-L1preadipocytes.The cell morphology of pcDNA.3.1empty vector transfected3T3-L1preadipocytes did not change.Three groups of cells cultured for8days, pcDNA.3.1-LI2transfected3T3-L1preadipocytes and MDI-induced3T3-L1preadipocytes can be seen visible large areas of bright red lipid droplets by Oil red O staining under both low and high magnification, while blank pcDNA.3.1vector transfected3T3-L1preadipocytes had only a very small number of lipid droplets;(2) The cell viability of blank control group, transfection pcDNA.3.1-LI2plasmid group and pcDNA.3.1vector group were detected by MTT, which suggested that the survival of pcDNA.3.1-LI2transfection plasmid group cells was significantly decreased compared with the other two groups on4,6,8days after transfection (P<0.05);(3) Flow cytometry analysis of cell cycle distribution showed significant increase of cell quantity in S and G2/M peroid24h or36h after serum treatment in blank pcDNA.3.1vector transfected3T3-L1,while in contrast, transfection of pcDNA3.1-LI2cells had little change (P<0.05);(4) Flow cytometry assay showed that the percentages of apoptosis in three groups were all higher in the4th day than that in the2nd day after transfection,but had no significant differences among the groups;(5) The two key adipogenic differentiation related genes PPARy and C/EBPa mRNA levels of pcDNA3.1-LI2transfected3T3-L1preadipocytes were significant higher than the empty vector control group (P<0.05), and the mRNA levels of genes gradually increased with the culture days; The protein levels of PPARy, C/EBPa, PREF-1, GADD153, aFABP, and FAS in3T3-L1fat cells transfered by pcDNA3.1-LI2or pcDNA3.1empty vector were analysed by western blot in the4th day and the8th day after transfection,respectively.The results suggested that over-expression of LI-2in cells contributed to the expression of PPARy, C/EBPa and the increase of aFABP and FAS protein levels for the transport and synthesis of fatty acid,which was significantly higher than empty vector control group, and the protein levels of Pref-1and GADD153which inhibit the differentiation of preadipocyte were significantly lower than the empty vector control group.Conclusions:LI-2increases the expression of the lipid regulators as well as inhibits the expression of adipogenic negative regulators,which is in favor of adipogenic differentiation. Moreover,LI-2can inhibit the proliferation of preadipocytes, which may promote the former cells into the adipogenic differentiation.But the change of apoptosis rate due to LI-2was not observed in this experiment, thus we need further study the influence of the silent LI-2to cells’apoptosis. Part Ⅳ The effects of LI-2gene silencing and mechanism on apoptosis and differentiation of3T3-L1cells Objective:By the silence of IL-2gene to further explore specific mechanisms and the relationship between LI-2and differentiation and apoptosis of3T3-L1preadipocytes, from positive and negative aspects to clarify this topic.Methods:(1) siRNA interference fragment was used to transfect3T3-L1preadipocytes. Cell morphology changes of transfection with siRNA transfection group and transfected with the negative Non-siRNA plasmid group were observed using an inverted microscope,and the lipid droplets in cells were observed by oil red staining;(2)We used Hoechst33342reagent staining the three groups of cells and observed apoptosis of cells in the fluorescence microscope. Flow cytometry was used to detecte apoptosis rate of the cells;(3) Ac-DEVD-AMC analysed caspase-3activity of the cells in each group at different time points and its protein levels were detected by Western blot;(4) CREB dual-luciferase reporter gene assay analysed the effect of LI-2gene silencing on CREB activity.At the same time the protein levels of CREB and p-CREB were detested by Western blot;(5)Real time-PCR and Western blot were used to detecte mRNA and protein levels of the adipogenic genes of3T3-L1cells after silence of the LI-2gene.Result:(1)The2groups of LI-2silencing3T3-L1preadipocytes induced by MDI showed that cell morphology had no significant changes, while MDI-induced cells of negative control group gradually changed from the shape of fiber cells to oval fat cells. Oil red O staining showed that after cultured for8days, siLI2-l, siLI2-2transfected3T3-L1preadipocytes only had a very small number of lipid droplets,while MDI-induced the negative control group cells had large areas of bright red lipid droplets both in low and high magnification;(2)3T3-L1preadipocytes were induced by MDI for4days after transfected with siLI-2-1, siLI-2-2or the Non-siRNA for2days.We observed nucleus of cells transfected siLI2-l or siLI2-2shrunk, formed apoptotic bodies, and became marginal and flake-shaped while the cell nucleuses’ shape of non-siRNA transfected cells were complete by fluorescence microscopy; Flow cytometry assay detected the percentage of apoptosis of cells transfected diefferent plasmids in three groups after2days or4days.The results suggested that the apoptosis rate of LI-2silencing cells in two groups induced by MDI compared to the negative control group was significantly increased (3) The above mentioned three groups for4days after the MDI induction, according to the size of the release of AMC fluorescence intensity about caspase-3activity,which suggested that caspase-3activity of two groups of LI-2silencing cells induced by MDI compared with negative control group was significantly increased. Protein levels for caspase were also significantly higher than the negative control group by Western blot;(4) CREB luciferase trans-reporting system showed that relative fluorescence luciferase activity of LI-2silencing cells was significantly lower than the negative control group of cells induced by MDI for2days or4days (P<0.05); pCREB protein levels of LI-2silencing cells were significantly lower than the negative control group (P<0.05)by western blotting,but there was no significant difference on CREB protein levels in the three groups;(5) LI-2gene silencing inhibited mRNA and protein expression levels of PPARγ and C/EBPα gene in the3T3-L1preadipocytes and the expressions of aFABP, FAS for transportion and synthesis of fatty acid, but protein levels of negative regulator for the adipogenic differentiation—pref-1and GADD153were significantly higher than the negative control group.Conclusions:The one hand, LI-2promotes adipogenic differentiation by regulating the expression of transcription factors, on the other hand,LI-2maintains the adipogenic differentiation by inhibiting proliferation and apoptosis of cells.