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Although an increasing number of small interfering RNA (siRNA) therapies are reaching the market, the challenge of efficient extra-hepatic delivery continues to limit their full therapeutic potential. Drug delivery vehicles and hydrophobic conjugates are being used to overcome the delivery bottleneck. Previously, we reported a novel dendritic conjugate that can be appended efficiently to oligonucleotides, allowing them to bind albumin with nanomolar affinity. Here, we explore the ability of this novel albumin-binding conjugate to improve the delivery of siRNA in vivo. We demonstrate that the conjugate binds albumin exclusively in circulation and extravasates to various organs, enabling effective gene silencing. Notably, we show that the conjugate achieves a balance between hydrophobicity and safety, as it significantly reduces the side effects associated with siRNA interactions with blood components, which are commonly observed in some hydrophobically conjugated siRNAs. In addition, it reduces siRNA monocyte uptake, which may lead to cytokine/inflammatory responses. This work showcases the potential of using this dendritic conjugate as a selective albumin binding handle for the effective and safe delivery of nucleic acid therapeutics. We envision that these properties may pave the way for new opportunities to overcome delivery hurdles of oligonucleotides in future applications.

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP-Lbp UNA-siRNA) on the development of fatty liver. Weekly LNP-Lbp UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD). In mice fed a high-fat and high-sucrose diet, which displayed induced liver lipogenesis, LBP downregulation led to reduced liver lipid accumulation, lipogenesis (mainly stearoyl-coenzyme A desaturase 1 [Scd1]) and lipid peroxidation-associated oxidative stress markers. LNP-Lbp UNA-siRNA also resulted in significantly decreased blood glucose levels during an insulin tolerance test. In mice fed a standard chow diet or an MCD, in which liver lipogenesis was not induced or was inhibited (especially Scd1 mRNA), liver LBP downregulation did not impact on liver steatosis. The link between hepatocyte LBP and lipogenesis was further confirmed in palmitate-treated Hepa1-6 cells, in primary human hepatocytes, and in subjects with morbid obesity. Altogether, these data indicate that siRNA against liver Lbp mRNA constitutes a potential target therapy for obesity-associated fatty liver through the modulation of hepatic Scd1.

The objective of this study was to generate small interfering RNA (siRNA) to knockdown antiviral chemokine-related genes in fetal rhesus monkey kidney (FRhK-4) cells. We generated siRNA duplexes to suppress antiviral chemokines like CXCL10 and CCL4 in FRhK-4 cells by downregulating interferon regulatory factor (IRF) 3 and IRF7. Three siRNA duplexes (si-F-IRF3-1, si-F-IRF3-2, and si-F-IRF3-3) targeting IRF3, and one siRNA duplex (si-F-IRF7) targeting IRF7 were generated. A nontarget siRNA duplex was used as the negative control. The nontarget or target siRNA duplexes (si-F-IRF3-1, si-F-IRF3-2, si-F-IRF3-3, and si-F-IRF7) were transfected into FRhK-4 cells using transfection reagents, and they were then incubated at 37°C for 6 h with 5% CO2. After 6 h, the medium was removed, and fresh medium was added to each cell, and they were then incubated at 37°C for 48 h with 5% CO2. The transfected FRhK-4 cells were infected with hepatitis A virus (HAV) HM-175/18f (viral titer: 105 PFU/mL) and incubated at 37°C for 3 h with 5% CO2 for HAV propagation. The expression levels of chemokines, including CXCL10 and CCL4, under the regulation of IRF3 and IRF7 in the transfected FRhK-4 cells were measured using quantitative real-time polymerase chain reaction after 3 h of HAV infection. The results indicated that CXCL10 and CCL4 expression levels were decreased in FRhK-4 cells transfected with si-F-IRF3-1, si-F-IRF3-3, or si-F-IRF7 (p

Background: A new coronavirus was identified in Jeddah, Saudi Arabia in 2012 and designated as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). To date, this virus has been reported in 27 countries. The virus transmission to humans has already been reported from camels. Currently, there is no vaccine or antiviral therapy available against this virus. Methods: The siRNAs were in silico predicted, designed, and chemically synthesized by using the MERS-CoV-orf1ab region as a target. The antiviral activity was experimentally evaluated by delivering the siRNAs with Lipofectamine™ 2000 and JetPRIMER as transfection reagents in both Vero cell and HEK-293-T cell lines at two different concentrations (10.0 nM and 5.0 nM). The Ct value of quantitative Real-Time PCR (qRT-PCR) was used to calculate and determine the reduction of viral RNA level in both cell supernatant and cell lysate isolated from both cell lines. Results: The sequence alignment resulted in the selection of highly conserved regions. The orf1ab region was used to predict and design the siRNAs and a total of twenty-one siRNAs were finally selected from four hundred and twenty-six siRNAs generated by online software. Inhibition of viral replication and significant reduction of viral RNA was observed against selected siRNAs in both cell lines at both concentrations. Based on the Ct value, the siRNAs # 11, 12, 18, and 20 were observed to be the best performing in both cell lines at both concentrations. Conclusion: Based on the results and data analysis, it is concluded that the use of two different transfection reagents was significantly effective. But the Lipofectamine™ 2000 was found to be a better transfection reagent than the JetPRIMER for the delivery of siRNAs in both cell lines.

Objectives: The COVID-19 was identified for the first time from the sea food market, Wuhan city, China in 2019 and the pathogenic organism was identified as SARS-CoV-2. Currently, this virus has spread to 223 countries and territories and known as a serious issue for the global human community. Many vaccines have been developed and used for immunization. Methods: We have reported the insilico prediction, designing, secondary structure prediction, molecular docking analysis, and in vitro assessment of siRNAs against SARS-CoV-2. The online bioinformatic approach was used for siRNAs selection and designing. The selected siRNAs were evaluated for antiviral efficacy by using Lipofectamine 2000 as delivery agent to HEK-293 cells. The MTT assay was used for cytotoxicity determination. The antiviral efficacy of potential siRNAs was determined based on the Ct value of q-RT-PCR and the data analysis was done by Prism-GraphPad software. Results: The analyzed data resulted in the selection of only three siRNAs out of twenty-six siRNAs generated by online software. The secondary structure prediction and molecular docking analysis of siRNAs revealed the efficient binding to the target. There was no cellular toxicity observed in the HEK-293 cells at any tested concentrations of siRNAs. The purification of RNA was completed from inoculated cells and subjected to q-RT-PCR. The highest Ct value was observed in siRNA 3 than the others. The results offered valuable evidence and invigorated us to assess the potency of siRNAs by using alone or in combination in other human cells. Conclusion: The data generated from this study indicates the significance of in silico prediction and narrow down the potential siRNA' against SARS-CoV-2, and molecular docking investigation offered the effective siRNAs binding with the target. Finally, it is concluded that the online bioinformatics approach provided the prediction and selection of siRNAs with better antiviral efficacy. The siRNA-3 was observed to be the best for reduction of viral RNA in cells.

Objective: The acute cases of pneumonia (COVID-19) were first reported from China in December 2019, and the pathogen was identified as SARS-CoV-2. Currently, many vaccines have been developed against this virus by using multiple genes, applying different platforms, and used for the vaccinations of the human population. Spike protein genes play an important role in host cell attachment and viral entry and have been extensively used for the development of vaccine and antiviral therapeutics. Short interfering RNA is also known as silencing RNA and contribute a significant role to regulate the expression of a specific gene. By using this technology, virus inhibition has been demonstrated against many viral diseases. Methods: In this work, we have reported the Insilico prediction, designing, and experimental validation of siRNAs antiviral potency against SARS-CoV-2-S-RBD. The siDirect 2.0 was selected for siRNAs prediction, and secondary structure was predicted by RNAfold while the HNADOCK was used for molecular docking analysis and specific binding of siRNAs to the selected target. We have used and evaluated four siRNAs for cellular toxicity and determination of antiviral efficiency based on the Ct value of q-real-time PCR in Vero E6 cells. Results: Based on the experimental evaluation and analysis of results from generated data, we observed that there is no cytotoxicity for any tested siRNAs in Vero E6 cells. Total four siRNA were filtered out from twenty-one siRNAs following the strict selection and scoring criteria. The better antiviral efficiency was observed in 3rd siRNAs based on the Ct value of q-real-time PCR. The results that emerged from this study encouraged us to validate the efficiency of these siRNAs in multiple cells by using alone and in a combination of two or more siRNAs to inhibit the SARS-CoV-2 proliferation. Conclusion: The Insilico prediction, molecular docking analysis provided the selection of better siRNAs. Based on the experimental evaluation only 3rd siRNA was found to be more effective than others and showed better antiviral efficiency. These siRNAs should also be evaluated in other cell lines either separately or in combination against SARS-CoV-2 to determine their antiviral efficiency.

The Middle East Respiratory Syndrome Coronavirus is well known to cause respiratory syndrome and this virus was identified and isolated for the first time from Jeddah, Saudi Arabia in 2012 from infected patient. In this report, we have conducted the in-silico prediction, designing and evaluation of siRNAs targeting Middle East Respiratory Syndrome Coronavirus orf1ab gene to inhibit the virus replication. By using bioinformatics software, total twenty-one functional, off-target reduced siRNA were selected from four hundred and sixty-two siRNAs based on their greater potency and specificity. We have evaluated only seven siRNAs to analyze their performance and efficacy as antivirals by reverse transfection approach in Vero cells. There was no cytotoxicity of siRNAs at various concentrations was observed in Vero cells. Based on the real-time PCR results, better inhibition of viral replication was observed in the siRNA-1 and 4 as compared to other siRNAs. The results generated from this work provided suitable information about the efficacy of siRNAs which encouraged us to further evaluate the remaining siRNAs to determine their inhibitory effect on the virus replication. We concluded that the insilico prediction and designing resulted in the screening of potential siRNAs with better efficiency, and strength. This can be used to develop oligonucleotide-based antiviral therapeutics against MERS-CoV in the near future.

Background: The MERS-CoV was identified for the first time from Jeddah, Saudi Arabia in 2012 from a hospitalized patient. This virus has now been spread to 27 countries with a total of 858 deaths and 2494 confirmed cases and has become a serious concern for the human population. Camels are well known for the transmission of the virus to the human population. Methods: In this report, we have discussed the designing, prediction, and evaluation of potential siRNAs against the orf1ab gene of MERS-CoV. The online software was used to predict and design the siRNAs and finally, total twenty-one siRNA were filtered out from four hundred and sixty-two sIRNAs as per their scoring and specificity criteria. We have used only ten siRNAs to evaluate their cytotoxicity and efficacy by reverse transfection approach in HEK-293-T cell lines. Results: Based on the results and data generated; no cytotoxicity was observed for any siRNAs at various concentrations in HEK-293-T cells. The ct value of real-time PCR showed the inhibition of viral replication in siRNA-1, 2, 4, 6, and 9. The data generated provided the preliminary information and encouraged us to evaluate the remaining siRNAs separately as well as in combination to analyses the replication of MERS-CoV inhibition in other cell lines. Conclusion: Based on the results obtained; it is concluded that the prediction of siRNAs using online software resulted in the filtration of potential siRNAs with high accuracy and strength. This technology can be used to design and develop antiviral therapy not only for MERS-CoV but also against other viruses.

Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo. Here, we systematically evaluated the impact of number, structure, and sequence context of PS-modified backbones on cellular uptake and RNAi-mediated silencing efficacy. We find that PS enhances cellular internalization in a sequence-dependent manner but only when present in a single-stranded but not double-stranded region. Furthermore, the observed increase in cellular internalization did not correlate with functional silencing improvement, indicating that PS-mediated uptake may drive compounds to non-productive sinks. Thus, the primary contributing factor of PS modifications to functional efficacy is likely stabilization rather than enhanced cellular uptake. A better understanding of the relative impact of different chemistries on productive versus non-productive uptake will assist in improved design of therapeutic RNAs.

Primary and secondary prevention protocols aim at reducing the plasma levels of lipids - with particular reference to low-density lipoprotein cholesterol (LDL-C) plasma concentrations – in order to improve the overall survival and reduce the occurrence of major adverse cardiovascular events. The use of statins has been widely considered as the first-line approach in lipids management as they can dramatically impact on the cardiovascular risk profile of individuals. The introduction of ezetimibe and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors overcame the adverse effects of statins and ameliorate the achievement of the target lipids levels. Indeed, advances in therapies promote the use of specific molecules – i.e. short strands of RNA named small-interfering RNAs (siRNAs) – to suppress the transcription of genes related to lipids metabolism. Recently, the inclisiran has been developed: this is a siRNA able to block the mRNA of the PCSK9 gene. About 50% reduction in low-density lipoprotein cholesterol levels have been observed in randomized controlled trials with inclisiran. The aim of this review was to summarize the literature regarding inclisiran and its possible role in the general management of patients with lipid disorders and/or in primary/secondary prevention protocols.

The objective of this study was to generate small interfering RNA (siRNA) to knockdown antiviral chemokine-related genes in fetal rhesus monkey kidney (FRhK-4) cells. We generated siRNA duplexes to suppress antiviral chemokines like CXCL10 and CCL4 in FRhK-4 cells by downregulating interferon regulatory factor (IRF) 3 and IRF7. Three siRNA duplexes (si-F-IRF3-1, si-F-IRF3-2, and si-F-IRF3-3) targeting IRF3, and one siRNA duplex (si-F-IRF7) targeting IRF7 were generated. A nontarget siRNA duplex was used as the negative control. The nontarget or target siRNA duplexes (si-F-IRF3-1, si-F-IRF3-2, si-F-IRF3-3, and si-F-IRF7) were transfected into FRhK-4 cells using transfection reagents, and they were then incubated at 37°C for 6 h with 5% CO 2 . After 6 h, the medium was removed, and fresh medium was added to each cell, and they were then incubated at 37°C for 48 h with 5% CO 2 . The transfected FRhK-4 cells were infected with hepatitis A virus (HAV) HM-175/18f (viral titer: 10 5  PFU/mL) and incubated at 37°C for 3 h with 5% CO 2 for HAV propagation. The expression levels of chemokines, including CXCL10 and CCL4, under the regulation of IRF3 and IRF7 in the transfected FRhK-4 cells were measured using quantitative real-time polymerase chain reaction after 3 h of HAV infection. The results indicated that CXCL10 and CCL4 expression levels were decreased in FRhK-4 cells transfected with si-F-IRF3-1, si-F-IRF3-3, or si-F-IRF7 (p < 0.05) compared to those in the negative control. These results indicate that si-F-IRF3-1 and si-F-IRF3-3, and si-F-IRF7 successfully knocked down IRF3 and IRF7 in FRhK-4 cells, respectively and suppressed antiviral chemokines. These siRNAs could be used to suppress antiviral chemokines in FRhK-4 cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels. As expected, the MCD increased hepatocyte vacuolation, liver inflammation and fibrosis markers, also increasing liver Lbp mRNA. In this model, liver Lbp gene knockdown resulted in a pronounced worsening of the markers of liver inflammation (also including CD68 and MPO activity), fibrosis, ER stress and protein carbonyl levels, all indicative of non-alcoholic steatohepatitis (NASH) progression. At cellular level, Lbp gene knockdown also increased expression of the proinflammatory mediators (Il6, Ccl2), and markers of fibrosis (Col1a1, Tgfb1) and protein carbonyl levels. In agreement with these findings, liver LBP mRNA in humans positively correlated with markers of liver damage (circulating hsCRP, ALT activity, liver CRP and KRT18 gene expression), and with a network of genes involved in liver inflammation, innate and adaptive immune system, endoplasmic reticulum stress and neutrophil degranulation (all with q-value<0.05). In conclusion, current findings suggest that a significant downregulation in liver LBP levels promotes liver oxidative stress and inflammation, aggravating NASH progression, in physiological and pathological non-obesogenic conditions., Competing Interests: Conflict of interest Ramon Díaz-Trelles, Rajesh Mukthavaram, Priya P. Karmali, Kiyoshi Tachikawa and Pad Chivukula are employees of Arcturus Therapeutics. The authors declared no additional conflict of interest., (Copyright © 2022. Published by Elsevier Ltd.)

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are crucial for plant growth and development via mediating post-transcriptional gene silencing. In wild-type Arabidopsis, DICER-LIKE 2 (DCL2)-dependent 22-nt siRNAs are rare, whereas DCL1 and DCL4-dependent 21-nt miRNAs and siRNAs are highly abundant. DCL4 naturally inhibits DCL2 in producing abundant 22-nt siRNAs from endogenous transcripts, but whether DCL1 suppresses endogenous 22-nt siRNA production and the extent of repression are still unknown. Here, we report that DCL1 and DCL2 cleaved both miRNA precursors and coding transcript-derived double-stranded RNAs. In a dcl1 dcl4 double mutant, massive 22-nt siRNAs were produced from endogenous protein-coding genes (genic siRNAs). Compared with wild-type, the 22-nt genic siRNAs derived from the Nitrate Reductase 1 (NIA1), NIA2, DIACYLGLYCEROL ACYLTRANSFERASES 3 (DGAT3), SUPPRESSOR OF MAX2 1-LIKE 5 (SMXL5), and SMXL4 in dcl1 dcl4 increased up to 95%. Our analysis further indicated that the 22-nt genic siRNAs in dcl1 dcl4 were mainly loaded into ARGONAUTE 1 (AGO1) or AGO2. Thus, our results demonstrated that both DCL1 and DCL4 safeguard post-transcriptional gene silencing, preventing the production of DCL2-dependent 22-nt genic siRNAs from disrupting plant growth and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

COVID-19 is a newly emerged viral disease that is currently affecting the whole globe. A variety of therapeutic approaches are underway to block the SARS-CoV-2 virus. Among these methods, siRNAs could be a safe and specific option, as they have been tested against other viruses. siRNAs are a class of inhibitor RNAs that act promisingly as mRNA expression blockers and they can be designed to interfere with viral mRNA to block virus replication. In order to do this, we designed and evaluated the efficacy of six highly specific siRNAs, which target essential viral mRNAs with no predicted human genome off-targets. We observed a significant reduction in the copy number viral mRNAs after treatment with the siRNAs, and are expected to inhibit virus replication. We propose siRNAs as a potential co-therapy for acute SARS-CoV-2 infection., Competing Interests: This work has been partially funded by the Natural Sciences and Engineering Research Council of Canada (NSERC). All other authors declare no competing interests., (© 2021 Elsevier B.V. All rights reserved.)

Liquid chromatography-mass spectrometry has been widely implemented as a powerful tool for providing in-depth characterization of nucleic acid therapeutic modalities, such as anti-sense oligonucleotides and small interfering RNAs (siRNAs). In this study, we developed a generic hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry method in the absence of ion-pairing reagents and demonstrated its capability as an attractive and robust alternative for oligonucleotide and siRNA analysis. HILIC separation of mixtures of unmodified and fully phosphorothioate-modified DNA oligonucleotides and their synthetic 3' exonuclease-digested metabolites were also assessed. High-resolution mass spectrometric (HRMS) analysis was used to determine the deconvoluted masses of oligonucleotide and siRNA standards and their impurities. To enable unbiased sequence characterization with tandem mass spectrometry (MS/MS), we also optimized higher-energy C-trap dissociation (HCD) on improving the sequence coverage of DNA and RNA oligonucleotides. Lastly, we evaluated on-column sensitivity for a phosphorothioate oligonucleotide by performing targeted analysis with either targeted selected ion monitoring (tSIM) or parallel reaction monitoring (PRM). Higher on-column sensitivity of 13 ng, equivalent to 2.0 pmol, of a phosphorothioate oligonucleotide was achieved by tSIM analysis as compared to PRM analysis., Competing Interests: Declaration of Competing Interest All authors were employees of Regeneron Pharmaceuticals, Inc while engaged in the study and may hold stock and/or stock options in the company. All authors have patent applications that were based on the research in the paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The MERS-CoV was identified for the first time from Jeddah, Saudi Arabia in 2012 from a hospitalized patient. This virus has now been spread to 27 countries with a total of 858 deaths and 2494 confirmed cases and has become a serious concern for the human population. Camels are well known for the transmission of the virus to the human population., Methods: In this report, we have discussed the designing, prediction, and evaluation of potential siRNAs against the orf1ab gene of MERS-CoV. The online software was used to predict and design the siRNAs and finally, total twenty-one siRNA were filtered out from four hundred and sixty-two sIRNAs as per their scoring and specificity criteria. We have used only ten siRNAs to evaluate their cytotoxicity and efficacy by reverse transfection approach in HEK-293-T cell lines., Results: Based on the results and data generated; no cytotoxicity was observed for any siRNAs at various concentrations in HEK-293-T cells. The ct value of real-time PCR showed the inhibition of viral replication in siRNA-1, 2, 4, 6, and 9. The data generated provided the preliminary information and encouraged us to evaluate the remaining siRNAs separately as well as in combination to analyses the replication of MERS-CoV inhibition in other cell lines., Conclusion: Based on the results obtained; it is concluded that the prediction of siRNAs using online software resulted in the filtration of potential siRNAs with high accuracy and strength. This technology can be used to design and develop antiviral therapy not only for MERS-CoV but also against other viruses., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Epigenetic gene regulation is important for proper development and gene expression in eukaryotes. Maize has a large and complex genome that includes abundant repetitive sequences which are frequently silenced by epigenetic mechanisms, making it an ideal organism to study epigenetic gene regulation. Epigenetic modifications are chromosome-bound, heritable changes to the genome that do not affect the DNA sequence, and can include DNA methylation, histone modification, and RNA processing. Our appreciation and understanding of epigenetic regulation has grown with the field since its inception ∼65 years ago. Early examples of epigenetic regulation were often associated with transposable elements, starting with McClintock's early work in the 1950s. The observation of other intriguing phenotypes segregating in non-Mendelian ratios in the 1950s provided material for genetic screens that allowed for mechanistic studies of epigenetic regulation that have come to fruition within the past 20 years. The relationship between epigenetic mechanisms and genome organization has become clear with the application of new technologies to characterize maize epigenomes. Our understanding of epigenetic control of gene expression now encompasses the context of genes relative to DNA methylation, chromatin structure, and transposable element content., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background & Aims: Oncogene polycomb group protein enhancer of zeste homolog 2 (EZH2) has been proposed to be a target gene of putative tumor suppressor microRNA-101 (miR-101). The aim of our study was to investigate the functional role of both miR-101 and EZH2 in human hepatocellular carcinoma (HCC)., Methods: MiR-101 and EZH2 expressions were evaluated in tumor tissues of 99 HCC patients and 7 liver cancer cell lines by real-time PCR. Luciferase reporter assay was employed to validate whether EZH2 represents a target gene of miR-101. The effect of miR-101 on HCC growth as well as programmed cell death was studied in vitro and in vivo., Results: MiR-101 expression was significantly downregulated in most of HCC tissues and all cell lines, whereas EZH2 was significantly overexpressed in most of HCC tissues and all cell lines. There was a negative correlation between expression levels of miR-101 and EZH2. Luciferase assay results confirmed EZH2 as a direct target gene of miR-101, which negatively regulates EZH2 expression in HCC. Ectopic overexpression of miR-101 dramatically repressed proliferation, invasion, colony formation as well as cell cycle progression in vitro and suppressed tumorigenicity in vivo. Furthermore, miR-101 inhibited autophagy and synergized with either doxorubicin or fluorouracil to induce apoptosis in tumor cells., Conclusion: Tumor suppressor miR-101 represses HCC progression through directly targeting EZH2 oncogene and sensitizes liver cancer cells to chemotherapeutic treatment. Our findings provide significant insights into molecular mechanisms of hepatocarcinogenesis and may have clinical relevance for the development of novel targeted therapies for HCC., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Centromere protein F (CENPF) is an essential nuclear protein associated with the centromere-kinetochore complex and plays a critical role in chromosome segregation during mitosis. Up-regulation of CENPF expression has previously been detected in several solid tumors. In this study, we aim to study the expression and functional role of CENPF in hepatocellular carcinoma (HCC). We found CENPF was frequently overexpressed in HCC as compared with non-tumor tissue. Up-regulated CENPF expression in HCC was positively correlated with serum AFP, venous invasion, advanced differentiation stage and a shorter overall survival. Cox regression analysis found that overexpression of CENPF was an independent prognosis factor in HCC. Functional studies found that silencing CENPF could decrease the ability of the cells to proliferate, form colonies and induce tumor formation in nude mice. Silencing CENPF also resulted in the cell cycle arrest at G2/M checkpoint by down-regulating cell cycle proteins cdc2 and cyclin B1. Our data suggest that CENPF is frequently overexpressed in HCC and plays a critical role in driving HCC tumorigenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)