The Effects Of SREBP-1c Gene Silencing On Fat Deposit In Calf Hepatocytes Cultured In Vitro

The fatty liver is a common nutritional and metabolic diseasethat is characterized by negative energy balance and fat deposit in liver of dairy intransition period, and it often occurred in high yield cows. Sterol regulatory elementbinding protein-1c (SREBP-1c) is an important lipogenic gene transcription factor,also known as adipocyte determination and differentiation factor1. SREBP-1c, whichis mainly found in liver and fat cells, is an important nuclear transcription factor, canbe related to fat metabolism by regulating the expression of fat synthesis genes inanimal. Recent researches have showed that synthesis of lipid can be regulated bytranscript factor family-Sterol regulatory element binding proteins.REBP-1is aisomer of SREBPs，it is an important gene for synthesis of fatty acid，and it is affinityto formation of fatty liver. Excessive expression of SREBP-1c will lead to disorder ofglucolipid metabolism，and then cause accumulation of non-adipose tissue such asliver and pancreas,it is an important step for insulin resistance（IR）, visceral obesityand fat ectopic deposition.Since，we infer that reducing the expression of SREBP-1ccan decrease the fat deposition in liver. RNA interference (RNAi) is induced bydouble-stranded RNA and results in specific degradation of double-stranded mRNA incell, and it belongs to post-transcriptional gene silencing mechanism. RNAi has beenextensively used as a novel and effective tool for functional genomic studies, and hasdisplayed great potential in treating human and animal diseases.In this experiment, we used RNAi to study the effect of SREBP-1c on to fatdeposition in hepatocytes in vitro. First we screened four interfere with target ofSREBP-1c, and then selected a relevant promoter to make up plasmid of shRNA, atlast shRNA was transfected into calf hepatocytes through Lipofectamine-2000,applying real-time PCR to select a suitable fragment which can maximatily reduce theexpression of SREBP-1c. After enzyme digestion with the useful plamid, we linked itto adenovirus vector to construct Ad-GFP-SREBP-1C,through transfect Ad-GFP-SREBP-1C to293cells to obtain adenovirus, infect it again to augment theamount of adenovirus, at last titre of adenovirus reach to1010PFU/ml.After the cellsinfected with adenovirus, applying real-time PCR, western blot, ELISA, biochemicalkits, enzyme activity kit and so on detected enzyme and biochemical indicator relatedto fatty acid metabolism. The result showed that the expression of SREBP-1c wassuppressed by Ad-GFP-SREBP-1C，resulting in up-regulating the mRNA expression offat acid synthesis genes and enzyme activities, down-regulating the mRNA expressionof fat acid oxidation genes and enzyme activities, down-regulating the mRNAexpression of VLDL assembly factors such as ApoB, ApoE and MTP, and decreasingthe concentration of VLDL and TG in hepatocyes. It is concluded that suppressingSREBP-1c gene could strengthen the ability of fatty acid metabolism and reduce TGconcentation in hepatocytes through up-regulating the mRNA expression of fat acidsynthesis genes and enzyme activities, and down-regulating the mRNA expression offat acid oxidation genes and enzyme activities. However, suppressing SREBP-1c genealso could decrease VLDL assembly in hepatocytes.The results lay on the base of prevention and cure for cows’fatty liver with genetreatment.