Silencing of DLGAP5 by siRNA Significantly Inhibits the Proliferation and Invasion of Hepatocellular Carcinoma Cells

Background

The dysregulation of oncogenes and tumor suppressor genes plays an important role in many cancers, including hepatocellular carcinoma (HCC), which is one of the most common cancers in the world. In a previous microarray experiment, we found that DLGAP5 is overexpressed in HCCs. However, whether the up-regulation of DLGAP5 contributes to hepatocarcinogenesis remains unclear.

Methodology/Principal Findings

In this study, we showed that DLGAP5 was significantly up-regulated in 76.4% (168 of 220) of the analyzed HCC specimens when compared with adjacent liver tissue. DLGAP5 overexpression was evident in 25% (22 of 88) of the HCC specimens without AFP expression, suggesting that DLGAP5 may be a novel biomarker for HCC pathogenesis. The silencing of DLGAP5 gene expression by RNA interference significantly suppressed cell growth, migration and colony formation in vitro. The expression level of DLGAP5 was also found to be related to the methylation level of its promoter in the HCC specimens.

Conclusions/Significance

Taken together, these data suggest that the expression of DLGAP5 is regulated by methylation and that the up-regulation of DLGAP5 contributes to HCC tumorigenesis by promoting cell proliferation.     

Wnt5a抑制Wnt3a促黑素细胞黑素生成的作用探究

目的：研究WntSa对Wnt3a处理过的melan—a细胞分泌黑色素的影响。方法：体外培养黑色素细胞（melan-a细胞）,分别进行GFP、Wnt3a、Wnt3a＋WntSa处理,比较细胞的突起,酪氨酸酶的活性以及黑素合成相关基因（TYR、TRP2、MITF）表达情况。结果：Wnt3a促进黑色素细胞突起的生长和TYR、TRP2、MITF的表达,而Wnt5a逆转了Wnt3a对黑色素细胞的作用。结论：Wnt5a抑制Wnt3a促黑素细胞黑素生成的作用,表明在melan．a黑素细胞中Wnt5a可有效抑制wnt经典通路。     

siRNA沉默Myh3基因抑制C2C12细胞糖酵解

肌球蛋白重链3(myosin heavy chain 3,Myh3)基因为肌肉细胞分化的标志基因,调节肌肉细胞能量的利用,但其是否会影响肌肉细胞不同状态下的糖酵解过程尚鲜有报道。本文以成肌和成脂分化不同阶段的小鼠C2C12细胞为模型,利用qRT-PCR方法研究Myh3与糖酵解相关基因Pkm(M-type pyruvate kinse)、Prkag3(protein kinase adenosine monophosphate-activated γ3-subunit)和Gsk3β(glycogen synthase kinase-3β)的表达模式。发现在C2C12细胞成肌分化过程中,Myh3与糖酵解基因Prkag3和Pkm的相对表达趋势基本一致,都呈现相对表达水平先上升,分化第2 d达到峰值,之后下降的趋势;糖原合酶抑制基因Gsk3β的表达趋势相对平稳。而在C2C12细胞成脂分化过程中,Myh3依然与糖酵解基因Prkag3和Pkm的相对表达趋势基本一致,相对表达量逐渐上升,在分化第8 d达到最高值;糖原合酶抑制基因Gsk3β的表达保持稳定状态。在C2C12细胞成肌分化状态下,qRT-PCR和Western 印迹检测干扰Myh3对细胞糖酵解相关基因Pkm、Prkag3和Gsk3β mRNA和蛋白质表达的影响。结果显示,干扰Myh3后,糖酵解基因Pkm和Prkag3的mRNA表达量极显著降低(P<0.01),糖原合酶抑制基因Gsk3β的mRNA表达无明显变化(P>0.05);Myh3干扰组中Myh3和Pkm的蛋白质水平显著低于空白组和NC组细胞。在C2C12细胞成脂分化状态下,干扰Myh3,糖原合酶抑制基因Gsk3β和糖酵解基因Prkag3的mRNA表达量极显著升高(P<0.01),糖酵解基因Pkm的mRNA表达下降;Myh3干扰组中Myh3和Pkm的蛋白质水平也低于空白组和NC组细胞。综合以上研究,C2C12细胞成肌和成脂状态下糖酵解水平存在明显差异,Myh3与酵解基因的表达模式相似,进一步研究发现,干扰Myh3可以抑制C2C12细胞成肌状态下的糖酵解,不影响糖原合成。与成肌状态不同,在C2C12细胞成脂状态下干扰Myh3,抑制了糖原合成和糖酵解。     

Silencing of MDR 1 gene in cancer cells by siRNA

Inhibition of p-glycoprotein (PGP) expression and reverse of multidrug resistance (MDR) phenotype in KB-8-5 cells by synthetic 21-bp double-stranded oligoribonucleotides were investigated. siRNA constructs for the efficient down regulation of MDR1 that are active in nanomolar concentrations and cause reversal of MDR phenotype in cells were developed.     

An Ultrastructural Study of Melanocytes and Melanosomes in the Skin and Hair Bulbs of Rufous Albinos

We have examined hair bulb and skin melanocytes of rufous albinos from Southern Africa to further characterize this form of albinism. In the skin melanocytes we find both eumelanosomes and pheomelanosomes at various stages of melanization and, in addition, there appeared to be many aberrant incompletely melanized melanosomes. On average, rufous melanosomes are 30% smaller than normal black skin melanosomes. In the keratinocytes, the melanosomes are packaged into distinct aggregations, whereas in normal black skin, they occur singly. We suggest that the reddish skin color of these albinos is a consequence of an increase in the pheomelanin synthesis resulting in a raised pheomelanin/eumelanin ratio and that the aggregation of melanosomes results in a skin color slightly lighter than normal. In hair bulb melanocytes, only eumelanosomes were seen and these were mostly incompletely melanized. These findings correlate with our visual observations that the hair color of Southern African albinos is very pale (light brown or ginger). Based on our observations, we speculate on the possible cause of rufous albinism.     

siRNA与miRNA在生物基因调控中的沉默机制比较

siRNA和miRNA的沉默机制是生物基因调控的重要手段之一. 小干扰RNA（small interfering RNA,siRNA）是RNA干扰的引发物,激发与之互补的目标mRNA沉默. 非编码RNA中的微小RNA（microRNA,miRNA）,能够识别特定的目标mRNA,通过与mRNAs的3′ 非翻译区结合,影响该目标蛋白的翻译水平. siRNA和miRNA的基因调控机制对生物学研究及疾病的病因和治疗等有直接影响. 本文主要对siRNAs和miRNAs的生物起源及沉默机制进行比较性论述：提出Dicers酶蛋白、Ago蛋白以及20 nt~25 nt的双链RNAs的 3类大分子是RNA沉默的特征结构,并进行了说明性论述|总结性叙述了siRNA和miRNA的2类小分子经典沉默机制,并提出其异同点. 最后,本文根据近期研究进展,对siRNA和miRNA的生物起源及沉默机制提出了新的疑问.     

miR-411a-3p抑制羊驼黑色素细胞黑色素的生成

microRNA-411a-3p（miR-411a-3p）在不同毛色羊驼皮肤中差异表达,提示其可能在毛色形成中起调控作用。为证明miR-411a-3p在羊驼皮毛黑色素形成中的作用,本研究通过生物信息学预测及靶基因搜索,发现胰岛素样生长因子1受体（IGF1R）是miR-411a-3p的靶基因,继而构建了含Igf1r mRNA 3′-UTR的荧光素酶报告基因载体。报告基因转染结合报告酶活性测定证明,Igf1r是miR-411a-3p的靶基因。原位杂交显示,miR-411a-3p主要在羊驼黑色素细胞胞质中表达,提示其可能在黑色素细胞中具有重要作用。qRT-PCR揭示,棕色和黑色羊驼皮肤中miR-411a-3p表达量明显低于白色羊驼。qRT-PCR联合蛋白质印迹分析显示,在羊驼黑色素细胞过表达miR-411a-3p,伴随Igf1r下调,小眼畸形相关转录因子（Mitf）依赖的毛色基因酪氨酸酶（Tyr）、酪氨酸酶相关蛋白-2（Tyrp2）及黑色素表达水平明显下调。上述结果提示,miR-411a-3p可通过靶向抑制Igf1r负调控羊驼黑色素黑色素合成。该结果将加深我们对羊驼毛色形成机制的认识。     

siRNA沉默socs3对红系发育的影响

为了研究细胞因子信号转导分子3(suppressor of cytokine signals-3,SOCS-3)对造血发育的影响,构建了SOCS-3慢病毒siRNA干涉载体,并转染人红白血病细胞株K562.根据绿色荧光蛋白的表达进行流式分选后,获得了高表达慢病毒干涉载体的细胞.实时荧光定量PCR和Western-blot检测了转染细胞中SOCS-3基因的干涉效率,结果显示,与对照组相比,siRNA干涉后K562细胞SOCS-3基因的表达量仅为其相对表达量的22.1%,干涉效率77.9%;Western-blot结果显示,SOCS-3在蛋白质水平表达也明显受抑制.进一步对SOCS-3基因沉默后的K562细胞进行了诱导分化,并采用联苯胺染色法检测K562细胞向红系分化比例变化,免疫荧光染色检测细胞表面抗原的变化,RT-PCR检测造血相关基因的变化.结果发现,SOCS-3沉默后K562细胞向红系的发育能力显著提高.研究结果证明,SOCS-3在造血发育中有重要调控作用,而对其表达进行干涉或沉默将在规模化的红细胞诱导研究中发挥重要作用.     

Silencing of Bcl-XL Expression in Human MGC-803 Gastric Cancer Cells by siRNA

To investigate the inhibitory effect of the Bcl-XL small interfering RNA(siRNA)on BcI-XLgene expression in the human gastric cancer cell line MGC-803,green fluorescent protein(GFP)siRNAwas constructed and transfected into MGC-803 ceils,together with GFP expression vector pTrace SV40.GFP expression levels were observed using fluorescence microscopy.Bcl-XL siRNA and negative siRNAwere then constructed and stably transfected into MGC-803 cells.RT-PCR and immunofluorescence wereused to detect the expression of Bcl-XL.Spontaneous apoptosis was detected by acridine orange(AO)andflow cytometry.Results were as follows:(1)48 h after GFP expression vector and GFP siRNA co-transfection,the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group,according to fluorescence microscopy results.The mRNA and protein levels of Bcl-XL in Bcl-XL siRNAstable transfectants were reduced to almost background level compared with negative siRNA transfectantsor untreated cells.(2)Changes in nucleus morphology was observed by AO staining nucleic and flowcytometry analysis,which showed that stable Bcl-XL siRNA transfectants have an increased spontaneousapoptosis (21.17%+1.26% vs.1.19%+0.18% and 1.56%+0.15% respectively,P<0.05 vs.negative siRNAor untreated control),siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or BcI-XLexpression in MGC-803 cells,and Bcl-XL siRNA can increase spontaneous apoptosis.Bcl-XL siRNA maybe a beneficial agent against human gastric adenocarcinoma.     

Silencing of Bcl-XL expression in human MGC-803 gastric cancer cells by siRNA

To investigate the inhibitory effect of the Bcl-XL small interfering RNA （siRNA） on Bcl-XL gene expression in the human gastric cancer cell line MGC-803, green fluorescent protein （GFP） siRNA was constructed and transfected into MGC-803 cells, together with GFP expression vector pTrace SV40.GFP expression levels were observed using fluorescence microscopy. Bcl-XL siRNA and negative siRNA were then constructed and stably transfected into MGC-803 cells. RT-PCR and immunofluorescence were used to detect the expression of Bcl-XL. Spontaneous apoptosis was detected by acridine orange （AO） and flow cytometry. Results were as follows： （1） 48 h after GFP expression vector and GFP siRNA co-transfection,the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group,according to fluorescence microscopy results. The mRNA and protein levels of Bcl-XL in Bcl-XL siRNA stable transfectants were reduced to almost background level compared with negative siRNA transfectants or untreated cells. （2） Changes in nucleus morphology was observed by AO staining nucleic and flow cytometry analysis, which showed that stable Bcl-XL siRNA transfectants have an increased spontaneous apoptosis （21.17%± 1.26% vs. 1.19%±0.18% and 1.56%±0.15% respectively, P〈0.05 vs. negative siRNA or untreated control）, siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or Bcl-XL expression in MGC-803 cells, and Bcl-XL siRNA can increase spontaneous apoptosis. Bcl-XL siRNA may be a beneficial agent against human gastric adenocarcinoma.     

Silencing of sodium/hydrogen exchanger in the heart by direct injection of naked siRNA

Cardiac Na(+)/H(+) exchanger (NHE1) hyperactivity is a central factor in cardiac remodeling following hypertension, myocardial infarction, ischemia-reperfusion injury, and heart failure. Treatment of these pathologies by inhibiting NHE1 is challenging because specific drugs that have been beneficial in experimental models were associated with undesired side effects in clinical practice. In the present work, small interference RNA (siRNA) produced in vitro to specifically silence NHE1 (siRNA(NHE1)) was injected once in vivo into the apex of the left ventricular wall of mouse myocardium. After 48 h, left ventricular NHE1 protein expression was reduced in siRNA(NHE1)-injected mice compared with scrambled siRNA by 33.2 ± 3.4% (n = 5; P < 0.05). Similarly, NHE1 mRNA levels were reduced by 20 ± 2.0% (n = 4). At 72 h, siRNA(NHE1) spreading was evident from the decrease in NHE1 expression in three portions of the myocardium (apex, medium, base). NHE1 function was assessed based on maximal velocity of intracellular pH (pH(i)) recovery (dpH(i)/dt) after an ammonium prepulse-induced acidic load. Maximal dpH(i)/dt was reduced to 14% in siRNA(NHE1)-isolated left ventricular papillary muscles compared with scrambled siRNA. In conclusion, only one injection of naked siRNA(NHE1) successfully reduced NHE1 expression and activity in the left ventricle. As has been previously suggested, extensive NHE1 expression reduction may indicate myocardial spread of siRNA molecules from the injection site through gap junctions, providing a valid technique not only for further research into NHE1 function, but also for consideration as a potential therapeutic strategy.     

siRNA Silencing of Gene Expression in Trabecular Meshwork: RhoA siRNA Reduces IOP in Mice

Few reports described efficient transfection in the trabecular meshwork (TM) in vivo. In the present study, we investigated the distribution of cy3-labeled siRNAs after giving injection into the anterior chamber (AC) and explored the use of RhoA siRNA (siRhoA) to modulate intraocular pressure (IOP) through downregulation of RhoA gene and protein expression. Cy3-labeled siRNAs were injected into the AC to investigate the distribution. In addition, siRhoA was applied to normal and DEX-induced elevated IOP mice. The RhoA gene was detected at 1d post-injection (PI) using real-time RT-PCR. Proteins were examined using immunofluorescence staining at 1, 2, and 3 day PI. IOP was measured pre- and post-injection using a TONOPEN. Toxicity was preliminarily assessed using clinical observation and hematoxylin-eosin staining. The study demonstrated that cy3-labeled siRNAs accumulated in mouse TM in a dose-dependent manner, with a peak at 24h PI. There was no visible siRNA fluorescence in the corneal endothelium, and little in the iris. siRhoA caused large decreases in RhoA mRNA and protein expression in mouse TM (p < 0.01). In normal mice, injections of siRhoA induced decreases in IOP, by 2d, with recovery to baseline by 3d PI. For DEX-treated animals, IOP significantly decreased from 2d to 5d PI (p < 0.05). There was no obvious toxicity after the siRhoA application. These results suggest that (1) siRNA injection into the AC leads to transient gene transfection in TM; (2) inhibiting RhoA expression in TM with siRNA is effective in suppressing elevated IOP in mice, suggesting that siRhoA is a potential pharmaceutical intervention for glaucoma.     

Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA

Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2′-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.     

Identification of a cDNA Coding for a Ca-Binding Phosphoprotein (p90), Calnexin, on Melanosomes in Normal and Malignant Human Melanocytes

In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca²⁺-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in λ Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M_r 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with β-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca²⁺-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an anti-body against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.     

干扰OVA66基因表达对移植瘤生物学功能的影响

目的探讨干扰OVA66基因表达对移植瘤细胞生物学功能的影响。方法采用脂质体法,以重组的pSUPER-shRNA-OVA66和空载体转染HeLa细胞获得稳定表达细胞株,经RT-PCR和Western blot鉴定OVA66基因的表达。将两组细胞接种于BALB／cnu／nu裸鼠的前肢腋下,连续观察干扰OVA66表达对肿瘤的生长影响。并于接种4周后,应用RT-PCR,免疫组化、免疫荧光等方法检测移植瘤细胞中OVA66的蛋白表达。组织病理分析肿瘤细胞生长和转移的特征。结果经检测pSUPER-shRNA-OVA66能有效抑制目的基因的表达。比较两组荷瘤小鼠中肿瘤的生长曲线发现,OVA66实验组的肿瘤生长速度明显低于对照组,并且肿瘤细胞体内转移和浸润能力显著降低。结论干扰OVA66基因和蛋白表达,可以抑制肿瘤细胞的生长,浸润和侵袭能力。     

csrA Inhibits the Formation of Biofilms by Vibrio vulnificus

PCR screening of the shellfish-borne pathogen Vibrio vulnificus revealed csrA-negative strains, and these strains formed increased biofilm compared to csrA-positive strains. Complementation in trans with csrA resulted in reduced biofilm formation, similar to that by csrA⁺ strains. Our results provide evidence that csrA inhibits biofilm formation in V. vulnificus.     

miRNA-338-3p通过靶向抑制MC1R基因表达抑制羊驼黑色素细胞黑色素的生成

黑色素皮质素1受体MC1R）是在黑色素细胞内表达的G蛋白耦合受体（G protein coupled receptor, GPCR）家族成员,参与黑色素细胞中黑色素的生成。微RNAs（miRNAs）是一类非编码RNA,通过与靶基因3′-UTR结合抑制基因表达。已有研究证明,miR-338-3p 在多种人类肿瘤细胞中（过）表达,可通过下调靶基因表达抑制肿瘤细胞的侵袭迁移能力。然而,有关miR-338-3p对羊驼皮肤黑色素细胞的黑色素合成影响却罕见报道。本研究证明,miRNA-338-3p通过靶向抑制MC1R基因表达,抑制羊驼黑色素细胞黑色素的生成。采用生物信息学预测MC1R基因是miRNA-338-3p的靶基因,其基因表达抑制羊驼黑色素细胞黑色素合成。随后构建miR-338-3p真核表达载体。其基因转染结合qPT-PCR和Western印迹结果揭示,与对照细胞比较,过表达miRNA-338-3p的羊驼黑色素细胞的MC1R基因,及其下游与黑色素生成相关的小眼相关性转录因子（MITF)、酪氨酸酶(TYR)、酪氨酸酶相关蛋白1（TYRP1）、酪氨酸酶相关蛋白2（TYRP2）编码基因mRNA及蛋白质表达水平明显下调。酶联免疫吸附分析显示,过表达miRNA-338-3p的羊驼皮肤黑色素细胞的黑色素产量,较对照细胞显著下降（P＜0.01）。综上结果,miR-338-3p可通过抑制靶基因MC1R表达,下调其下游基因MITF、TYR、TYRP1和TYRP2基因的表达,从而抑制羊驼皮肤黑色素细胞黑色素的合成。miRNA-338-3p在羊驼生长发育过程中,是否参与调控体内皮肤黑色素细胞的黑色素生成尚待进一步研究。