FAM213B基因启动子在猪子宫内膜细胞的转录调控

Regulation of the Promoter of FAM213B Gene in Porcine Endometrial Cells

【目的】分析猪FAM213B基因启动子转录活性区域,并检测转录因子NFκB对启动子活性及猪FAM213B基因表达影响,为深入了解FAM213B基因的转录调控机制影响前列腺素合成、母猪妊娠等繁殖活动奠定基础。【方法】采集卵泡期子宫,分离子宫内膜上皮组织,经胶原酶方法获得猪原代子宫内膜细胞,用于猪FAM213B基因启动子活性检测。参照笔者所在课题组前期研究获得的猪FAM213B基因m RNA全序列(Gen Bank登录号:KX444503)以及5￠调控区序列(Gen Bank登录号:100134955),通过PCR扩增猪FAM213B基因较长启动子序列,并进行测序鉴定;在此基础上,PCR扩增带有Mlu I和Xho I限制性酶切位点的FAM213B基因启动子7个5'端缺失片段,并通过双荧光素酶报告基因载体系统构建猪FAM213B基因启动子7个不同的5'端缺失载体。将7个载体经无内毒素处理后与p RL-TK质粒用阳离子脂质体法一起转染猪子宫内膜细胞,用双荧光素酶报告基因系统进行Luciferase活性检测,比较各启动子片段转录活性。生物信息学分析FAM213B基因启动子潜在转录因子结合位点,通过Ch IP试验验证FAM213B基因启动子与潜在转录因子NFκB的相互作用。构建NFκB1和Rel A的超表达载体,化学合成NFκB1和Rel A的干扰si RNA片段,分别转染猪子宫内膜细胞,通过双荧光素酶报告基因系统进行Luciferase活性检测和荧光定量PCR分别检测超表达和干扰表达NFκB1和Rel A对猪FAM213B基因启动子活性和m RNA表达影响。【结果】PCR和测序获得猪FAM213B基因启动子长度为2 261bp(-2178/+83)。生物信息学预测结果表明猪FAM213B基因启动子区存在潜在的CREB、CCAAT增强子结合蛋白、E-box因子的结合位点,炎性因子NFκB潜在结合位点分别位于-1143/-1132和-664/-655区间。启动子报告基因活性检测结果表明:重组载体P2(-1352/+30)荧光活性最高,极显著高于P1(-1 760/+30)(P<0.01),在-1 760/-1 352区域存在负调控元件;而P2显著高于P3(-919/+30)(P<0.05),表明在-1352/-919区域存在正调控元件;P3(-919/+30)活性极显著高于P4(-604/+80)(P<0.01),表明在-919/-604区域存在正调控元件;P4、P5、P6和P7活性差异不显著,-1352/-919区域为核心启动子区,-1352/-604对于维持该启动子较高转录活性起着重要的作用。Ch IP结果表明启动子区-1143/-1132存在NFκB1结合位点,-664/-655存在Rel A结合位点。超表达载体pc DNA3.1-NFκB1与P2(-1352/+30)启动子片段重组载体共转染猪子宫内膜细胞后,P2启动子活性极显著高于对照组(P<0.01),pc DNA3.1-NFκB1载体单独转染猪子宫内膜细胞后FAM213B基因m RNA表达量显著高于对照组(P<0.05);超表达载体pc DNA3.1-Rel A与P3(-919/+30)启动子片段重组体共转染猪子宫内膜细胞,P3启动子活性极显著低于对照组(P<0.05),pc DNA3.1-Rel A载体单独转染猪子宫内膜细胞后,FAM213B基因的mRNA表达量显著低于对照组。转染NFκB1和Rel A的si RNA片段干扰片段,FAM213B基因启动子活性和m RNA表达量则表现与超表达相反的结果。【结论】获得了猪FAM213B基因启动子核心启动子序列为-1352/-919区域,NFκB是FAM213B基因启动子的转录因子,NFκB成员NFκB1和Rel A在猪子宫内膜细胞中对FAM213B基因的表达起调控作用。

【Objective】To interpret partially the role of FAM213 B gene expression in prostaglandin synthetise and sow pregnancy through the identification of the transcription region of porcine FAM213 B gene promoter and the effection of NFκB on the promoter. 【Method】 The porcine endometrium from the uterus in follicular phase was digested by collagenase and the isolated endometrial cells were cultured for the detection of the FAM213 B promoter activity. Based on the m RNA and promoter sequences of FAM213 B gene obtained in our former work（Gen Bank ID： KX444503 and 100134955）, the longer 5′ regulationary sequence was amplified and sequenced. Then, seven promoter fragments with 5′ terminal deletion, containing Mlu I and Xho I sites, were linked into the Dual-luciferase Reporter vectors. Seven constructed vectors treated by endotoxin free and p RL-TK plasmid were co-transfected into endometrial cells through liposome method. The core region of transcriptional activity of the gene promoter was identified through the Dual-luciferase Reporter Assay System. The putative transcription factors binding sites were analyzed by bioinformatics, and the binding of NFκB with FAM213 B promoter were detected by Ch IP（Chromatin immunoprecipitation）. The over-expression vectors of NFκB1 and Rel A and interference fragments of their own were transfected into endometrial cells. Then, the transcription activity of the promoter and m RNA expression of the gene were detected by the Dual-luciferase Reporter system and fluorescent quantitative, respectively. 【Result】 Through the PCR and sequencing, one fragment of 2 261 bp（-2178/＋83） of porcine FAM213 B gene were obtained. The bioinformatics analysis showed that there were putative binding sites of CREB, CCAAT, E-box, and NFκB in the promoter. And the putative binding sites of NFκB were found in-1143/-1132 and-664/-655 regions. The result of the Dual-luciferase Reporter showed the region of P2（-1352/＋30） exhibited the strongest transcriptional activity, and it was significantly higher than that of P1（-1760/＋30）（P〈0.01）, which showed there were negative regulation elements in-1760/-1352 region. And the transcriptional activity of P2（-1352/＋30） was significantly higher than that of P3（-919/＋30）（P〈0.05）, implying the existence of positive elements in-1352/-919 region. The significant stronger activity of P3（-919/＋30） than P4（-604/＋80）（P〈0.01） meant the existence of positive elements in-919/-604 region. No significant differences were observed between P4, P5, P6, and P7. The region of-1352/-919 was the core element of the promoter. The results of Chromatin Immunoprecipitation（Ch IP） demonstrated that NFκB1 binds to one site around-1143/-1132, and Rel A site around-664/-655. The co-transfection of the over expression of pc DNA3.1-NFκB1 and P2 vector into endometrial cells increased the activity of the promoter（P〈0.01）, and the transfection of pc DNA3.1-NFκB1 enhanced the m RNA expression of FAM213 B（P〈0.05）. While the co-transfection of the over expression of pc DNA3.1-Rel A and P3 vector into endometrial cells decreased the activity of the promoter（P〈0.05）, and the transfection of pc DNA3.1-Rel A weakened the m RNA expression of the gene. At the same time, the transfection of inference si RNA fragments of NFκB1 and Rel A into endometrial cells led the contrary results for the activity of the promoter and the m RNA expression of the gene.【Conclusion】The core region of porcine FAM213 B gene promoter was identified round-1352/-919. NFκB was the trancription factor of FAM213 B gene. NFκB1 and Rel A, two members of NFκB family, regulate the expression of FAM213 B gene in endometrial cells.