Effects Of Lentivirus-mediated SOCS-3 RNA Interference On Rat Adipocyte Metabolism

IntroductionObesity is one of the risk factors in various chronic diseases and malignancy, which has been a serious threat to human physical and mental health.With the discovery of obese gene and its encoded gene product:leptin,the obesity research has laid foundation level in molecular biology.Previous studies demonstrated that in the majority of obese subjects and rodents exist leptin resistance,that is,to diminish the leptin signal response which may reduce body weight.Suppressor of cytokine signaling 3(SOCS-3) is a negative feedback inhibition factor of leptin signal transduction,induced by leptin.SOCS-3 is also the inhibitory of insulin,growth hormone and other hormones, which central inhibition may affect other hormones playing their normal physiological functions.Therefore,in our study,we first establish high-fat diet-induced obesity rat model and detected SOCS-3 mRNA expression in adipose tissue,then infected by SOCS-3 shRNA lentivirus vector in DIO rat adiopcytes,to observed SOCS-3-knockdown in DIO adipocytes reduce the SOCS-3 expression,whether may affect the cell metabolism.Materials and methods1.Diet-induced obesity rat model and detection(1) the establishment of diet-induced obesity rat model:after 1 week feeding adaptation,according to body weight 40 male Wistar rats were randomly divided into 2 groups:7 rats for control group,which were fed with the,and the other 33 rats feeding with high-fat diet,high fat diet based containing common basis diet 73%,lard 20%and 7%casein protein,were called high fat group.After 8 weeks,according to the weight increment was greater than the control group(?)+s as the standard of obesity,10 rats from high-fat group were randomly selected as the obese group,5 rats were killed for the detection of serum lipids and SOCS-3 mRNA expression in epididymal fat organization,and the other 5 were used for primary cultures of rat adipose stromal cells.At the end of the experiment,blood samples were collected via abdominal aorta from the anaesthetized rats.(2) Determination of serum lipids:note by the kit,using semi-automatic biochemical analyzer for determination of serum total cholesterol and triglyceride content.(3) SOCS-3 mRNA expression in adipose tissue:according to TRIzol manual steps,to gain total RNA from approximately 100mg of rat epididymal adipose tissue. Then use 1μg total RNA,to carry out RT-PCR reaction by Takara RNA PCR kit.Used 2μl cDNA as a template,and usedβ-actin as internal control.Both theβ-actin and SOCS-3 gene had the same reaction conditions,the reverse transcription reaction conditions is 30℃10min,42℃30min,99℃5min,5℃5min,while the PCR reaction conditions is 94℃2min predegeneration,35 cycle of amplification including denaturation 94℃30s,annealing 56℃30s,extension 72℃1min.and then 1 cycle of extension 72℃10min.Took 5μl target gene products,plus pre-stained liquor reacted 3min,then ran on 2%agarose gel electrophoresis and photographed under gel imaging camera analyzer.Compare with the ratio ofβ-actin gene expression as relative content.2.Isolation and differentiation of rat adipose stromal cellsStrictly sterile conditions are required,and the crudely prepared fat pads were carefully liberated from remaining connective tissue and blood vessel from obese group rat epididymal tissue.After repeated washing with PBS,the digestion step was followed by bovine serum albumin and collagenaseâ…¡mixture.The mixture was filtered through a 250μm nylon mesh,to collect the disaggregated cells,samples were centrifuged at 50×g for l0min,then centrifuged at 200×g for l0min.The pellet was resuspended in erythrocyte-lysing buffer,and incubated for 5-10 min at room temperature.Than filtered through a 25μm nylon mesh,centrifuged at 200×g twice for 5min.Obtained cells after resuspension in basal medium then incubated to 12-well plates.Cultured under the condition of 37℃with 5%CO₂.After cell adhesion into a single-layer,cultures were washed twice with PBS.The cells were incubated in differentiation cocktail medium for the first 3 d,then changed post differentiation medium every other day.After 10 d,the lipid droplets in adipocytes and the rate of differentiation were identified by oil red O stain.3.Construction of SOCS-3 shRNA lentivirus vectorLentivirus vectors were commissioned by Genechem Chemical limited company.4.Infected by lentivirus and incubated with leptinInfected by lentivirus after differentiation for 10d,and divided into three groups: the blank control group(CON group),infected by negative control lentivirus(NC group),infected by SOCS-3 shRNA lentivirus(KD group).After 96h,the infection rate was observed by fluorescence microscopy.Infecting 5d,randomly selected 2 wells from each group to incubate with rat leptin for 6 h.5.The expression of SOCS-3,ACC and ACO mRNAPCR reaction conditions of annealing,SOCS-3 and GAPDH:56℃,ACC and ACO:50℃.GAPDH was used as internal.6.Statistical analysisData were expressed as mean±standard deviation.Statistical analysis was performed by Student's t test or one-way ANOVA using SPSS 13.0 statistical software. P＜0.05 is considered as significant.Results1.The body weight changes Before feeding high fat diet,there were no abnormal situation in each group(P＞0.05),but after 1 week,the body weight began to be differentiation,and the obesity group has been always higher than those of the control group,at the end of 8 weeks,compared with the control group,obesity rat body weight and weight increment had statistically significant differences(P＜0.05).2.The serum lipids and SOCS-3 mRNA expression in adipose tissueAt the end of 8 weeks,compared with control group,both of the serum total cholesterol and triglyceride levels of the obesity group were significantly higher(P＜0.05),and the same to the expression of SOCS-3 mRNA(P＜0.05).3.The appearance of r rat adipose stromal cells and differentiationThere appeared to be round or approximation round of new incubate cells.With the increase of culture time,cells become elongated,showing a typical fibroblast-shaped,growing better with the potential of multi-differentiation.After induced by adipogenic differentiation agent,the cells became round,with volume increased,formed lots of lipid droplets intracellular.Lipid droplets can be stained red by oil red O.Counts of differentiation rate was about 70%.4.Rat adipocytes infected by lentivirus-RNA-interference systemAfter 72h infection,there were no fluorescents in CON group,but the expression of green fluorescence in both of NC group and KD group,which could be observed by fluorescence microscopy,the infection rate was about 80%.5.SOCS-3 mRNA level after infectionSOCS-3 mRNA expression levels,the rat adipocytes infected by SOCS-3 shRNA lentivirus(KD group) were higher than that infected by negative control lentivirus (NC group)(P＜0.05).The effective SOCS-3-knockdown rate was about 54%.6.ACC and ACO mRNAAfter SOCS-3-knockdown adipocytes incubated with rat recombinant leptin,the expression of ACC mRNA was much lower in KD cells than that in NC cells(P＜0.05), and the expression of ACO mRNA in KD cells was increased as compared to NC cells(P＜0.05).Conclusions1.There may be existence of some leptin signal transduction pathways inhibition in diet-induce obesity rats.2.Our results suggested that after SOCS-3-knockdown,the inhibition of SOCS-3,an excessive negative regulator of leptin signal in DIO adipocytes,at least partly relieved.