Deficiency in the 15 kDa selenoprotein inhibits human colon cancer cell growth

Selenium is an essential micronutrient for humans and animals, and is thought to provide protection against some forms of cancer. These protective effects appear to be mediated, at least in part, through selenium-containing proteins (selenoproteins). Recent studies in a mouse colon cancer cell line have shown that the 15 kDa selenoprotein (Sep15) may also play a role in promoting colon cancer. The current study investigated whether the effects of reversing the cancer phenotype observed when Sep15 was removed in mouse colon cancer cells, were recapitulated in HCT116 and HT29 human colorectal carcinoma cells. Targeted down-regulation of Sep15 using RNAi technology in these human colon cancer cell lines resulted in similarly decreased growth under anchorage-dependent and anchorage-independent conditions. However, the magnitude of reduction in cell growth was much less than in the mouse colon cancer cell line investigated previously. Furthermore, changes in cell cycle distribution were observed, indicating a delayed release of Sep15 deficient cells from the G(0)/G(1) phase after synchronization. The potential mechanism by which human colon cancer cells lacking Sep15 revert their cancer phenotype will need to be explored further.     

Using drug-excipient interactions for siRNA delivery

SiRNA is the trigger of RNA interference, a mechanism discovered in the late 1990s. To release the therapeutic potential of this versatile but large and fragile molecule, excipients are used which either interact by electrostatic interaction, passively encapsulate siRNA or are covalently attached to enable specific and safe delivery of the drug substance. Controlling the delicate balance between protective complexation and release of siRNA at the right point and time is done by understanding excipients-siRNA interactions. These can be lipids, polymers such as PEI, PLGA, Chitosans, Cyclodextrins, as well as aptamers and peptides. This review describes the mechanisms of interaction of the most commonly used siRNA delivery vehicles, and looks at the results of their clinical and preclinical studies.     

Silencing of the SNARE protein NAPA sensitizes cancer cells to cisplatin by inducing ERK1/2 signaling, synoviolin ubiquitination and p53 accumulation

We found earlier that NAPA represents an anti-apoptotic protein that promotes resistance to cisplatin in cancer cells by inducing the degradation of the tumor suppressor p53. In the present study, we investigated the cellular mechanism underlying the degradation of p53 by NAPA. Knockdown of NAPA using short-hairpin RNA was shown to induce p53 accumulation and to sensitize HEK293 cells to cisplatin. On the other hand, this sensitization effect was not found in H1299 lung carcinoma cells which lack p53. Expression of exogenous p53 in H1299 cells was increased following knockdown of NAPA and these cells showed increased sensitivity to cisplatin-induced apoptosis. Notably, knockdown of NAPA induced the ubiquitination and degradation of the E3 ubiquitin ligase synoviolin and the accumulation of p53 in unstressed HEK293 cells. Conversely, NAPA overexpression decreased the ubiquitination and degradation of synoviolin, and reduced p53 protein level. Knockdown of NAPA disrupted the interaction between synoviolin and proteins that form the endoplasmic reticulum-associated degradation (ERAD) complex and in turn decreased the ability of this complex to ubiquitinate p53. In addition, knockdown of NAPA induced the activation of the MAPK kinases ERK, JNK and p38, but only inhibition of ERK reduced synoviolin ubiquitination and p53 accumulation. These results indicate that NAPA promotes resistance to cisplatin through synoviolin and the ERAD complex which together induce the degradation of p53 and thus prevent apoptosis. Based on these findings, we propose that the combination of cisplatin and knockdown of NAPA represents a novel and attractive strategy to eradicate p53-sensitive cancer cells.     

Development of microRNA-145 for therapeutic application in breast cancer

MicroRNAs, small non-coding RNAs, are key regulators of tumorigenesis and cancer metastasis through inhibition of gene expression. Therefore, there is increasing interest in developing anti-cancer therapies using microRNAs. In this study, we determined the therapeutic potency of microRNA-145(miR-145) against breast cancer. We found a reverse-correlation between the expression of miR-145 and its target genes, such as fascin-1, c-myc, SMAD2/3 and IGF-1R in breast cancer cell lines and breast cancer patient tissues. Transfected miR-145 mimicking double-stranded oligonucleotides was directly reduced cell proliferation and motility via interaction with 3′UTR of target gene and also indirectly regulates Wnt signaling. An inhibitor of miR-145 nullified this decreasing effect of miR-145 on cell proliferation and motility. We prepared an adenoviral constructed miR-145(Ad-miR-145) and subjected it to breast cancer cells in vitro and orthotopic breast cancer mice in vivo. Ad-miR-145 suppressed cell growth and motility in both the in vitro and in vivo systems. Furthermore, a treatment combining Ad-miR-145 with 5-FU significantly showed anti-tumor effects, compared to treating alone. In conclusion, this study demonstrated that miR-145 suppresses tumor growth by inhibition of multiple tumor survival effectors, and more we suppose that miR-145 is potentially useful in the therapy of breast cancers.     

红色荧光蛋白核迁移蛋白shRNA干扰载体的构建

目的:利用红色荧光蛋白(RFP),建立一种直观、快速筛选有效RNA干扰片段的方法.方法:通过分子克隆技术将核迁移蛋白(NUDC)与RFP构建成融合基因,克隆至真核表达载体pDs中,以实现其融合表达;同时,将人U6启动子及9个NUDC的发夹结构分别克隆至上述同一真核载体中,构建成一系列针对NUDC不同干扰位点的RNA干扰载体,通过采用酶切及DNA序列鉴定,然后转染293T细胞,通过荧光显微镜观察293T细胞的荧光发光强度及荧光细胞数.结果:酶切及测序结果证实质粒为所需的序列;荧光显微镜观察结果显示,shNUDC-A的干扰效果最好.结论:成功构建了含RFP的NUDC真核shRNA干扰载体.     

Construction, Stable Expression of Survivin shRNA Vector

Objective: To construct survivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin shRNA. Methods: The siRNA sequence targeting survivin mRNA was synthesized and cloned into pEGFP-H1. The constructed plasmid and pEGFP-H1 were transfected into GBC-SD cells respectively via liposome, and the transfecting effect was detected with Flow Cytometry. Then the transfected cells were selected with G418. Results: The recombinant plasmid was successfully constructed, named pEGFP-survivin. The gene transfection efficiencies in pEGFP-H1-transfected group and pEGFP-survivin- transfected group were the 80.29%±2.71% and 83.85%±2.34%(P>0.05), which was successful to get the cells that are stable expressing shRNA, named GBC-SD/EGFP and GBC-SD/survivin. Conclusion: Survivin shRNA expression vector was constructed successfully and got GBC-SD cells which are stable expression shRNA.     

RNAi技术干扰人喉癌Hep-2细胞PTTG1基因的初步研究

目的:探索对人PTTG1基因mRNA序列有较高干扰效率的位点,构建对PTTG1基因的表达有较高抑制效率的重组质粒。方法:筛选、合成1对互补DNA单链,退火为双链后与双酶切后的质粒载体连接,构建了表达短发夹RNA的1种重组质粒plk0.1-puro/PTTG1。重组质粒以不同浓度转染,并于转染后不同时间收集细胞,以β-actin为内参,采用RT-PCR和Western blot技术分别检测PTTG1基因的mRNA、蛋白质的相对表达水平。结果:与空白对照组相比,6孔板中每孔转染质粒plk0.1-puro/PTTG 11000ng以上、转染超过12d时,PTTG1基因mRNA、蛋白质的相对表达水平下降70%以上。结论:重组质粒plk0.1-puro/PTTG1,在6孔细胞培养板中以1000ng/孔转染12d以上时,有效抑制了人PTTG1基因的表达,在关于PTTG1基因的研究中具有较好的应用价值。     

Knockdown of growth-arrest-specific gene 7b (gas7b) using short-hairpin RNA desensitizes neuroblastoma cells to cisplatin: Implications for preventing apoptosis of neurons

Efficient control of cell survival and cell proliferation is critical for the development of neuron cells. Earlier, we observed that growth arrest-specific gene 7 (Gas7) plays a role in controlling neuritogenesis in mammals. In the present study, we report that the Gas7b isoform is involved in controlling growth arrest and apoptosis of neuroblastoma cells in response to various stimuli. Accordingly, knockdown of Gas7b using small-hairpin RNA (shRNA) was shown to reduce apoptosis induced either by serum starvation or by the antineoplastic agents cisplatin and nocodazole in human neuroblastoma SH-SY5Y cells. Gas7b knockdown also enhanced the ability of the treated cells to form clones in response to cisplatin. On the other hand, forced expression of Gas7a or Gas7b isoform in mouse neuroblastoma Neuro2A cells, which express a defective Gas7 gene, rendered the cells proapoptotic and vulnerable to cisplatin-induced apoptosis. In addition, Neuro2A cells that overexpressed Gas7 showed a reduced ability to form clones. Overexpression of Gas7 produced similar but less extensive effects in nonneuronal HEK293 cells. Taken together, our observations suggest that Gas7b is involved not only in neuritogenesis but also in the regulation of neuronal cell death.     

靶向人SIRT1基因的shRNA真核表达质粒的构建及鉴定

目的构建针对人SIRT1基因的shRNA真核表达质粒,并筛选出对胰腺癌细胞系PANC-1基因沉默效果最明显的shRNA质粒表达载体。方法针对SIRT1基因的mRNA序列设计,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠杆菌扩增,酶切,PCR,测序鉴定,转染胰腺癌PANC-1细胞,实时荧光定量PCR和Western blot检测SIRT1 mRNA和蛋白的被抑制情况。结果经测序证实,成功构建SIRT1-shRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致。重组质粒转染PANC-1细胞后,SIRT1 mRNA和蛋白水平明显下调;其中以1号重组质粒效应最强。结论成功构建了携带以SIRT1为靶向的shRNA的重组质粒。其对胰腺癌PANC-1细胞SIRT1的表达具有显著抑制效应。该实验为进一步研究SIRT1的功能和肿瘤的基因治疗提供了实验基础。