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Diagnostics 2013, 3(1), 170-191; doi:10.3390/diagnostics3010170
Published: 15 March 2013
Abstract: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is the third leading cause of cancer-related deaths worldwide. Treatment options for HCC are very limited, as it is often diagnosed at a late stage. Recent studies have demonstrated that microRNAs (miRNAs), a class of non-coding RNAs, are aberrantly expressed in HCC. Some of these were shown to be functionally involved in carcinogenesis and tumor progression, suggesting that miRNAs can serve as novel molecular targets for HCC therapy. Several promising studies have recently demonstrated the therapeutic potential of miRNAs in animal models and in reducing the viral load in hepatitis C patients. In this review, these advances and strategies for modulating miRNAs for in vivo therapeutic delivery and replacement therapy are discussed.
1. Introduction
Human liver cancer can be classified either as hepatoblastoma, hepatocellular carcinoma (HCC), angiosarcoma or cholangiocarcinoma. Among these cancer types, HCC is the most common type that accounts for approximately 70–80% of all primary liver cancer cases [1]. HCC normally develops as a consequence of underlying liver disease and is often associated with cirrhosis. A variety of risk factors contribute to the initiation of HCC [2]. Surgical resection and whole liver transplantation are the current best options for treatment. Recurrence is a common occurrence in patients who have had a resection, and the survival rate is 30–40% at five years post-surgery [3].
Hepatocarcinogenesis is a multistep process involving a number of factors, such as mutations that cause genetic alterations, aberrant expression of cellular proteins, inhibition of tumor suppressors, overexpression of oncogenes and molecules that regulate these events, including microRNAs (miRNAs) and various cellular proteins [2]. Initial studies on HCC pathogenesis have identified a number of critical signaling pathways, as well as the mutations that activate oncogenes (β-catenin, Axin1, PI-3-kinase, K-ras) and inactivate tumor suppressors (p53, Rb1, CDKN2A, IGF2R, PTEN) [2,4]. Subsequent efforts to identify potential prognostic and diagnostic biomarkers have resulted in the development of the first FDA approved drug for HCC: sorafenib, a multi-kinase inhibitor [5]. Sorafenib has been successful, because it targets proteins of multiple signaling pathways simultaneously—VEGFR2, VEGFR3, PDGFR, Flt-3, c-kit and the Raf/MEK/ERK pathway—to inhibit tumor growth and to induce apoptosis of tumor cells [6,7]. The success of sorafenib has resulted in the development of a number of novel therapeutic compounds. These include antibodies, small peptides, heparin-degrading endosulfatases and oncolytic viral agents [2].
Another emerging area of HCC therapeutics is based on miRNAs. miRNAs are small (~21–23 nucleotides long), non-coding RNAs that regulate post-transcriptional gene expression of their target genes either by inducing translational repression via their binding to partially complementary sequences or by directing mRNA degradation through their binding to perfectly complementary sequences in the 3' untranslated region (UTR) of messenger RNAs (mRNAs) [8]. Each mature miRNA potentially controls many gene targets, and each mRNA is regulated by multiple miRNAs. To date, more than 17,000 distinct mature miRNA sequences have been identified from over 140 species [9]. A number of recent studies have documented the involvement of miRNAs in HCC in tumor progression and metastasis [10,11,12,13,14,15]. In humans, more than 50% of miRNA genes are located at fragile sites or in cancer-associated genomic regions that are frequently involved in chromosomal abnormalities, such as loss of heterozygosity, amplification and breakpoints [2], indicating that they might be useful as novel diagnostic and/or prognostic markers and could constitute potential molecular targets in cancers.
miRNAs are transcribed by RNA polymerase II to form primary transcripts (pri-miRNAs), capped with 7-methylguanosine and polyadenylated. These pri-miRNAs are processed in the nucleus by the RNase III enzyme Drosha and its cofactor, Pasha/DGCR8, into ~60–70 nucleotide precursors (pre-miRNAs), which form an imperfect stem-loop structure and are exported into the cytoplasm by the RAN GTP-dependent transporter, Exportin 5. RNase III enzyme Dicer then processes pre-miRNAs into mature miRNAs, which are subsequently loaded onto the RNA-induced silencing complex (RISC). The miRNA/RISC complex then binds to the 3'-UTR of target mRNAs and downregulates their expression [16,17]. Thus, miRNAs modulate various cellular signaling pathways involved in cell growth, proliferation, motility and survival. miRNAs are also subjected to regulation by epigenetic mechanisms, and mutations in their promoter and coding regions were shown to contribute to tumorigenesis [18,19,20,21,22].
2. Involvement of MicroRNAs in HCC
During the past decade, it has been well established that specific miRNAs modulate various cellular processes in the liver and that their aberrant expression correlates with the severity and poor prognosis of HCC [23,24,25,26,27]. For example, in one study, the expression of miR-199a, miR-92, miR-106a, miR-222, miR-17-5p, miR-18 and miR-20 correlated with the degree of tumor differentiation, suggesting the involvement of these miRNAs in disease progression [23], while in another study, miRNAs associated with viral infection and the stage of liver disease were identified by comparing miRNA expression profiles of patients with hepatitis B virus (HBV)-related HCC with those from HCV-related HCC [1,25]. Further analysis of HBV-associated miRNAs revealed that their targets included genes involved in pathways related to cell death, DNA damage, recombination and signal transduction, and the targets for HCV-associated miRNAs included genes involved in immune response, antigen presentation, cell cycle, proteasome and lipid metabolism [25]. This finding demonstrated that HBV and HCV induce different sets of miRNAs during infection.
miRNA-122, an abundant liver-specific miRNA that modulates hepatic lipid metabolism [28], is often downregulated in human HCC [29,30]. Loss of its expression correlates with loss of mitochondrial metabolic function and is detrimental to sustaining critical liver function, thereby contributing to the morbidity and mortality of liver cancer patients [31]. In the HBV-related HCC cell line, HepG2.2.15, miR-122 inhibited viral replication by targeting NDRG3, a member of the N-myc downstream-regulated gene family [32], suggesting that both miRNA-122 and NDRG3 are viable therapeutic targets for HBV-related HCC. During HCV infection, however, miR-122 binds directly to two sites in the 5' non-coding region of HCV genome and positively regulates the viral life cycle [29]. Thus, inhibition of miR-122 presents an attractive treatment option for HCV infection.
In another study, examination of miRNAs in HCC with cirrhotic background revealed that members of the let-7 family, miR-221 and miR-145 were downregulated [33]. In these tissues and in HCC cell lines, miR-122 was also downregulated, and its target gene product, cyclin G1, was highly expressed, promoting the growth of cancer cells. However, when miR-122 expression was restored, it significantly reduced in vitro migration, invasion and anchorage-independent growth of Mhalavu and SK-HEP-1 cells, as well as in vivo tumorigenesis, angiogenesis and intrahepatic metastasis in an orthotopic liver cancer model by inhibiting ADAM17, a protein involved in metastasis [34]. Interestingly, miR-122 also inhibited tumorigenic properties of HCC and sensitized tumor cells to sorafenib [32]. Collectively, these studies demonstrate that miR-122 can function both as a positive and negative regulator in liver cancer. Other studies have shown that miRNAs associated with cell cycle inhibition (miR-34a, miR-101, miR-199-a-5p and miR-223) were downregulated in HCC [22,35,36,37,38,39,40], and those involved in cell proliferation and inhibition of apoptosis (miR-17-92 polycistron, miR-21, miR-96, miR-221 and miR-224) were upregulated [41,42,43,44]. Furthermore, miRNA-221 is associated with tumor multifocality [45].
As demonstrated in these examples (Table 1), aberrant miRNA expression leads to the dysregulation of critical cellular mechanisms and activation of tumorigenic pathways involved in tumor differentiation, diagnosis, staging, progression, prognosis and response to therapy [28,29,30,31,32,33,34,35,36,37].
|Table 1. Differentially expressed miRNAs in liver tissues and hepatocellular carcinoma (HCC) cell lines.
|Sample type
|miRNAs *
|Cellular Target/Mechanism
|Tumor tissues
|Microarray, qPCR
|miR-199a, miR-92, miR-106a, miR-222, miR-17-5p, miR-18, miR-20
|Tumor progression
|Tumor tissues, rat model of hepatoma
|Microarray, Northern blot
|miR-122, let-7a, miR-21, miR-23, miR-130, miR-190, miR-17-92 family
|Tumor tissue
|Loss of mitochondrial metabolism
|HCC cell lines
|Tumor tissues, HCC cell lines
|Microarray, qPCR Northern blot
|miR-122 let-7 family, miR-145
|Cyclin G1
|HCC cell lines
|Western blot, Soft agar assay
|ADAM17, migration, invasion, anchorage-dependence, angiogenesis, metastasis
|Tumor tissues, HCC cell lines
|qPCR, Western blot
|c-Met, apoptosis, cell cycle arrest, Senescence
|Tumor tissues, HCC cell lines
|Microarray, Northern blot, Western blot
|Mcl-1, apoptosis, tumor suppression
|Tumor tissues
|Microarray, qPCR
|miR-199-a-5p, miR-223
|Cell cycle inhibition
|Human and Woodchuck HCC cell lines
|qPCR, Northern blot
|miR-17-92, miR-21
|Cell proliferation, apoptosis
|Tumor tissues
|qPCR, Northern blot
|CDKN1C/p57, CDKN1B/p27
|Tumor tissues
|Microarray, qPCR, Northern blot
|Tumor tissues
|Apoptosis inhibitor-5
* Downregulated miRNAs were shown in italics.
3. Strategies for the Modulation and Delivery of miRNAs
The application of miRNAs in cancer therapeutics normally involves one of the two strategies: (1) gain of function to inhibit tumor-inducing miRNAs (also termed as "oncomiRs") using miRNA antagonists, such as locked nucleic acids (LNA), antagomiRs and antimiRs; and (2) miRNA replacement by re-introducing miRNAs with tumor suppressor functions to restore the loss of function [46]. While miRNA antagonists are oligonucleotides containing the complementary sequences of endogenous miRNAs, miRNAs used for replacement therapy can either be oligonucleotides or those expressed from transfected/transduced vectors. Endogenous miRNAs can be modulated by transient or stable transfection or by viral delivery of a small RNA molecule that can either be a pri-miRNA transgene, a pre-miRNA, a mature miRNA, a miRNA/miRNA*, a small interfering RNA (siRNA) or a short hairpin RNA (shRNA) [17]. The primary challenge for gene therapy is to develop a method that can deliver a therapeutic miRNA to target cells where proper gene expression can be achieved. An ideal delivery method should: (1) allow efficient entry of miRNA and its carrier into the target cell; (2) protect the RNA against degradation by nucleases in intercellular matrices; and (3) be nontoxic. Exogenous RNA ("miRNA modulator") thus expressed with an appropriate delivery method can regulate the activity of miRNAs to alleviate tumorigenicity.
To function effectively in vivo, however, miRNA modulators must cross the membranes of tumor cells first and then bind their targets stably in the cytoplasm. In general, viruses are able to mediate gene transfer with high efficiencies, as they can infect every cell in a tumor tissue and may induce long-term expression of miRNAs in the infected cell. They can also be manipulated to alter their tropism so that they can infect the given cell type by binding to the cell surface receptor of choice. Therefore, viral vectors are the preferred choice for therapeutic studies of human diseases. However, acute immune responses, immunogenicity and insertion mutagenesis uncovered in gene therapy clinical trials have raised serious safety concerns on therapeutic application of commonly used viral vectors [47].
When compared with viral methods, nonviral delivery approaches are less efficient. These methods can be classified either as physical or chemical approaches. Physical methods employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer, such as needle injection, electroporation, gene gun, ultrasound and hydrodynamic delivery; and chemical approaches use synthetic or naturally occurring compounds as carriers to deliver the transgene into cells [47]. These methods have encountered serious obstacles in recent years, because naked RNAs are highly unstable in biofluids and are degraded rapidly by cellular nucleases [11,32,33]. To overcome these challenges, efforts are being made to stabilize the miRNA by chemical modification, conjugation and encapsulation; and to improve its delivery to the target tissues and tumor cells in vivo. Some of the promising methods successfully employed in cancer research (Table 2) are discussed below.
|Table 2. Successful miRNA-based therapeutic studies reported for various types of cancer.
|Therapeutic strategy
|miRNA modulator/overexpression
|Delivery method ( in vitro/in vivo)
|miRNA inhibition
|i.v. injection
|s.c. injection
|Anti-oncomiRs miR-143/miR-145/liposome complexes
|i.v. injection
|miR-145/PU-PEI complexes
|s.c. injection
|Lung adenocarcinoma
|s.c. injection
|MUC1 aptamer-miR-29b
|Nucelolin-miR-122 aptamer
|miRNA replacement therapy
|scFV antibody coupled to miR-34a nanoparticles
|i.v. injection
|miR-34a mimic
|s.c. injection
|miR-33a/PEI and miR-145/PEI complexes
|i.p./i.v. injection
|miR-34a mimic
|miR-34a and let-7 mimics miR-26a
|AAV vector
AAV, adeno-associated viruses.
3.1. Stabilizing miRNA Inhibitors by Chemical Modifications
The base-pair interaction between miRNAs and their target mRNAs is essential for the function of miRNAs. Therefore, to silence miRNAs, nucleic acids that were antisense to the target miRNA have been developed. These antisense oligodeoxyribonucleotides (AMOs) specifically and stoichiometrically bind to their targets and silence them efficiently and irreversibly [50]. The AMO approach has recently been used successfully for miRNA therapy of human diseases [65,66]. By modifying this approach and by engineering various antisense miRNAs into a one single unit, Lu et al. showed that multiple target miRNAs can be silenced simultaneously in cell culture studies with the breast cancer cell line MCF-7 [50]. Despite this success, dose-response actions and in vivo stability of AMOs is largely unknown. Further studies are needed to further evaluate their potential in cancer therapy.
One of the first modifications to be carried out to enhance the stability of small RNAs was the substitution of 2'-OH in the ribose ring with 2'-O-methyl or 2'-fluorin. In in vivo experiments with rodents, antisense miRNA inhibitors carrying this substitution displayed enhanced stability and had a higher binding affinity to the miRNA target [67,68]. The use of phosphorothioate bond, that replaces the oxygen atom in the phosphodiester linkage with a sulfur atom, also improved stability of these small RNA to nuclease degradation [69]. AntagomirRs against miR-17-5p and miR-221/miR-222 containing the 2-O-methyl modification and terminal phosphothioates, together with a 3'-conjugated cholesterol group, significantly reduced tumor growth when administered subcutaneously in mouse models of neuroblastoma and prostate cancer, respectively [52,53]. This approach has since faced setbacks because of the requirement for high doses of antagomiRs [70].
The most commonly used miRNA inhibitors in cancer studies are the locked nucleic acid (LNA) antisense oligonucleotides [71]. LNAs are conformational RNA analogs that bind complementary RNA with extremely high affinity making them ideal candidates for cancer detection and analysis of cancer diagnostics. LNA-antimiRs have locked ribose rings that connect 2'-O and 4'-C atoms through methylene bridges, thus conferring unprecedented specificity, due to a significant increase in the RNA:RNA melting temperature [71]. The advantage of LNA-antimiRs is that they can be used in low doses, as they are relatively more resistant to nuclease digestion. Successful use of the LNA-based approach was also shown by high affinity inhibition of miR-91a and miR-21 in animal models of breast cancer [72,48].
Double-stranded RNA molecules containing the same sequence as the depleted, naturally occurring miRNA are commonly used as "miRNA mimics" for replacement therapy. They are expected to target the same set of mRNAs that is also regulated by the natural miRNAs [46]. To protect these dsRNAs from nuclease digestion and to avert immune responses, they were modified with the addition of inverted bases and alkyl or biotin groups [70,73]. While designing these dsRNAs, it is important to preserve target specificity and to ensure miRISC loading [69]. In a mouse model of human colorectal cancer, miR-143 duplex with the modified passenger strand and 3'-overhangs displayed greater activity and stability to nucleases in a mouse xenograft model of human colorectal cancer [54]. This chemically modified synthetic miR-143 also showed a significant tumor suppressive effect than the endogenous miR-143 [54].
More recently, a novel class of small molecule inhibitors of miRNAs have been developed [74]. By screening over 1,000 compounds at the 10 μM concentration, Gumireddy et al. identified two diazobenzene compounds that can inhibit miR-21 in three tumor cell lines [74]. It would be interesting to see if any of these could inhibit miR-21 in vivo.
3.3. Modulating Virus-Mediated Delivery of miRNAs
Viral vectors commonly used in human gene therapy are modified, noninfectious adenoviruses, adeno-associated viruses (AAV), herpes simplex viruses (HSV) and lentiviruses. Each of these can encode miRNA mimics and express them in infected cells. Since viral vectors can be engineered to alter their tropism for transduction into any cell type, they are the most preferred vectors for gene therapy. Recombinant adenoviral (Ad) and AAV vectors were commonly used in such studies, because they do not integrate into the host genome, but replicate as episomes in the nucleus and stably express miRNAs. Both Ad and AAV vectors are ideal for transient expression of transgenes in tumor cells or in cells that divide slowly [81]. Moreover, these vectors are of greater relevance, because random integration of lentiviral vectors may result in the activation of oncogenes and induce tumorigenesis. It was shown recently that AAV vectors transduce hepatocytes efficiently in vivo when injected through the intra-portal or intra-hepatic artery of healthy and cirrhotic rat livers, suggesting that they are convenient for transgene expression in the liver [82]. A major drawback of viral vectors, in general, is that they elicit strong immune responses in transduced cells, which reduces gene transfer and limits re-administration. Therefore, chemical or physical modification of Ad vectors with polymers has been used as a strategy for cancer therapy. Lately, to endow targeting moiety to polymer-coated Ad vectors, a diversity of ligands, such as tumor-homing peptides, growth factors or antibodies, have also been introduced to avoid unwanted transduction and to enhance therapeutic efficacy [83]. In addition, efforts are being made to encapsulate Ad vectors into poly (lactide-co-glycolide) (PLG) microspheres prior to administration [84]. When compared to free Ad vectors, PLG-encapsulated Ad vectors are known to have a significantly higher transduction efficiency rate in both E1 complementing and non-complementing cells [84]. When Ad was enveloped by cationic lipids, significantly high levels of viral uptake was observed in cultured HepG2 cells [85]. When administered in mice, these artificial envelopes can significantly alter the interactions with blood components and divert viral particles from their natural liver tropism, resulting in reduced hepatotoxicity [85]. Another added advantage of encapsulated viral vectors is that their level of transduction can be controlled by varying the quantity of encapsulated viral particles in the microspheres and the amount of microspheres administered.
AAV vectors, on the other hand, are single stranded adenoviruses devoid of critical genes involved in viral infection, making them relatively safe to administer in gene therapy trials. However, because of the difficulties in the construction of AAV vectors, self-complementary AAVs (scAAVs) that contain inverted repeats of its genome were developed. scAAVs can fold into dsDNA without the need for additional DNA synthesis or base-pairing between multiple vector sequences [68]. Successful therapeutic delivery of miR-26a-expressing AAV vector has recently been reported in a murine liver cancer model [64].
Efforts to knock-down miRNAs by using the classical RNA interference (RNAi) techniques were tested for inhibiting miRNAs. However, these approaches have largely been unsuccessful. Therefore, to induce the knockdown of miRNAs, genetic "sponges" were developed. These "miRNA sponges" are competitive inhibitors expressed from vectors containing multiple, tandem binding sites to a miRNA of interest [86]. When transiently transfected into cultured cells, miRNAs expressed from a strong promoter on these sponges will compete with endogenous miRNAs and sequester mature miRNAs away from their natural targets. This approach has been successfully used recently to knockdown miR-223 using a lentiviral vectors [87].
All these diverse methods of miRNA delivery and encapsulation would allow us to perform both loss-of-function and gain-of-function assays and to modulate the expression of miRNAs in vivo, thereby facilitating the application of miRNAs for cancer therapy.
4. Strategies for the Development of miRNA Therapeutics for HCC
The field of miRNA therapeutics for HCC is still in its infancy. So far, only a handful of successful outcomes have been reported. By using the above approaches that were successful with other cancers, experiments can be designed to suppress oncomiR expression and to restore the expression of downregulated miRNAs in tumor tissues of the liver. Some of the promising candidates for these miRNA therapeutics are discussed below.
4.1. Inhibition of OncomiRs
OncomiRs promote tumor growth and proliferation by blocking the activities of cellular tumor suppressors, cell cycle regulators and pro-apoptotic genes. For instance, miR-224 increases tumor cell progression by inhibiting the expression of apoptosis inhibitor-5 [18,44], and miR-221 promotes cell proliferation by controlling the cell cycle inhibitors (CDKI) CDKN1C/p57 and CDKN1B/p27 [42]. miR-519d, on the other hand, contributes to hepatocarcinogenesis by targeting CDKNA/p21, PTEN, AKT3 and TIMP2 [88]. Intratumoral administration of miR-143 showed that high levels of miR-143 can significantly promote HCC metastasis in an athymic nude mouse model by repressing the expression of fibronectin type III domain containing 3B (FNDC3B), which regulates cell motility [89]. miR-222, a frequently overexpressed miRNA in HCC, also regulates cell motility by enhancing Akt signaling [90], whereas miR-423 directly binds to 3'-UTR of p21Cip1/Waf1 to suppress its expression and to promote cell cycle progression and tumorigenesis [91].
While miR-146a increases the resistance of HCC cells from the cytotoxic effects of interferon-α by inhibiting the expression of Smad4 [92], upregulation of hepatic transforming growth factor (TGF)-β and its downstream mediators, Smads 2, 3 and 4, was found to be correlated with an increased expression of miR-181 in the livers of mice fed with a choline-deficient L-amino acid-defined (CDAA) diet [93]. Depletion of miR-181b inhibited tumor growth in nude mice, whereas its expressed enhanced resistance of HCC cells to the anticancer drug, doxorubicin [93]. miR-17-5p upregulates the migration and proliferation of HCC cells by activating the p38 mitogen-activated protein kinase MAPK pathway and increasing the phosphorylation of heat shock protein 27 [94], suggesting its potential application in HCC therapy. The miR-34 family members are direct transcriptional targets of tumor suppressor, p53, and loss of miR-34 function can impair p53-mediated cell cycle arrest and apoptosis [19].
miR-210, which is often upregulated in HCC, promotes hypoxia-mediated tumor cell metastasis by targeting the vacuole membrane protein 1 [95]. Therefore, inhibition of miR-210 expression could lead to reduction in HCC metastasis and should facilitate the development of novel therapeutic strategy against hypoxic tumor cells. Similarly, miR-30d, a miRNA associated with intrahepatic metastasis of HCC, promotes tumor cell migration and invasion in vitro and intrahepatic and distal pulmonary metastasis in vivo by targeting the Galphai2 (GNAI2) protein [96]. The expression of miR-148a is elevated in HepG2 and Hep3B cells, where it promotes cell proliferation, cell cycle progression and cell migration. When anti-miR-148a was overexpressed from a lentivirus, it inhibited the activity of miR-148a, in addition to blocking the Akt signaling pathway, in these cells, suggesting that it could serve as an early diagnostic marker and/or therapeutic target [97]. Additionally, miR-1 and miR-499 inhibited invasion and migration of HepG2 cells by targeting the ets1 proto-oncogene, which causes degradation of extracellular matrix [98]. Another antagomiR, miR-219-5p, induced tumor suppressive effects in HCC cell lines by targeting glypican-3 and by causing cell cycle arrest at the G1 to S transition [99].
While successful strategies to inhibit oncomiRs for HCC therapy have not been reported so far, each of these miRNAs represent a strong candidate for therapeutic intervention towards achieving a gain of function with the use of miRNA antagonists, RNAi and small molecule inhibitors. A recent study has showed the effects of a modified AMO against miR-221 in reducing the number and size of HCC tumors in a miR-221 transgenic mouse model [51]. While this study is promising, further in-depth studies on candidate miRNAs will allow us to ascertain their potential as therapeutic targets for HCC treatment.
4.2. miRNA Replacement Therapy
The first successful demonstration of miRNA replacement to restore the expression levels of a downregulated miRNA by delivery to the tumor was reported using a miR-26a-encoding AAV vector in a mouse model of HCC [64]. miR-26a is normally downregulated in HCC, and its overexpression in mouse livers results in the inhibition of cancer cell proliferation and induction of tumor-specific apoptosis [64]. More recently, another miRNA downregulated in HCC, miR-34a, has been successfully tested using this approach and by delivering it with NOV340 liposomes in an orthotopic model of HCC [100]. In both cases, significant tumor reduction, dramatic protection from disease progression without toxicity and prolonged survival of animals has been reported. While these reports were aimed to restore a loss of function, similar success has not yet been achieved with approaches to inhibit miRNA expression levels. All these studies are built on the regulation of multiple cellular pathways associated with human disease, which now appears to be a requirement for successful cancer therapy [101].
Along these lines, another downregulated miRNA in HCC, miR-375, was overexpressed in liver cancer cells. It decreased cell proliferation, clonogenicity, migration/invasion, induced G1 arrest and apoptosis [102] in vitro, indicating that it could be a good candidate for in vivo studies. Indeed, when cholesterol-conjugated 2'-O-methyl-modified miR-375 mimics (Chol-miR-375) were administered in nude mice, they were able to significantly suppress the growth of hepatoma xenografts [103].
In other promising studies, overexpression of miR-376a suppressed cell proliferation in HuH7 cells [101] and miR-138 induced cell cycle arrest by targeting cyclin D3 [103]. In an in vitro experiment with HCC cell lines transfected with miR-637 mimics or transduced with Lv-miR637 vectors, miR-637 suppressed tumor growth by negatively regulating the phosphorylation of STAT3 protein [104].
Downregulation of miR-29b correlates with rapid recurrence and poor survival of individuals with HCC. miR-29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro and in mouse xenografts where it regulated the expression of matrix metalloproteinase 2 [105]. Restoration of miR-29b expression resulted in the deregulation contributes to angiogenesis, invasion and metastasis of HCC [105], suggesting it can be used as a novel therapeutic target in anti-HCC therapy.
Intratumoral injection of cholesterol-conjugated miR-99a mimics was recently demonstrated to significantly inhibit tumor growth and to reduce α-fetoprotein levels in HCC-bearing nude mice. In this study, miR-99a expression inversely correlated with protein levels of insulin-like growth factor 1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR), inducing cell cycle arrest at the G1 phase [106]. This finding suggested a potential tumor suppressor role for miR-99a. Another miRNA often downregulated in HCC is miR-199a-3p. By transfecting pre-miR-199a-3p and anti-miR-199a-3p oligonucleotides, it was shown to target mammalian target of rapamycin (mTOR) and c-Met in HCC cells [107]. Restoring attenuated levels of miR-199a-3p in these cells led to cell cycle arrest at the G1 phase, reduced invasive capability, enhanced susceptibility to hypoxia and increased sensitivity to doxorubicin-induced apoptosis, suggesting that enhancement of miR-199a-3p levels may have a therapeutic benefits in HCC [107].
miR-122 binds to the 3'-UTR of Bcl-2 family member, Bcl-w, an anti-apoptotic protein, to induce cell death in HepG2 and Hep3B cells [108]. When expressed from an Ad vector, miR-122 sensitized HCC cells to adriamycin and vincristine treatment by causing cell cycle arrest at the G2/M phase and by modulating the expression of multidrug resistant protein, MDR-1 [109]. Using several 3' cholesterol-conjugated, 2'-O-Me oligonucleotides and an unconjugated 2'-O-methoxyethyl-phosphorothioate-modified oligonucleotide, miR-122 was inhibited in mouse livers to demonstrate its function in the lipid metabolism [110,111]. Elmen et al. also subsequently reported the inhibition of miR-122 in the mouse liver after the systemic administration of a 16-mer LNA-modified antimiR oligonucleotide [112]. These studies provide strong evidence that miR-122-based HCV therapy can be developed with antagomiRs. To pursue this lead, Lanford et al. administered a LNA-modified phosphorothioate oligonucleotide (SPC3649) complementary to the 5'-end of miR-122 in chimpanzees with chronic HCV infection and showed that SPC3649 suppression of miR-122 had long-lasting suppression of HCV viraemia with no evidence for viral resistance or side effects in the treated animals [113]. In addition to demonstrating the feasibility and safety of prolonged administration of a LNA oligonucleotide drug in vivo, this study showed that miR-122 is essential for HCV accumulation and that the miR-122 seed sites were conserved in all HCV genotypes and subtypes, suggesting the genotype independent nature of this therapy. More recently, Janssen et al., further extended this approach and reported successful therapy in HCV patients with an oligonucleotide drug, MiraVirsen, that targets miR-122 [114]. This was the first miRNA-based therapeutic drug developed to treat a liver disease and is currently being tested by Santaris Pharma (Hørsholm, Denmark) in phase 2 clinical trials [28].
These are some of the promising candidates for miRNA replacement therapy for HCC. It is important to exercise caution while designing clinical trials with miRNA mimics for targeting multiple genes relevant to human disease, because of the concerns about potential toxicity in normal tissues, especially under conditions where the therapeutic delivery of miRNA mimics will also lead to an accumulation of exogenous miRNA in normal cells. These toxic effects might be the result of overloading RISC with the exogenous miRNA, thereby competing with endogenous miRNAs necessary for normal cellular welfare and/or hyperactivating cellular pathways that will also reduce the viability of normal cells [46].
An important aspect to consider for miRNA-based therapy is the assessment of off-target effects of miRNA modulators. When using these oligonucleotides, there is a potential risk of affecting cellular RNAs other than the target miRNA [115]. Therefore, proper understanding on the effects of unwanted interactions between antimiR molecules and endogenous RNAs and appropriate designing of antimiRs to minimize the off-target effects is critical. As discussed earlier, the incorporation of chemical modifications, such as LNAs, into antimiRs have been shown to improve mismatch discrimination between a given antimiR and its target miRNA. However, these exogenous miRNAs compete with endogenous ones for miRISC, causing the saturation of miRISC complexes, resulting in the loss of endogenous miRNA regulation and fatality, due to liver toxicity, as demonstrated in animal studies [116,117].
5. Perspectives
Because of their functionality in diverse cellular events, miRNA are widely studied in cancer research, as evidenced by hundreds of clinical trials that are currently underway [118]. Moreover, there is a surge in the filing of patent applications worldwide on the use of miRNA in cancer therapeutics [119]. HCC is a multifactorial disease. Therefore, the expression of a large number of genes, proteins and other molecules from diverse cellular processes and pathways are altered in HCC. Hence, the use of a combination therapy that targets multiple different steps and pathways, rather than a single test or a set of tests, might be an appropriate strategy to combat human HCC. miRNAs fit this bill perfectly, as they are capable of targeting many mRNAs simultaneously. Positive therapeutic outcomes achieved with sorafenib that can inhibit receptor tyrosine kinases of multiple signaling cascades, as well as evidence that a single molecule miR-26a can significantly reduce HCC without any toxicity, demonstrate the success of this multi-pronged approach. The main challenge for successful translation of these strategies into the clinic to harness the full potential of miRNAs for therapeutic development remains in vivo delivery. Novel approaches to enhance the stability of miRNA modulators in vivo and methods to deliver them efficiently to the target tissues are urgently needed to overcome this challenge.
I apologize to authors whose work could not be cited due to space limitations and refer the readers to papers cited here and the references therein for more in-depth discussions.
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- He, X.X.; Chang, Y.; Meng, F.Y.; Wang, M.Y.; Xie, Q.H.; Tang, F.; Li, P.Y.; Song, Y.H.; Lin, J.S. MicroRNA-375 targets AEG-1 in hepatocellular carcinoma and suppresses liver cancer cell growth in vitro and in vivo. Oncogene 2012, 31, 3357–3369, doi:10.1038/onc.2011.500.
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- Zhang, J.F.; He, M.L.; Fu, W.M.; Wang, H.; Chen, L.Z.; Zhu, X.; Chen, Y.; Xie, D.; Lai, P.; Chen, G.; Lu, G.; Lin, M.C. Primate-specific microRNA-637 inhibits tumorigenesis in hepatocellular carcinoma by disrupting signal transducer and activator of transcription 3 signaling. Hepatology 2011, 54, 2137–2148.
- Fang, J.H.; Zhou, H.C.; Zeng, C.; Yang, J.; Liu, Y.; Huang, X.; Zhang, J.P.; Guan, X.Y.; Zhuang, S.M. MicroRNA-29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression. Hepatology 2011, 54, 1729–1740, doi:10.1002/hep.24577.
- Li, D.; Liu, X.; Lin, L.; Hou, J.; Li, N.; Wang, C.; Wang, P.; Zhang, Q.; Zhang, P.; Zhou, W.; Wang, Z.; Ding, G.; Zhuang, S.M.; Zheng, L.; Tao, W.; Cao, X. MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma. J. Biol. Chem. 2011, 286, 36677–36685.
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© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


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November 25, 2014
People in a Fruita neighborhood describe hearing loud explosions as a shed burst into flames Sunday afternoon.
Officials with Lower Valley Fire Department say that a controlled burn hit a shed and sparked numerous 55 gallon drums of waste oil on fire.
Lower Valley Fire, the homeowners and neighbors all worked together to extinguish the blaze. The homeowners' daughter says that while she's glad the house didn't catch on fire, many of her childhood memories went up in smoke with the shed. Debris from the fire even knocked out a power line.
Officials say no one was injured in the blaze, damage to the shed and its contents are estimated at one-hundred-and fifty-thousand dollars.
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Herb Sennett
About me
Herb Sennett writing novels at the ripe young age of 65. His first novel is awaiting publication early in 2013. He writes in two genres: historical novels and murder mysteries located in South Florida. His first novel is set in Ancient Canaan (or Palestine). His second will be set in pre-Golden Age Greece.


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Hello Everyone!
Or as we say in Chinese, ni hao ma?
Welcome to A Little Piece of Me!
Please enjoy my blogs and photos, most of which are taken by the extraordinary Laura Marie Duncan and the wonderful Peter Gibbons.
Other Media:
Theatre Showreel: Celia Mei Rubin Showreel 2012
On Camera Reel : Celia Mei Rubin On Camera Reel 2012
SATURDAY NIGHT FEVER reel (as Stephanie Mangano): SNF
More videos can be found here: Celia's Videos
I am currently making my Broadway debut in Matilda, The Musical at the Shubert Theater.
You can follow me on Twitter: @Celia_Mei
You can follow my blog by clicking here: "Memoirs of a Chinese-Jew."
Feel free to contact me — I would love to hear from you, even if all you want to do is say hi. You can find contact details on my Contact page. Enjoy this little piece of me, and have a wonderful day filled with all good things!
– Celia Mei Rubin


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No one could ever accuse designer Mary Quant of letting fun times pass her by – one look at her Mod-ish collections from the Swinging Sixties tells you that. Micro miniskirts, bright colour-blocking and over-the-knee socks were all her doing (and is it just us, or do they feel quite du jour right now too?). And at 75, the fun will certainly not be stopping. She's still working away with her cosmetics company 'Mary Quant Cosmetics' and penning her second autobiography, but she noticed one particular aspect of the fashion industry in the current climate has changed for the worse – '[What's] most annoying: that other people take things so seriously...' she explains. 'I hate the effect the recession's having because I like everything to be bouncing.' Well, luckily for any sour-faced fashionistas out there, Mary's hoping that her zingy outlook will naturally cheer them up! We think that sounds like an invite round to her place for tea and pick-me-up biscuits, non?
Mary Quant's not taking life too seriously...
22 June 2009
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Old Lyme House Rental Photos and Description
3 Bedrooms,1 Full Bathroom, Outdoor Shower, Sleep 6
Summer vacation rental - Private Old Colony Beach, Old Lyme CT. 3 bedroom home, sleeps 6. Very spacious, full kitchen, one full bathroom, washer/dryer, outdoor shower, gas grill, cable TV, DVD player, and air conditioning. Beautiful enclosed front porch and an enlarged deck off the back of the house. A two Minute walk to the beach. Great for families! Enjoy fishing, fun in the sun, and volleyball on the beach. Close to Essex Steam train, Mystic Seaport and Aquarium, Westbrook and Clinton Outlet stores, New London Ferry to Block Island, and Foxwoods and Mohegan Sun Casino. One hour to Newport Rhode Island, Hartford CT, and New York City. Excellent Get Away!
Our second floor is now available for rental. Please inquire for details.
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- children welcome
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WASHINGTON -- Police have arrested a teenager in connection with the violent mugging of a local actor early Tuesday morning.
According to a release by the Montgomery County Police Department, detectives arrested Elijah Jeremiah Smith, 18, of Washington, D.C., on charges of assault and robbery in downtown Silver Spring, Md. A search warrant had been conducted at the suspect's residence, and "items of evidence related to the assault and robbery were recovered."
A photo of the suspect is not available at this time.
Meanwhile, victim Frank Britton suffered a shattered bone on the side of his face, requiring immediately surgery this week. The attack also resulted in a contusion on the back of his head and swelling to an eye.
Britton is a local stage actor, and after performing in the Forum Theater's opening night of "The Last Days of Judas Iscariot," he and the cast went out to celebrate on Colesville Road Silver Spring.
About 2:30 a.m. Tuesday, four men attacked and robbed him by a taxi stand close to the Silver Spring Metro station. They took his iPhone, iPad and wallet.
Friends and family established a GoFundMe account to help cover his medical bills. In the first day, the account showed more than $40,000.
An understudy played his role in Wednesday's performance.
No word yet on the other suspects.
Follow @WTOP on Twitter and on the WTOP Facebook page.
© 2014 WTOP. All Rights Reserved.


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|Regulatory Story
|Go to market news section
Securities Trust of Scotland plc
The board of Securities Trust of Scotland plc (the "Company") announces that on 4th December 2012 it issued 500,000 new ordinary shares in the Company at 125 pence per share. The shares were issued at a premium to the prevailing net asset value. These shares will rank pari passu with the existing ordinary shares in issue.
It is expected that the new ordinary shares will be admitted to listing on the Official List of the UK Listing Authority and to trading on the main market of the London Stock Exchange on Friday, 7th December 2012.
The shares in issue following this notification are 105,259,771.
William Simmonds
J.P. Morgan Cazenove Limited
020 7588 2828
Victoria Timlin
Martin Currie Investment Management Ltd
0131 229 5252
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Book Description:
Horatius Bonar was born into a long line of ministers who have served a total of 364 years in the Church of Scotland. voluminous and highly popular author. He also served as the editor for "The Quarterly journal of Prophecy" from 1848 to 1873 and for the "Christian Treasury" from 1859 to 1879. In addition to many books and tracts wrote a number of hymns, many of which, e.g., "I heard the voice of Jesus say" and "Blessing and Honor and Glory and Power," became known all over the English-speaking world. ContentsPage1. The Importance of a Living Ministry....................................32. The Minister s True Life and Walk.......................................73. Past Defects .....................................................................114. Ministerial Confession.....................................................165. Revival in the Ministry ....................................................30


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NCGA Corn Board selects Schott as next first vice president
The National Corn Growers Association Corn Board has elected Bart Schott of Kulm, N.D., to the position of first vice president for the 2010 fiscal year, which begins Oct. 1. Schott and his family are third- and fourth-generation farmers raising no-till corn, soybeans and wheat. He also runs a seed business.
"I am honored and humbled to be selected by my fellow board members to serve as the next first vice president of NCGA," said Schott. "We live in dynamic times and I'm excited to be a part of the change that can bring both opportunities and challenges for corn farmers and our customers in producing food, feed and fuel for our nation and the world."
Schott has served as chairman of the North Dakota Corn Council. He is a member of the NCGA Finance Committee as well as a board liaison for the Grower Services Action Team. Schott is also chairman of NCGA's Bylaws Committee and a past chairman of the Association Relations Committee. Schott previously served as the NCGA Corn Board liaison for the National Cattlemen's Beef Association and a member of the NCGA Biotechnology Working Group and the Research and Business Development Action Team. He and his wife, Linsey, have three children, Peter, Andrew and Micah.


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Carey shows off her latest blingin' cast in New York City. Plus: Viola Davis & Terrence Howard, Jake Gyllenhaal, Dakota Johnson and more
Credit: Gregg DeGuire/WireImage
Published: Friday Sep 13, 2013 | 05:30 PM EDT
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Also at the Prisoners premiere: costars Terrence Howard and Viola Davis, who glam up the red carpet together.
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what makes a Croque Monsieur? [moved from Home Cooking board]
I had one theother day in a belgium restaurant and it was basically a grilled ham and chees sandwhich. Whem my mom used to make them for me they were grilled cheese and ham dipped in egg as like french toast and then cooked
what is the real thing?
re: tacostacoseverywhere
Actually the sandwich with the egg is called a "Croque Madame."
To respond to the OP..... A traditional Croque Monsieur has thinly sliced ham, Gruyere cheese, Bechamel sauce, Dijon mustard, French bread.
The components of the sandwich are layered between buttered slices of bread then the whole sandwich is toasted in a frying pan with melted butter. There are Croque Monsieur sandwich presses that can be used instead of the fry pan.
re: Gio
Actually a Croques Madame has a fried egg on top of the sandwich. OP meant her mom dipped the bread in a beaten egg when making Croques Monsieur. I have heard of it made this way before, however, it is not the "proper" way to make a Croques Monsieur. The method Gio mentions is more of a traditional CM, though I don't think they go in a sandwich press, I have only ever seen them baked.
This is the recipe I have used by Jacques Pepin and Julia Child...
re: SweetPea914
Julia has several varations on the theme of the tradiditional CM. In one she uses mozzarella, of all things. I remember her PBS show on the CM, and in her wonderful vocal vibrato proclaimed the Croque Monsieur the king of sandwiches. It seems to me that she used a simple sandwich press - not the panini press we're used to seeing now-a-days, tho
re: SweetPea914
I know, odd!!.... here's the link to the Boston Globe article about Julia's updated mozz CM:
Nice photo of her, too.
re: SweetPea914
gio-- what she said. believe me, i'm aware of the difference between those two sammies. virtually everytime i've seen a croque monsieur it has been dipped in egg like pain perdu, then sauteed. croque madames have both an egg, and sometimes hollandaise (if you feel like getting totally heart healthy) as an accompaniment.
When I've had them they usually have cheese or toasted cheese sauce on the top too, and are meant to be eaten with a knife and fork. But yes, essentially a baked ham and cheese. I don't think they are usually dipped in a batter, but they are also usually softer than a typical soaked-in-butter American-style grilled cheese sandwich. So make toast, put ham and cheese in between with either mustard or butter, bake or grill until hot, top with cheese or cheese sauce, broil that until melty and a crust (croque) forms and eat.
Hi, it may not be completely traditional, but I do enjoy the type of croque monsieur they serve at L'Express (corner of 20th and Park Avenue South). This is (I believe) ham, gruyere, swiss, and a bechamel sauce between two pieces of bread (probably slices of a french Boule) The mixture is then dredged through a bit of egg and a lot of grated cheese (not sure what type of cheese, though it tastes pretty sharp, I might even say parmigiano, but I'm not sure). Then it's pan seared to create a crusty cheesy outside. Then I have the dijon mustard on the side. Really lovely.
after having my frist croque madame at a little patisserie by an alsatian baker... i tend to veer towards his component selection.
slice of brioche, spread a thick layer of bechamel, paris ham, swiss/emmenthal. i put two layers of this and an extra piece of brioche into the oven/toaster oven and let broil until melty and neary good crustiness and then layer up. croque monsieur. but we're gluttons and put on a very yolky fried egg to make it a madame.
from the sounds of it... the battering with the egg makes it closer to a monte cristo, which is suspected to be an american version of the croque monsieur.
As far as I know, a croque-monsieur CAN be dipped in egg like French toast, but it's not necessary.
A croque-madame has a fried egg on top, as mentioned by others.
re: Megiac
A monte cristo also also has turkey and is topped with powdered sugar.
Croque monsier has ham, gruyere/swiss and bechamel on the inside of two slices of bread which are buttered and fried. I like to take it the extra step and add more cheese to the top and then briol.
The recipe I got from Williams-Sonoma doesn't call for dipping the bread in the egg mixture ala french toast. That and the version I had at the Girl & the Fig at Sonoma is more an open faced sandwich - you don't press the slices of bread together.
So basically it's brioche, ham, bechamel sauce, grated gruyere layers, in that order. We usually add egg on top - love the runny yolk all over.
Kind of reminds me in some way to our other favorite breakfast food - eggs benedict (english muffin, canadian bacon, poached egg, hollandise).
This sounds more like a montechristo, minus the turkey.
The croque-monsieurs from my childhood consisted of ham, gruyere and bechamel sauce layered between toasted slices of white bread, more bechamel and cheese on top of it, then broiled until golden.
Croque-madame differs according to where you are in France. In some regions, it's a croque-monsieur with a sunny side up on top, in others it's a croque monsieur where the ham is replaced by a thinly sliced hot dog or frank.
This was an interesting discussion to read. I never realized there were so many variaitions. For a brunch crowd, I'll make it a crosque monsieur casserole where I take (hamd and gruyere) sandwiches, put it in a buttered 9x13 pan, pour the egg mixture over and let it sit overnight and then bake--kind of a strata idea. Quick and easy when you're having a lot of people over.
re: chowser
Chowser, do you use mustard inside the sandwich? I'm going to try your casserole very soon. Sounds like just the thing for a Football Sunday!
re: chowser
Oh, me too.......
Interesting about the maple syrup. I never would have thought to add it to an egg mixture. Many thanks!


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Pump Patrol: Save 18 cents a gallon
Published On: Feb 29 2012 02:18:42 PM CST
Updated On: Jul 25 2012 12:40:05 PM CDT
Updated On: Jul 25 2012 12:40:05 PM CDT
The average cost of a gallon of gas in Houston is $3.36. But some stations around town sold regular gas for 18 cents less.
The following three stations sold gas for less on Wednesday, according to www.GasBuddy.com.
- Buc-ee's, Crabb River Road near Sansbury Boulevard in Richmond: $3.18
- Valero, Barker Cypress Road near Longenbaugh Drive in Cypress: $3.18
- Kroger, Highway 6 near Airport Boulevard in Sugar Land: $3.19
More Information:
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text: Jihadists 'Plot to Take over Birmingham Schools' Print article Send a Tip by Nick Hallett 7 Mar 2014 post a comment Education chiefs in Birmingham are investigating an alleged plot by Islamic fundamentalists to take over schools by ousting head teachers and staff members through a dirty tricks campaign. The Birmingham Mail reports that it has received documents purporting to show that jihadists are launching an operation called 'Trojan Horse' through which they plan to orchestrate false allegations against staff members. Once the original staff members are ousted, the group allegedly plan to install their own supporters in positions of influence and encourage the schools to educate children in strict Islamic principles. They would then apply for academy status, allowing them to be taken out of local authority control, and thus be freer to run the school along strict Islamic lines. The plot to oust existing staff is said to involve recruiting hard-line parents to spread false allegations. One document says: "We have caused a great amount of organised disruption in Birmingham and as a result we now have our own Academies and are on our way to getting rid of more head teachers and taking over their schools. "Whilst sometimes the practices we use may not seem the correct way to do things you must remember this is a 'Jihad' and as such all means possible to win the war is acceptable." Ofsted inspectors launched a surprise inspection this week at Park View Academy in Birmingham after complaints of discrimination against non-Muslim staff. Meanwhile, the Birmingham Mail also reported that a fraud investigation has been reopened at a local primary school after allegations of 'faked' resignation letters. This school is also mentioned in the documents. The documents also mention an alleged plot to oust the head teacher at another school by 'planting the seed' of allegations of cheating in SAT tests. The head teacher and her deputy both resigned last year after education chiefs rejected the school's SAT results due to exactly those allegations. Also mentioned are plans to roll out the 'Trojan Horse' scheme to other areas including Manchester and Bradford. In correspondence between one Islamist in Birmingham and another in Bradford, the Birmingham fundamentalist writes: "Operation 'Trojan Horse' has been very carefully thought through and is tried and tested within Birmingham, implementing it in Bradford will not be difficult for you." It states that 'Trojan Horse' is "totally invisible to the naked eye and allows us to operate under the radar. I have detailed the plan we have in Birmingham and how well it has worked and you will see how easy the whole process is to get the whole process is to get the head teacher out and our own person in." It also adds: "The poor performing schools are easy to disrupt, the better performing with strong head teachers is much harder and so we have to manufacture a strong enough reason, but rest assured we have not failed yet, no matter how difficult removing the head teacher may be. You just have to be clever and find the most appropriate way to deal with the school."


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Just clearing out some laptops dating from 2002 to 2005, was going to send to recycling place (free) but questions have been asked about making money from them!
I can't see that we'd make anything more than a few quid by selling parts, plus I'd have to store them somewhere whilst up for sale.
Any thoughts?


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Arizona State Board of Nursing http://www.azbn.gov/
Applying for certification and licensure http://www.azbn.gov/Applications.aspx
Best Nursing Degree http://www.bestnursingdegree.com/
National League for Nursing http://www.nln.org/
National League for Nursing Accrediting Commission http://www.nlnac.org/
Maricopa Community Colleges Healthcare Education HIPAA Assessment (not HIPPA)
Arizona Nurses Foundation Academic Scholarship Program.
Up to $25,000 in scholarships awarded each semester!
The Arizona Nurses Foundation provides scholarships to help support entry into professional nursing and for career mobility within nursing. Scholarships are based on the applicant's merit and financial need. Students may be enrolled part-time or full-time.
Scholarships are available for nursing students who are enrolled or accepted for enrollment or currently enrolled in nursing schools and are committed to nursing practice in Arizona.
Applications forms and guidelines are available at www.aznurse.org; click on the Foundation tab.
Application Deadlines: Fall: March 1 Spring: October 1
Arizona Nurses Foundation
1850 E. Southern Avenue, Suite 1
Tempe, AZ 85282
480.831.0404 x 100
www.aznurse.org (Flyer)
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LEE COUNTY, Fla.- A Lee County Deputy who shot and killed a woman this summer will not face criminal charges. Deputies say the suspect, Donna Weaver of Bonita Springs threatened them and refused to put down her pistol.
The State Attorneys Office released its review of the sheriff's office investigation and cleared the deputy.
State Attorney Steve Russell wrote in part, "I am convinced that the actions of the officer involved in this incident were a legally justifiable use of deadly force, in defense of himself or others."
The shooting happened on July 29, 2013. Deputies responded to a home on Okeana Street in Bonita Springs after several calls to 911.
Callers reported Donna Weaver was drunk, destroying property, and "beating on" her boyfriend.
Toxicology result show Weaver had a BAC of .219.
In a sworn statement, Deputy Christopher Meyer, who shot Weaver, said he could hear someone say she was coming out of the home with a gun.
Deputy Meyer described Weaver standing outside her door brandishing a pistol and saying "I've got one chambered for you. You guys are gonna have to (expletive) kill me."
The deputy also told prosecutors "I was in fear for my safety. I thought she was gonna shoot me."
The report states a surveillance camera from a neighbors back yard picked up audio from the incident. The report states deputies could be heard ordering Weaver to put the gun down more than 14 times, with Weaver responding with expletives, followed by the sound of three gunshots.
Investigators said Deputy Meyer fired three times. Weaver died of a gunshot wound to the chest.
In an earlier interview with WINK News, Lee County Sheriff Mike Scott said Weaver had every opportunity to step down peacefully.
"Our deputies ordered her several times to drop a weapon and she refused to do that so deadly force was met with deadly force," he said.
The State Attorneys Office said it found nothing that would contradict the sheriff's office investigation, and Deputy Meyer's use of deadly force was justified.
The case is considered closed.


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21-year-old wounded in drive-by shooting in Orlando
Published On: Mar 28 2014 09:07:07 AM EDT
Updated On: Mar 28 2014 09:45:54 AM EDT
Updated On: Mar 28 2014 09:45:54 AM EDT
A 21-year-old was shot in a drive-by shooting Friday morning in Orlando, according to police.
The victim was taken to Orlando Regional Medical Center in stable condition after the shooting, which occurred around 3:40 a.m. at 5255 Cinderlane Parkway at The Reserve At Rosemont apartments.
Orlando police said someone inside a silver vehicle with tinted windows fired shots as it drove by the area. The victim was struck in the right leg and taken to the hospital.
No other details, including the gender of the victim, were released by police.
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Sunday, April 15, 2012
From the small screen to the red carpet… The glitz, glamour and for some – kinda weird fashion was out in force at Crown Casino for the 2012 TV Week Logie Awards...
Take a look through these Red Carpet snaps and tell us – Who was the best or worst dressed?
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Hells Bells! AC/DC is hitting their Australian homeland's highway to hell to tour Down Under.More


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INSIDIOUS CHAPTER 2 contest winner directs DEAD afterlife teaser trailer
Michael James Grise, winner of the INSIDIOUS CHAPTER 2 contest, directed the latest DEAD afterlife teaser trailer. Michael was hand picked by James Wan for the video he had made for the INSIDIOUS CHAPTER 2 contest and won a walk on role to that highly anticipated sequel. Now, Michael has crafted a great concept video for DEAD afterlife, one that is sure to make James Wan proud… again. Directed by Michael James Grise Special thanks … Continue reading


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No one gets to the top of the pop charts by being a complete idiot. Well, of course it happens for some folks once or twice. But besides a knack for hooks, it takes some serious sharp thinking to stay there. So don't make the mistake of thinking the rapper/producer/all-around svengali Lil Jon is ... More >>
via lilwayne-online.comDid Weezy make the cut?Self-proclaimed "greatest rapper alive" Lil Wayne performs Sunday at the BankAtlantic Center in Sunrise. That boast had serious clout when Tha Carter III dropped last year. But with Weezy's ruinous rock album Rebirth in delay hell -- latest news has i ... More >>
The South Florida native is the new producer king.
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Dirty South documents the triumph of down-home hip-hop
Stankonia (Arista/LaFace)


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Added Oct 17, 2012
by sweetharte
Toronto, Canada
This is a dress essentially made from a t-shirt pattern. I added a waist band and serged a 1 1/2" wide elastic in to it, and for the skirt I improved a simple aline. Use a twin needle to complete your sleeves, hem and neckline for a real professional finish. This dress has no zippers or buttons so it is as easy to make as it is to wear! It feels good to look luxurious in something so simple!
For this kind of dress you must have a stretch velvet, and I find it much easier to work with one that doesn't have a nap.
Nov 20, 2012, 07.40 PMby Kim Mullins
wow totally gorgeous dress. you look stunning in it. tutorial?? big smile please* best convincing big eye look* or if not a drawing of how you did it????
Oct 17, 2012, 07.38 PMby memoryseed
Very cute! Love the dress and the color is amazing on you!
Oct 17, 2012, 06.07 PMby Linda74Sews
Lovely dress. I'm not as experienced working with knits, no less stretch velvet, so I am impressed.
Oct 17, 2012, 05.23 PMby mwelland
Looks amazing! The colour is fantastic with your hair. I love your modeling too!
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