Transcriptional regulation of human thyroid hormone receptor β1 gene expression: effect of human retinoid X receptor and identification of a transcriptional silencer region

Effects of human retinoid X receptor (hRXR) and its ligand, 9-cis-retinoic acid, on T₃-mediated auto-regulation of hTRβ1 gene expression were examined using a chloramphenicol acetyltransferase (CAT) reporter system, and a deletional analysis of the promoter. hRXR enhanced T₃-dependent CAT induction mediated through the proximal (p) TRE in a ligand (9-cis-retinoic acid) independent manner. In a gel mobility shift assay, hRXR enhanced the binding of hTRβ1 to the pTRE by the formation of hRXR-hTRβ1 heterodimers. On the other hand, hRXR and 9-cis-retinoic acid did not show any effects on T₃-dependent CAT induction mediated through the distal (d) TRE or the binding of hTRβ1 to the dTRE. A four hundred-base pair (bp) fragment adjacent upstream of the dTRE showed a T₃ independent suppresser effect on the function of the pTRE and dTRE. Thus, this region may be an important regulator of the T₃ dependent up-regulation of the TRβ1 gene expression which is observed only under specific conditions.     

hPOT1 RNA干扰对人胃癌BGC823细胞TRF1、TRF2和Tankyrase1表达的影响

目的 观察人端粒保护蛋白(hPOT1)RNA干扰对胃癌BGC823细胞端粒重复序列结合因子1(TRF1)、端粒重复序列结合因子2(TRF2)、端锚聚合酶(Tankyrase1)转录水平的影响.方法 采用半定量RT-PCR方法检测TRF1、TRF2和Tankyrase1在mRNA表达水平的改变.结果 hPOT1RNA干扰抑制细胞中hPOT1表达后,TRF1表达明显上调,TRF2和Tankyrase1明显下调.结论 人胃癌BGC823细胞中hPOT1表达下调伴随TRF1表达明显上调、TRF2和Tankyrase1表达明显下调,提示hPOT1和TRF1、TRP2、Tankyrase1三个端粒相关蛋白之间具有较为密切的联系,并且相互制约、相互影响.     

内皮素和心钠素调节TRH受体基因的表达

目的研究神经肽内皮素（ＥＴ）和心钠素（ＡＮＰ）在细胞培养及体内实验中对促甲状腺激素释放激素受体（ＴＲＨＲ）基因表达的影响。方法Ｎｏｒｔｈｅｒｎ印迹和Ｓ１核酸酶保护实验检测ＴＲＨＲｍＲＮＡ量，新生肽链试验测定ＴＲＨＲ基因的转录。结果ＥＴ３２ｈ即增加胶质细胞ＴＲＨＲｍＲＮＡ量，６～８ｈ达高峰，增加３倍以上（３．０±０．５２）；相反，ＡＮＰ２ｈ即降低ＴＲＨＲｍＲＮＡ，６～８ｈ达最低值，并逆转６０～８０％因血浆所增高的ＴＲＨＲｍＲＮＡ。ＥＴ３在６ｈ时增加ＴＲＨＲ基因的转录达２倍（ｎｕｃｌｅａｒｒｕｎｏｎ试验），而ＡＮＰ对ＴＲＨＲ基因的转录无明显变化。体内实验结果显示ＥＴ３和ＡＮＰ对ＴＲＨＲｍＲＮＡ影响与体外实验相似。结论ＥＴ３可能通过调节ＴＲＨＲ基因的表达而影响ＴＲＨＲｍＲＮＡ，而ＡＮＰ则可能影响ＴＲＨＲｍＲＮＡ的降解而降低ｍＲＮＡ的水平。     

RNA干扰真核表达载体对前列腺癌细胞CXCR4基因表达的抑制作用

目的 研究RNA干扰(RNAi)真核表达载体对前列腺癌细胞趋化因子CXC受体4(CXCR4)基因的抑制作用.方法 构建针对CXCR4基因的RNA干扰质粒表达载体,采用脂质体法转染前列腺癌PC-3m、LNCaP细胞系,半定量逆转录聚合酶链反应(RT-PCR)、Western blot法检测其对CXCR4 mRNA及蛋白表达的影响.结果 构建的重组真核表达载体在前列腺癌PC-3m、LNCaP细胞系中均抑制了CXCR4 mRNA及蛋白表达.与空白载体组细胞作对照,PC-3m细胞中小干扰RNA(siRNA)对CXCR4 mRNA的抑制率24、48 h分别为(87.8±10.2)%、(56.1±9.3)%,LNCaP细胞中siRNA对CXCR4 mRNA的抑制率分别为(56.9±8.8)%、(49.2±11.2)%,siRNA对PC-3m和LNCaP细胞蛋白抑制率,于24 h,前者为(64.7±6.7)%,后者为(58.7±11.6)%,与阴性载体转染组细胞比较,两组差异有统计学意义(P＜0.05).结论 RNA干扰真核表达载体可以有效地抑制两种前列腺癌细胞PC-3m、LNCaP细胞中CXCR4基因的表达.     

RNA干扰在白血病基因治疗中的应用前景

RNA干扰(RNAi)作为一种进化保守的转录后基因沉默机制,是指双链RNA分子(dsRNA)被切割成21～23个核苷酸的小干扰RNA(siRNA),最终使其同源的mRNA特异性降解.RNAi现广泛用于多个生物体的功能基因组学研究,以及在基因表达异常导致的多种疾病的临床前模型中进行干预治疗.大约50%的白血病伴有异常基因的表达,利用RNAi肿瘤特异性基因靶向治疗为治疗白血病提供了一条新途径.随着siRNA在活体中的稳定性和转染效率的提高、非特异性作用的减少,正迅速应用于临床中.     

Regulation of gonadotropin receptor gene expression

The receptors for the gonadotropins differ from the other G protein-coupled receptors by having a large extracellular hormone-binding domain, encoded by nine or ten exons. Alternative splicing of the large pre-mRNA of approximately 100 kb can result in mRNA species that encode truncated receptor proteins. In this review we discuss the regulation of gonadotropin receptor mRNA expression and the possible roles of alternative splicing in gonadotropin receptor function.     

脂联素小干扰RNA(siRNA)对3T3-L1脂肪细胞代谢相关基因的影响

采用RNA干扰技术抑制3T3-L1脂肪细胞脂联素基因表达,干预后细胞的葡萄糖转运蛋白(GLUT)-1、GLUT-4、胰岛素受体底物1和激素敏感脂肪酶mRNA表达水平分别下调53%、26%、27%和38%(P＜0.05或P＜0.01).     

不同碘负荷对小鼠甲状腺钠碘转运体基因表达的影响

目的 旨在探讨不同碘负荷状态下钠碘转运体(NIS)基因表达的变化及其在甲状腺自身调节中的作用.方法 取断乳1月龄的Babl/c小鼠按碘摄入量不同分为低碘绀、正常碘组、5倍碘组、10倍碘组和50倍碘组,饲养3个月、6个月后处死动物.采用实时荧光定量PCR和免疫组化检测NIS mRNA和蛋白表达水平,采用过硫酸铵消化砷-铈催化分光光度法测定甲状腺组织碘含量,采用竞争结合放射免疫分析方法检测甲状腺组织激素水平.结果 与正常碘组比较.各月龄低碘组小鼠NIS mRNA和蛋白表达显著上调,NIS主要定位于细胞膜.具有运碘功能,甲状腺摄碘功能增强,但长期严重碘缺乏最终导致甲状腺组织中碘含量和甲状腺组织激素水平明显降低;各高碘组NIS mRNA和蛋白表达有所下降,呈现随碘摄入量增加表达逐渐减低的趋势,且NIS主要分布于胞浆内,不具备跨膜转运碘的能力.甲状腺摄碘能力显著下降,甲状腺组织中碘含量虽有所升高,但小与碘摄人成平行关系.结论 不同碘负荷状态下 NIS基因表达的调控可发生在转录、翻译和翻译后水平,NIS基因表达及其活性的调节是机体适应低碘、耐受高碘的一种重要保护机制,是甲状腺自身调节的关键所在.     

应用干扰RNA体外抑制严重急性呼吸综合征冠状病毒N基因的表达

目的构建绿色荧光蛋白（GFP）与严重急性呼吸综合征（SARS）冠状病毒N蛋白的融合表达载体pEGFP-C1-N以及针对SARS冠状病毒N基因的小发卡型shRNA表达载体pshRNAN，观察shRNA对SARS冠状病毒N基因复制和表达的影响。方法将构建成功的pEGFP-C1-N与psh RNA-N共转染293细胞，于转染后24、48、72h观察GFP表达的强弱，采用Western Blot分别检测GFP与N蛋白的表达，逆转录PCR检测N基因的mRNA。结果pshRNA—N作用组绿色荧光强度明显弱于未干扰组，Western Blot和逆转录PCR结果也证实pshRNA—N对N基因和N蛋白的表达有明显抑制作用，而无关序列的shRNA无此作用。结论针对SARS冠状病毒N基因的shRNA具有显著和特异的抑制N蛋白表达的作用。     

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.     

RNA干扰抗乙型肝炎病毒治疗新进展

RNA干扰（RNA interferencing,RNAi）是短双链RNA,即小干扰RNA（small interferencing RNA,siRNA）介导的对同源基因转录后水平或者翻译水平的抑制作用.近年来应用RNAi技术治疗病毒感染性疾病的实验研究取得了很大的进展.RNAi作为一种新型的抗乙型肝炎病毒（HBV）基因治疗技术受到广泛关注,其高效、特异的抗病毒作用,设计灵活的多靶位效应使其成为一种前景广阔的基因治疗手段.     

短发夹状双链RNA抑制丙型肝炎病毒IRES介导的基因表达

目的研究载体表达的短发夹状双链RNA（shRNA）对丙型肝炎病毒（HCV）IRES介导的基因表达的特异性抑制作用。方法构建HCV IRES调控的绿色荧光蛋白表达载体（pIRES—GFP）和虫荧光索酶表达载体（p5′ UTR—Luc），以及针对HCV IRES的shRNA表达载体（pshRNA-HCV）。共转染HepG2细胞，于转染后24、48、72h观察绿色荧光的强弱，用Western blot检测绿色荧光蛋白的表达，半定量逆转录聚合酶链反应法检测GFP的mRNA水平。双荧光索酶系统检测虫荧光索酶活性。结果pshRNA-HCV作用组绿色荧光强度明显弱于未干扰组，GFP蛋白表达量及虫荧光素酶活性降低60％～70％，半定量逆转录聚合酶链反应显示pshRNA-HCV导致了GFP基因mRNA水平的降低。结论针对HCV IRES的shRNA能够显著和特异地抑制该区域调控的蛋白表达水平及mRNA水平，该研究结果为利用RNA干扰技术治疗HCV感染进行了初步探索。     

Thyroid hormone and glucocorticoid regulation of receptor and target gene mRNAs in pituitary GH3 cells

We have examined the effects of triiodothyronine (T₃), in dose-response and time-course studies, on T₃ receptor (T₃R) and β and glucocorticoid receptor (GR) mRNAs in rate pituitary GH₃ cells, in parallel with T₃ actions on expression of the growth hormone (GH) target gene. Modulatory influences of dexamethasone (dex) on T₃ action were studied by treatment with dex before and during T₃ treatment. T₃ treatment (1–100 nM) for 24 h reduced T₃R mRNA, while the presence of dex (1 μM) enhanced the T₃ effect on T₃R mRNA and induced T₃ inhibition of T₃R β mRNA. Stimulatory effects of T₃ treatment on GH mRNA and release were seen in the face of inhibition of T₃R mRNAs; these effects on GH were also enhanced by the presence of dex. T₃ treatment for 24 h increased GR mRNA; this effect was inhibited by the presence of dex. We next examined the influence of dex on GR and T₃R and β mRNAs, in parallel with effects of dex on the prolactin (PRL) target gene. Modulatory influences of T₃ on dex action were studied by treatment of cells with T₃ before and during dex treatment. Treatment with dex (0.1–10 μM) for 24 h reduced GR mRNA, an action enhanced by the presence of T₃ (100 nM). Dex treatment resulted in inhibition of PRL mRNA and release despite parallel inhibition of GR mRNA by dex; these effects were enhanced by the presence of T₃. In contrast to actions on GR, dex has no effect on T₃R mRNAs. These effects of T₃ and dex on receptor mRNAs suggest that glucocorticoid modulation of T₃ action is not related to direct actions on T₃R synthesis. In contrast, the mechanism of T₃ modulation of glucocorticoid action may be due in part to alteration of GR mRNA expression. Effects of T₃ and dex on target gene expression were observed in the presence of parallel reduction of their respective receptor mRNAs. This provides new evidence that interactions between these hormones are likely to be mediated by mechanisms other than regulation of receptor gene expression.