Effect And Mechanism Of PLIN1 Gene Knockout On Lipolysis Of 3T3-L1 Adipocytes

Objective:The PLIN1 knockout vector was constructed to knockout the PLIN1 in 3T3-L1 preadipocytes and reduce the expression of PLIN1 protein.The effect of PLIN1 knockout on lipolysis in 3T3-L1 adipocytes was investigated and the mechanism of lipolysis was explored.Methods:1.The PLIN1 knockout vector was constructed by CRISPR/Cas9 technology and double-digested and identified by DNA sequencing technology.3T3-L1 preadipocytes were routinely cultured,and the target fragment of PLIN1 was amplified by PCR.The sgRNA was transcribed in vitro,and Cas9 was digested in vitro to identify sgRNA activity.2.3T3-L1 preadipocytes were routinely cultured.Transfection of cells by electroporation.The puromycin screening for successfully transfected cells.Optimized the hormonal cocktail to induce differentiation into mature adipocytes.Detection of PLIN1 protein expression in adipocytes by Western blot.RT-PCR was used to detect the relative expression of PLIN1 mRNA,and the knockout effect of PLIN1 was verified.3.Oil red O staining for lipid droplets in fat cells,measuring lipid droplet size and quantity.Enzymatic determination of TG and glycerol in cells,reflecting cell lipolysis.Detection of lipolytic enzyme,Fsp27 and PPAR? protein expression in cells by Western blot.Detection of relative expression of HSL and ATGL mRNA in cells by RT-PCR.Results:1.All three sgRNAs can recognize the PLIN1 target,and the in vitro digestion activity is effective.The DNA sequencing results of the PLIN1 knockout vector were consistent with expectations.2.The purity of each group was between 1.8 and 2.0,and the concentration was around 2 ?g/?L.On the fourth day,the lowest concentration of puromycin-killing cells was 4 ?g/ml,and the survival rate of puromycin-selected cells was 30%.After screening,the expression of PLIN1 protein was significantly decreased in the three positive knockout groups(P<0.05).There was no expression of PLIN1 mRNA in the three knockout cells.3.After the induction of differentiation,the volume of lipid droplets in fat cells gradually increases,and it is distributed around the nucleus,forming a distinct "ring-like" structure.Compared with the control group,the number of large lipid droplets in the PLIN1 knockout group was reduced,the distribution of small lipid droplets was extensive,and the rate of lipid droplet fusion was slowed down.The triglyceride content in the cells of the PLIN1 knockout group(0.0310±0.0053 mmol/g protein)were significantly higher than those of the control group(blank control group: 0.1078±0.0039 mmol/g protein,negative control group: 0.1256±0.0076 mmol/g protein)(P<0.05).The glycerol content in the cells of the PLIN1 knockout group(0.0984±0.0076 mmol/g protein)were significantly lower than those of the control group(blank control group: 0.0485±0.0131 mmol/g protein,negative control group: 0.0376±0.0124 mmol/g protein)(P<0.05).Compared with the control group,the expression of HSL and ATGL protein and the relative expression of mRNA in the PLIN1 knockout group increased(P<0.05).There was no difference in the expression levels of Fsp27 and PPAR? between the control group and the PLIN1 knockout group(P>0.05).Conclusion:1.The 3 PLIN1 knockout vectors were successfully constructed,and was effective in vitro activity assays.2.Successfully knockout the PLIN1 in 3T3-L1 preadipocytes.3.After the adipocytes PLIN1 is knockout,the rate of lipolysis in the cells is accelerated.4.Loss of surface barrier of lipid droplets,increased lipid droplet surface area due to slower fat droplet fusion rate and increased number of small lipid droplets and increased expression of ATGL and HSL proteins in adipocytes cause an increase in lipolysis in adipocytes.