固醇调节元件结合蛋白1c激活大鼠Patatin样磷酯酶结构域蛋白3基因转录

Sterol regulatory element-binding protein-lc activates patatin like phospholipase domain containing 3 gene transcription in rats

目的探讨固醇调节元件结合蛋白1c（SREBP-1e）对Patatin样磷酯酶结构域蛋白3（PNPLA3）基因的转录调控作用。方法构建7周龄雄性体重匹配的禁食（24h）以及禁食后再喂食（48h）SD大鼠（自由饮食组3只，饥饿组3只，再喂食组4只）和高脂及小剂量链脲佐菌素诱导的2型糖尿病sD大鼠（正常对照组5只，2型糖尿病组6只）。用逆转录聚合酶链反应（RT—PCR）和Western blotting法检测各组大鼠肝脏组织中SREBP一1c和PAPM3的表达水平。将大鼠PⅣP翻3启动子5’端上游-1000bp序列分成3段，分别构建荧光素酶报告载体（R—PⅣPM3．1、R—PM似3—2、R—PNPL43—3），转染人正常肝细胞株L02，比较3个载体基础荧光素酶活性以及SREBP-1e过表达诱导的荧光素酶活性。分析上述实验中荧光素酶活性最高的PNPLA3启动子片段可能的SREBP-1c结合位点（SRE），分别构建野生型和SRE突变型报告载体，比较两个载体荧光素酶活性。多组定量资料比较用方差分析，两组定量资料比较用t检验。结果与自由饮食组相比，饥饿组大鼠肝脏SREBP—lc、PNPLA3和脂肪酸合成酶FAS基因表达均下降，再喂食组三者表达显著升高，差异均有统计学意义（F=114．14，334．11，754．20，均P〈0．05）。与正常对照组相比，2型糖尿病组SREBP-1e、PNPLA3基因（t=-18．39，-30．07，均P〈0．05）及蛋白表达（t=4．58，6．81，均P〈0．05）均显著增高。R—PNPLA3-1报告载体基础荧光素酶活性较对照升高51．13倍（t=-28．93，P〈0．05），R一删PM3—2和R—PNPLA3—3无基础荧光素酶活性；在L02细胞中，转染SREBP-1c表达质粒的R—PⅣPL43—1组荧光比值较转染空质粒的组升高2．63倍（t=-7．64，P〈0．05），而R—PⅣPM3．2组及R—PNPLA3—3组转染SREBP-1e表达质粒荧光比值较转染空质粒组均无变化；PNPLA3启动子-100～-911）p存在SRE，SRE突变的报告载体（MUT—R—PNPLA３—1）荧光比值较野生型（R—PNPLA３-1）降低40．80％（t=4．99，P〈0．05）。结论SREBP-1c通过PNPLA３基因启动子－100～－91bp激活大鼠PNPLA3基因转录。

Objective To explore the effect of sterol regulatory element-binding protein-lc （SREBP-lc） on the transcription of patatin like phospholipase domain containing 3 （PNPLA3） in rat. Methods Seven-week male weight matching Sprague-Dawley rats were assigned to fed ad libitum group （ n = 3 ） , fasting group （ n = 3, fasting for 24 h ）, refeeding group （ n = 4, fasting for 24 h and refeeding 48 h） and normal control group（ n = 5 ）, high-fat-diet and streptozotocin-induced diabetic group（ n = 6）. Reverse transcription polymerase chain reaction （RT-PCR） and Western blotting were performed to determine the expression of SREBP-1 c and PNPLA3 in rats＇ liver. Three nested deletions of the 5＇ end of the 1.0 kb PNPLA3 promoter were generated and introduced into the luciferase reporter gene （R-PNPLA3-1, R-PNPLA3-2, R-PNPLA3-3 ）, and then transfeeted into human normal hepatic cell line LO2 respectively to compare the basal and post-stimulate luciferase activity after transfected with SREBP-lc plasmid. The PNPLA3 promoter which was shown the highest luciferase activity might contain a putative SREBP-1 c binding sites （SRE）. Then wild-type and mutant SRE reporter vectors were constructed to compare their luciferase activity. Statistical analysis was performed with one-way ANOVA of variance, followed by the Student＇s t test. Results Compared with the fed ad libitum group, the gene expression of SREBP-lc, PNPLA3 and fatty acid synthase （FAS） in liver decreased significantly in fasting group, and recovered in refeeding group （ F = 114. 14, 334. 11, 754. 20, all P 〈 0. 05 ）. Compared with normal control group, the gene and protein expression of SREBP-1 c and PNPLA3 increased markedly in diabetic group （t = - 18.39, -30. 07 and 4. 58, 6.81, all P 〈0. 05）. The basal luciferase activity of R-PNPLA3-1 increased for 51.13 folds when compared with that in the control group （ t = - 28.93, P 〈 O. 05 ）, while no notable changes was found with R-PNPLA3- 2 and R-PNPLA3-3; transfection of SREBP-lc plasmid into L02 cells caused 2. 63 folds activation of R- PNPLAJ-1 （t = -7.64, P 〈0. 01 ） , while no changes in R-PNPLA3-2 and R-PNPLA3-3 groups was found. A putative SRE located in - 97/- 88 bp of rat PNPLA3 genc promoter, the luciferase activity of mutant SRE reporter vectors （MUT-R-PNPLA3-1） decreased for 40. 8 % when compared with that in wild-type （R-PNPLA3- 1 ） （ t = 4. 99, P 〈 0. 05 ） . Conclusion SREBP-1 c trans-activates rat PNPLA3 via a proximal SRE （ - 100/-91 bp） of PNPLA3 promoter.