Publications by authors named "Qihong Huang"

85 Publications

Recognition of pathogens in food matrixes based on the untargeted in vivo microbial metabolite profiling via a novel SPME/GC × GC-QTOFMS approach.

Food Res Int 2021 Apr 14;142:110213. Epub 2021 Feb 14.

Guangdong Provincial Engineering Research Center for Ambient Mass Spectrometry, Guangdong Provincial Key Laboratory of Emergency Test for Dangerous Chemicals, Institute of Analysis, Guangdong Academy of Sciences(China National Analytical Center, Guangzhou), Guangzhou 510070, China. Electronic address:

Foodborne diseases incurred by pathogenic bacteria are one of the major threats in food safety, and thus it is important to develop facile and effective recognition methodology of pathogens in food. Herein, a new automatic approach for detection of in vivo volatile metabolites emitted from foodborne pathogens was proposed by coupling solid phase microextraction (SPME) technique with a comprehensive two-dimensional gas chromatography quadrupole time-of-flight mass spectrometry (GC × GC-QTOFMS). A novel polymer composite based SPME probe which possessed high-coverage of microbial metabolites was utilized in this contribution to realize the sensitive extraction of untargeted metabolites. As a result, a total of 126 in vivo metabolites generated by the investigated pathogens were detected and identified, with 33, 29, 25, 21 and 18 volatile metabolites belonging to Shigella sonnei, Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus and Staphylococcus aureus, respectively. Multivariate statistical analyses were applied for further analysis of metabolic data and separation of responsive metabolic features among different microbial systems were found, which were also successfully verified in foodstuffs contaminated by microorganisms. The growth trend of the potential volatile markers of each pathogen in food samples kept consistent with that of the pure strain incubated in medium during the whole incubation time. This study promotes the application of SPME technology in microbial volatile metabolomics and contributes to the development of new approaches for foodborne pathogens recognition.
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http://dx.doi.org/10.1016/j.foodres.2021.110213DOI Listing
April 2021

Efficient excitation of hybrid modes on a double-layer graphene with metallic slit grating.

Appl Opt 2021 Mar;60(8):2207-2212

The excitation of double-layer hybrid plasmonic modes is investigated by the finite element method. The hybrid modes, verified as the standing even order of both symmetric and anti-symmetric modes, are effectively generated. There are several advances in comparison with using the Si grating: the metallic grating not only compensates phase mismatch, but also acts as a magnetic polariton. The dependences of each hybrid mode on the geometric parameters are analyzed respectively. Interestingly, a second spectra splitting occurs at each hybrid resonant mode with an obliquely incident light. At last, the excitation efficiency can be further enhanced to 90% using the Salisbury screen. The proposed hybrid system can be utilized to design various double-layer graphene-based plasmonic devices, including tunable optical switches, thermal emitters, multiband absorbers, sensors, etc.
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http://dx.doi.org/10.1364/AO.416918DOI Listing
March 2021

An alkaline thermostable laccase from termite gut associated strain of Bacillus stratosphericus.

Int J Biol Macromol 2021 Mar 4;179:270-278. Epub 2021 Mar 4.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, Shandong 266237, China. Electronic address:

Laccase, an important oxidoreductase, is widely distributed in various organisms. Termites are known to decompose lignocellulose efficiently with the aid of gut microorganisms. However, few laccases have been characterized from termite or its gut microbes. We aimed to screen the strain capable of degrading lignocellulose from fungus-growing termites. In this study, Bacillus stratosphericus BCMC2 with lignocellulolytic activity was firstly isolated from the hindgut of fungus-growing termite Macrotermes barneyi. The laccase gene (BaCotA) was cloned both from the BCMC2 strain and termite intestinal metagenomic DNA. BaCotA was overexpressed in E. coli, and the recombinant BaCotA showed high specific activity (554.1 U/mg). BaCotA was thermostable with an optimum temperature of 70 °C, pH 5.0. Furthermore, BaCotA was resistant to alkali and organic solvents. The enzyme remained more than 70% residual activity at pH 8.0 for 120 min; and the organic solvents such as methanol, ethanol and acetone (10%) had no inhibitory effect on laccase activity. Additionally, BaCotA exhibited efficient decolorization ability towards indigo and crystal violet. The multiple enzymatic properties suggested the presented laccase as a potential candidate for industrial applications. Moreover, this study highlighted that termite intestine is a good resource for either new strains or enzymes.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.02.205DOI Listing
March 2021

Modulation of mTOR and epigenetic pathways as therapeutics in gallbladder cancer.

Mol Ther Oncolytics 2021 Mar 3;20:59-70. Epub 2020 Dec 3.

Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.

Gallbladder cancer (GBC) is the most common malignancy of the biliary tract, with extremely dismal prognosis. Limited therapeutic options are available for GBC patients. We used whole-exome sequencing of human GBC to identify the ErbB and epigenetic pathways as two vulnerabilities in GBC. We screened two focused small-molecule libraries that target these two pathways using GBC cell lines and identified the mTOR inhibitor INK-128 and the histone deacetylase (HDAC) inhibitor JNJ-26481585 as compounds that inhibited proliferation at low concentrations. Both significantly suppressed tumor growth and metastases in mouse models. Both synergized with the standard of care chemotherapeutic agent gemcitabine in cell lines and in mouse models. Furthermore, the activation of the mTOR pathway, measured by immunostaining for phosphorylated mTOR and downstream effector S6K1, is correlated with poor prognosis in GBC. Phosphorylated mTOR or p-S6K1 in clinical samples is an independent indicator for overall survival in GBC patients. Taken together, our findings suggest that mTOR inhibitors and HDAC inhibitors can serve as potential therapeutics for GBC, and the phosphorylation of mTOR and S6K1 may serve as biomarkers for GBC.
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http://dx.doi.org/10.1016/j.omto.2020.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851494PMC
March 2021

First record of gregarine protists (Apicomplexa: Sporozoa) in Asian fungus-growing termite Macrotermes barneyi (Blattaria: Termitidae).

Sci Rep 2021 Jan 13;11(1):989. Epub 2021 Jan 13.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, 266237, Shandong, China.

Macrotermes barneyi, widely distributed in southern China, is the major fungus-growing termite in the subfamily Macrotermitinae. It has no flagellated protists in the guts. Here, we report occurrence of gregarine, a protozoan parasite in the digestive tract of M. barneyi. The general morphology and ultrastructure of the gregarine gamonts and syzygies by light micrograph and scanning electron micrograph are presented. SSU rDNA sequence analysis showed that the termite gregarine has the highest identity (90.10%) to that of Gregarina blattarum from cockroaches. Phylogenetic analysis based on the SSU rDNA sequences from diverse insect eugregarines indicated that the gregarine from M. barneyi is phylogenetically close to G. blattarus, L. erratica and G. tropica from Gregarinidae and Leidyanidae families, and may represent a novel species. This study expands our knowledge about the diversity of terrestrial eugregarines parasitizing in termites.
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http://dx.doi.org/10.1038/s41598-020-79671-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806973PMC
January 2021

Phosphoproteomic Analysis Reveals Rio1-Related Protein Phosphorylation Changes in Response to UV Irradiation in REY15A.

Front Microbiol 2020 3;11:586025. Epub 2020 Dec 3.

CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, China.

DNA damage response (DDR) in eukaryotes is largely regulated by protein phosphorylation. In archaea, many proteins are phosphorylated, however, it is unclear how the cells respond to DNA damage through global protein phosphorylation. We previously found that Δ, a Rio1 kinase homolog deletion strain of REY15A, was sensitive to UV irradiation. In this study, we showed that Δ grew faster than the wild type. Quantitative phosphoproteomic analysis of the wild type and Δ, untreated and irradiated with UV irradiation, revealed 562 phosphorylated sites (with a Ser/Thr/Tyr ratio of 65.3%/23.8%/10.9%) of 333 proteins in total. The phosphorylation levels of 35 sites of 30 proteins changed with >1.3-fold in the wild type strain upon UV irradiation. Interestingly, more than half of the UV-induced changes in the wild type did not occur in the Δ strain, which were mainly associated with proteins synthesis and turnover. In addition, a protein kinase and several transcriptional regulators were differentially phosphorylated after UV treatment, and some of the changes were dependent on Rio1. Finally, many proteins involved in various cellular metabolisms exhibited Riol-related and UV-independent phosphorylation changes. Our results suggest that Rio1 is involved in the regulation of protein recycling and signal transduction in response to UV irradiation, and plays regulatory roles in multiple cellular processes in .
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http://dx.doi.org/10.3389/fmicb.2020.586025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744417PMC
December 2020

Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon REY15A.

Front Microbiol 2020 1;11:607431. Epub 2020 Dec 1.

CRISPR and Archaea Biology Research Center, State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, China.

EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the mismatch cleavage activity and function of an EndoMS homolog (SisEndoMS) from REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn was used as the co-factor instead of Mg, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆ by counter selection against 5-fluoroorotic acid (5-FOA). The knockout mutant had much higher spontaneous mutation rate (5.06 × 10) than that of the wild type (4.6 × 10). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in which have a single chromosome is discussed.
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http://dx.doi.org/10.3389/fmicb.2020.607431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736090PMC
December 2020

An Experimental Study on Rapid Localization of the Atrioventricular Node During Open-Heart Surgery.

Heart Surg Forum 2020 Oct 8;23(6):E725-E730. Epub 2020 Oct 8.

Department of Cardiothoracic Surgery, The Affiliated Hospital of Hangzhou Normal University, Hangzhou City, Zhejiang Province, China.

Background: To verify the validity and feasibility of using a mechanical compression method to locate the atrioventricular node in open-heart surgery.

Methods: Ten healthy miniature pigs were used to establish an animal model of the beating heart under cardiopulmonary bypass. During the operation, the atrioventricular node and its surrounding areas were stimulated by mechanical compression (mechanical compression method), and the occurrence of complete atrioventricular block was judged by real-time electrocardiograph monitoring and direct observation of the heart rhythm to identify the position of the atrioventricular node. The final localization of the atrioventricular node was determined using the iodine staining method, and the results were used as the "gold standard" to test the effectiveness and feasibility of the mechanical compression method for locating the atrioventricular node.

Results: With the beating heart model, complete atrioventricular block occurred after mechanical compression of the "atrioventricular node" area in 10 pigs. Nine pigs regained normal conduction immediately after the compression was released, and one pig failed to recover. No atrioventricular block or other arrhythmias occurred after mechanical compression of the "non-atrioventricular node" area. The sensitivity of the method was 86.6%, specificity was 100.0%, misdiagnosis rate was 0.0%, missed diagnosis rate was 13.4%, positive predictive value was 100.0%, negative predictive value was 97.9%, positive likelihood ratios were +∞, negative likelihood ratios were 13.4%, accuracy was 98.1%, and diagnostic odds ratio was +∞.

Conclusion: This study innovatively proposes the application of the mechanical compression method to locate the atrioventricular node during operation and preliminarily proves that this method is effective and feasible through animal experiments.
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http://dx.doi.org/10.1532/hsf.3123DOI Listing
October 2020

Clinical Significance of P16 Gene Methylation in Lung Cancer.

Adv Exp Med Biol 2020 ;1255:133-142

Center for Clinical Single Cell Biomedicine, Henan Provincial People's Hospital, Zhengzhou, Henan, China.

Lung cancer is the leading cause of death from cancer in China. The lack of early screening technologies makes most patients to be diagnosed at advanced stages with a poor prognosis which often miss the best treatment opportunities. Thus, identifying biomarkers for minimally invasive detection and prognosis of early stage disease is urgently needed. Genetic and epigenetic alterations that promote tumorigenesis and metastasis exist in multiple cancers. These aberrant alterations usually represent early events in cancer progression suggesting their potential applications as a biomarker for cancer prediction. Studies have shown that DNA methylation is one of the key factors in progression of lung cancer. P16 promoter methylation is one of the most common epigenetic change plays a key role in lung cancer. In this review, we highlight the p16 gene methylation and its clinical significance in lung cancer.
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http://dx.doi.org/10.1007/978-981-15-4494-1_11DOI Listing
September 2020

Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.

Mol Cell 2020 09 29;79(5):741-757.e7. Epub 2020 Jul 29.

Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3-B, 2200 Copenhagen, Denmark. Electronic address:

Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.
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http://dx.doi.org/10.1016/j.molcel.2020.07.008DOI Listing
September 2020

A Novel Digestive GH16 β-1,3(4)-Glucanase from the Fungus-Growing Termite Macrotermes barneyi.

Appl Biochem Biotechnol 2020 Dec 28;192(4):1284-1297. Epub 2020 Jul 28.

State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Qingdao, 266237, China.

β-1,3-glucanases are the main digestive enzymes of plant and fungal cell wall. Transcriptomic analysis of the fungus-growing termite Macrotermes barneyi revealed a high expression of a predicted β-1,3(4)-glucanase (Mbbgl) transcript in termite gut. Here, we described the cDNA cloning, heterologous expression, and enzyme characterization of Mbbgl. Sequence analysis and RT-PCR results showed that Mbbgl is a termite-origin GH16 β-1,3(4)-glucanase. The recombinant enzyme showed the highest activity towards laminarin and was active optimally at 50 °C, pH 5.5. The enzyme displayed endo/exo β-1,3(4)-glucanase activities. Moreover, Mbbgl had weak transglycosylation activity. The results indicate that Mbbgl is an endogenous digestive β-1,3(4)-glucanase, which contributes to the decomposition of plant biomass and fungal hyphae. Additionally, the multiple activities, pH, and ion stabilities make Mbbgl a potential candidate for application in the food industry.
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http://dx.doi.org/10.1007/s12010-020-03368-wDOI Listing
December 2020

Significance of clinical phenomes of patients with COVID-19 infection: A learning from 3795 patients in 80 reports.

Clin Transl Med 2020 Jan 4;10(1):28-35. Epub 2020 Apr 4.

Zhongshan Hospital Institute of Clinical Science, Fudan University Shanghai Medical School, Shanghai, China.

A new coronavirus SARS-CoV-2 has caused outbreaks in multiple countries and the number of cases is rapidly increasing through human-to-human transmission. Clinical phenomes of patients with SARS-CoV-2 infection are critical in distinguishing it from other respiratory infections. The extent and characteristics of those phenomes varied depending on the severities of the infection, for example, beginning with fever or a mild cough, progressed with signs of pneumonia, and worsened with severe or even fatal respiratory difficulty in acute respiratory distress syndrome. We summarized clinical phenomes of 3795 patients with COVID-19 based on 80 published reports from the onset of outbreak to March 2020 to emphasize the importance and specificity of those phenomes in diagnosis and treatment of infection, and evaluate the impact on medical services. The data show that the incidence of male patients was higher than that of females and the level of C-reaction protein was increased as well as most patients' imaging included ground-glass opacity. Clinical phenomes of SARS-CoV-2 infection were compared with those of SARS-CoV and MERS-CoV infections. There is an urgent need to develop an artificial intelligence-based machine learning capacity to analyze and integrate radiomics- or imaging-based, patient-based, clinician-based, and molecular measurements-based data to fight the outbreak of COVID-19 and enable more efficient responses to unknown infections in future.
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http://dx.doi.org/10.1002/ctm2.17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240842PMC
January 2020

Acute lung injury in patients with COVID-19 infection.

Clin Transl Med 2020 Jan 31;10(1):20-27. Epub 2020 Mar 31.

Zhongshan Hospital Institute of Clinical Science, Shanghai Medical School, Fudan University, Shanghai, China.

During the 2020 Spring Festival in China, the outbreak of a novel coronavirus, named COVID-19 by WHO, brought on a worldwide panic. According to the clinical data of infected patients, radiologic evidence of lung edema is common and deserves clinical attention. Lung edema is a manifestation of acute lung injury (ALI) and may progress to hypoxemia and potentially acute respiratory distress syndrome (ARDS). Patients diagnosed with ARDS have poorer prognosis and potentially higher mortality. Although no effective treatment is formally approved for COVID-19 infection, support of ventilation with oxygen therapy and sometimes mechanical ventilation is often required. Treatment with systemic and/or local glucocorticoids might be helpful to alleviate the pulmonary inflammation and edema, which may decrease the development and/or consequences of ARDS. In this article, we focus on the lung edema and ALI of patients with this widely transmitted COVID-19 infection in order to provide clinical indications and potential therapeutic targets for clinicians and researchers.
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http://dx.doi.org/10.1002/ctm2.16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240840PMC
January 2020

Correction: MiR-1 downregulation correlates with poor survival in clear cell renal cell carcinoma where it interferes with cell cycle regulation and metastasis.

Oncotarget 2019 Dec 24;10(67):7183-7184. Epub 2019 Dec 24.

Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

[This corrects the article DOI: 10.18632/oncotarget.3915.].
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http://dx.doi.org/10.18632/oncotarget.27357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935255PMC
December 2019

Crenarchaeal 3D Genome: A Prototypical Chromosome Architecture for Eukaryotes.

Cell 2019 09;179(1):56-58

CRISPR and Archaea Biology Research Center, Microbial Technology Institute and State Key Laboratory of Microbial Technology, Shandong University, 72 Binhai Road, Jimo, Qingdao, Shandong, 266237, China. Electronic address:

In this issue of Cell, Takemata et al. demonstrate that coalescin (ClsN), an archaeal condensin ortholog, facilitates higher-level organization of chromosomes in crenarchaea that bears greater similarity to metazoans than bacteria. Their study unravels biological function for chromosome organization in Archaea and provides insights into the evolution of eukaryotic chromosomal compartmentalization.
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http://dx.doi.org/10.1016/j.cell.2019.08.045DOI Listing
September 2019

The Lonely Guy (LOG) Homologue SiRe_0427 from the Thermophilic Archaeon REY15A Is a Phosphoribohydrolase Representing a Novel Group.

Appl Environ Microbiol 2019 10 1;85(20). Epub 2019 Oct 1.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, People's Republic of China

Lonely Guy (LOG) proteins are important enzymes in cellular organisms, which catalyze the final step in the production of biologically active cytokinins via dephosphoribosylation. LOG proteins are vital enzymes in plants for the activation of cytokinin precursors, which is crucial for plant growth and development. In fungi and bacteria, LOGs are implicated in pathogenic or nonpathogenic interactions with their plant hosts. However, LOGs have also been identified in the human pathogen , and the accumulation of cytokinin-degraded products, aldehydes, within bacterial cells is lethal to the bacterium in the presence of nitric oxide, suggesting diverse roles of LOGs in various species. In this study, we conducted biochemical and genetic analysis of a LOG homologue, SiRe_0427, from the hyperthermophilic archaeon REY15A. The protein possessed the LOG motif GGGxGTxxE and exhibited phosphoribohydrolase activity on adenosine-5-monophosphate (AMP), similar to LOGs from eukaryotes and bacteria. Alanine mutants at either catalytic residues or substrate binding sites lost their activity, resembling other known LOGs. SiRe_0427 is probably a homotetramer, as revealed by size exclusion chromatography and chemical cross-linking. We found that the gene encoding SiRe_0427 could be knocked out; however, the Δ strain exhibited no apparent difference in growth compared to the wild type, nor did it show any difference in sensitivity to UV irradiation under our laboratory growth conditions. Overall, these findings indicate that archaeal LOG homologue is active as a phosphoribohydrolase. Lonely Guy (LOG) is an essential enzyme for the final biosynthesis of cytokinins, which regulate almost every aspect of growth and development in plants. LOG protein was originally discovered 12 years ago in a strain of with a distinct floral phenotype of a single stamen. Recently, the presence of LOG homologues has been reported in , an obligate human pathogen. To date, active LOG proteins have been reported in plants, pathogenic and nonpathogenic fungi, and bacteria, but there have been no experimental reports of LOG protein from archaea. In the current work, we report the identification of a LOG homologue active on AMP from REY15A, a thermophilic archaeon. The protein likely forms a tetramer in solution and represents a novel evolutionary lineage. The results presented here expand our knowledge regarding proteins with phosphoribohydrolase activities and open an avenue for studying signal transduction networks of archaea and potential applications of LOG enzymes in agriculture and industry.
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http://dx.doi.org/10.1128/AEM.01739-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805079PMC
October 2019

Phosphorylation of the Archaeal Holliday Junction Resolvase Hjc Inhibits Its Catalytic Activity and Facilitates DNA Repair in REY15A.

Front Microbiol 2019 31;10:1214. Epub 2019 May 31.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, China.

Protein phosphorylation is one of the main protein post-translational modifications and regulates DNA repair in eukaryotes. Archaeal genomes encode eukaryotic-like DNA repair proteins and protein kinases (ePKs), and several proteins involved in homologous recombination repair (HRR) including Hjc, a conserved Holliday junction (HJ) resolvase in Archaea, undergo phosphorylation, indicating that phosphorylation plays important roles in HRR. Herein, we performed phosphorylation analysis of Hjc by various ePKs from . It was shown that SiRe_0171, SiRe_2030, and SiRe_2056, were able to phosphorylate Hjc . These ePKs phosphorylated Hjc at different Ser/Thr residues: SiRe_0171 on S34, SiRe_2030 on both S9 and T138, and SiRe_2056 on T138. The HJ cleavage activity of the phosphorylation-mimic mutants was analyzed and the results showed that the cleavage activity of S34E was completely lost and that of S9E had greatly reduced. strain expressing S34E in replacement of the wild type Hjc was resistant to higher doses of DNA damaging agents. Furthermore, SiRe_0171 deletion mutant exhibited higher sensitivity to DNA damaging agents, suggesting that Hjc phosphorylation by SiRe_0171 enhanced the DNA repair capability. Our results revealed that HJ resolvase is regulated by protein phosphorylation, reminiscent of the regulation of eukaryotic HJ resolvases GEN1 and Yen1.
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http://dx.doi.org/10.3389/fmicb.2019.01214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555300PMC
May 2019

Systemic toxicity of di (2-ethylhexyl) adipate (DEHA) in rats following 28-day intravenous exposure.

Regul Toxicol Pharmacol 2019 Jun 28;104:50-55. Epub 2019 Feb 28.

Baxter Healthcare Corporation, PreClinical, Roundlake, United States.

Di (2-ethylhexyl) adipate (DEHA) is a potential plasticizer alternative for di-2-ethylhexyl phthalate (DEHP). Toxicity of DEHA has been studied mostly via oral exposure but not assessed after repeated intravenous exposure. The present study shows the toxicity effects after intravenous administration for 28 consecutive days and the reversibility of the effects following a 14-day recovery period. The study was conducted under GLP conditions. Four groups of rats (15/sex/group) each received either vehicle or DEHA in vehicle (100, 200, or 450 mg/kg/day). Criteria for evaluation included clinical observations, body weight, food consumption, clinical pathology (hematology, serum chemistry, coagulation, urinalyses), gross (necropsy) evaluation, organ weight and histopathological evaluation. There were no DEHA-related changes in all the endpoints evaluated at 100 or 200 mg/kg/day. There were no test article-related changes in clinical pathology or gross necropsy observation at 450 mg/kg/day. At the high-dose, DEHA-related findings included clinical observations, decreased body weight gain and food consumption, increased liver weight in females associated with minimal hepatocellular hypertrophy, and decreased thymus weight in males and females without histopathology findings. All these findings were completely reversible within a 14-day recovery period. Therefore, the 200 mg/kg/day dose is considered to be the No-Observed-Effect Level (NOEL).
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http://dx.doi.org/10.1016/j.yrtph.2019.02.016DOI Listing
June 2019

RNA-binding protein LIN28B inhibits apoptosis through regulation of the AKT2/FOXO3A/BIM axis in ovarian cancer cells.

Signal Transduct Target Ther 2018;3:23. Epub 2018 Aug 31.

3Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Center for Stem Cell Biology and Tissue Engineering, Department of Biology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080 P.R. China.

LIN28B is an evolutionarily conserved RNA-binding protein that regulates mRNA translation and miRNA maturation in embryonic stem cells and developing tissues. Increasing evidence demonstrates that is activated in cancer and serves as a critical oncogene. However, the underlying molecular mechanisms of LIN28B function in tumorigenesis are still largely unknown. Here we report that LIN28B was expressed in over half of the patients with epithelial ovarian cancer who were examined ( = 584). Functional experiments demonstrated that LIN28B inhibited ovarian cancer cell apoptosis. Furthermore, we showed that the proapoptotic factor BIM played an essential role in the antiapoptotic function of LIN28B. RNA-IP microarray analysis suggested that LIN28B binds to mRNAs that are associated with the DNA damage pathway, such as , in ovarian cancer cells. By binding to mRNA and enhancing its protein expression, LIN28B regulated FOXO3A protein phosphorylation and decreased the transcriptional level of , which antagonized the antiapoptosis activity of LIN28B. Taken together, these results mechanistically linked LIN28B and the AKT2/FOXO3A/BIM axis to the apoptosis pathway. The findings may have important implications in the diagnosis and therapeutics of ovarian cancer.
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http://dx.doi.org/10.1038/s41392-018-0026-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117292PMC
February 2021

A Smart DNA Tweezer for Detection of Human Telomerase Activity.

Anal Chem 2018 03 22;90(5):3521-3530. Epub 2018 Feb 22.

Reliable and accurate detection of telomerase activity is crucial to better understand its role in cancer cells and to further explore its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially designed single-stranded oligonucleotides: a central strand dually labeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs' complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG) . TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/μL for HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.
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http://dx.doi.org/10.1021/acs.analchem.7b05373DOI Listing
March 2018

Long non-coding RNAs in metastasis.

Cancer Metastasis Rev 2018 03;37(1):75-81

Division of Oncogenomics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Long non-coding RNA (lncRNA) genes have recently been discovered as key regulators of developmental, physiological, and pathological processes in humans. Recent studies indicate that lncRNAs regulate every step of gene expression, and their aberrant expression can be found in the majority of cancer types. Particularly, lncRNAs were found to function in tumor development and metastasis, which is the major cause of cancer-related death. Thus, exploring key roles of lncRNAs in metastasis is predicted to enhance our knowledge of metastasis, and uncover novel therapeutic targets and biomarkers of this process. In this review, we discuss the molecular mechanisms of lncRNAs in gene expression regulation and their function in metastasis.
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http://dx.doi.org/10.1007/s10555-017-9713-xDOI Listing
March 2018

Autophosphorylation and Cross-Phosphorylation of Protein Kinases from the Crenarchaeon .

Front Microbiol 2017 7;8:2173. Epub 2017 Nov 7.

State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.

Protein phosphorylation, one of the most important post-translational modifications, regulates almost every cellular process. Although signal transduction by protein phosphorylation is extensively studied in Eukaryotes and Bacteria, the knowledge of this process in archaea is greatly lagging behind, especially for Ser/Thr/Tyr phosphorylation by eukaryotic-like protein kinases (ePKs). So far, only a few studies on archaeal ePKs have been reported, most of which focused on the phosphorylation activities , but their physiological functions and interacting network are still largely unknown. In this study, we systematically investigated the autophosphorylation and cross-phosphorylation activities of ePKs from REY15A using proteins expressed in or kinase assay showed that 7 out of the 11 putative ePKs have autophosphorylation activity. A protein Ser/Thr phosphatase, SiRe_1009, was able to dephosphorylate various autophosphorylated ePKs, confirming that these proteins are Ser/Thr kinases. Two ePKs, SiRe_2030 and SiRe_2056, homologs of typical eukaryotic PKs involved in peptide synthesis in response to various cellular stresses, exhibit highly efficient phosphorylation activities on both themselves and other ePKs. Overexpression of the protein kinases revealed that elevated level of either SiRe_1531 or SiRe_2056 inhibited the cell growth of cells. Finally, a phosphorylation network of the protein kinases was proposed and their putative physiological roles were discussed.
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http://dx.doi.org/10.3389/fmicb.2017.02173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682000PMC
November 2017

Androgen-receptor splice variant-7-positive prostate cancer: a novel molecular subtype with markedly worse androgen-deprivation therapy outcomes in newly diagnosed patients.

Mod Pathol 2018 01 27;31(1):198-208. Epub 2017 Oct 27.

Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Androgen-deprivation therapy has been the standard treatment for metastatic and locally advanced prostate cancer, but the majority of patients will progress to castration-resistant prostate cancer within 2-3 years. Unlike the case in breast cancer, no clinically validated biomarker has been used to predict the outcomes of androgen-deprivation therapy. To evaluate androgen-receptor splice variant-7 (AR-V7) detection in newly diagnosed advanced prostate cancer and describe the distinctive prognosis of this novel molecular subtype, this study retrospectively enrolled 168 newly diagnosed prostate cancer patients from 2003 to 2015 who received androgen-deprivation therapy. AR-V7 immunohistochemical staining was performed with a monoclonal antibody, and AR-V7 expression was determined using Immune-Reactive Score data. The association between nuclear AR-V7 expression and prognosis was determined. Multiple cause-specific Cox regression and stratified cumulative incidences were used to analyze the prognosis risk. Among the 168 patients, 32 (19%) were AR-V7-positive. Compared with the AR-V7-negative patients, the AR-V7-positive patients had significantly lower prostate-specific antigen response rates (P<0.001) to androgen-deprivation therapy and a much shorter time to castration-resistant prostate cancer (P<0.0001). In Kaplan-Meier analysis, the AR-V7-positive group showed markedly lower castration-resistant prostate cancer progression-free survival (P<0.0001) and much lower cancer-specific (P<0.0001) and overall survival (P<0.0001) both in all enrolled patients and in patients with metastases. AR-V7 positivity was a significant predictor of castration-resistant prostate cancer progression in multiple Cox regression (hazard ratio: 4.826; 95% CI: 2.960-7.869; P<0.001). AR-V7 immunohistochemical detection in newly diagnosed prostate cancer patients who are planning to receive androgen-deprivation therapy, especially those with metastases, is necessary and valuable for prognostic assessment. AR-V7-positive prostate cancer should be considered a novel prostate cancer subtype that should be distinguished upon initial biopsy. The main limitation of this study is its observational nature.
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http://dx.doi.org/10.1038/modpathol.2017.74DOI Listing
January 2018

Repression of BET activity sensitizes homologous recombination-proficient cancers to PARP inhibition.

Sci Transl Med 2017 07;9(400)

Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia, PA 19104, USA.

Strategies to enhance response to poly(adenosine diphosphate-ribose) polymerase inhibitor (PARPi) in primary and acquired homologous recombination (HR)-proficient tumors would be a major advance in cancer care. We used a drug synergy screen that combined a PARPi, olaparib, with 20 well-characterized epigenetic drugs and identified bromodomain and extraterminal domain inhibitors (BETis; JQ1, I-BET762, and OTX015) as drugs that acted synergistically with olaparib in HR-proficient cancer cells. Functional assays demonstrated that repressed BET activity reduces HR and thus enhances PARPi-induced DNA damage in cancer cells. We also found that inhibition or depletion of BET proteins impairs transcription of and , two genes essential for HR. Moreover, BETi treatment sensitized tumors to PARP inhibition in preclinical animal models of HR-proficient breast and ovarian cancers. Finally, we showed that the gene was focally amplified across 20 types of common cancers. Combination with BETi could greatly expand the utility of PARP inhibition to patients with HR-proficient cancer.
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http://dx.doi.org/10.1126/scitranslmed.aal1645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705017PMC
July 2017

miR-125b-5p enhances chemotherapy sensitivity to cisplatin by down-regulating Bcl2 in gallbladder cancer.

Sci Rep 2017 03 3;7:43109. Epub 2017 Mar 3.

Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Dongfang Road, Shanghai 200127, China.

Gallbladder cancer represents the most common malignancy of the biliary tract and is highly lethal with less than 5% overall 5-year survival rate. Chemotherapy remains the major treatment for late-stage patients. However, insensitivity to these chemotherapeutic agents including cisplatin is common. MicroRNAs (miRNAs) have been shown as modulators of drug resistance in many cancer types. We used genome-wide gene expression analysis in clinical samples to identify miR-125b-5p down-regulated in gallbladder cancer. miR-125b-5p up-regulation promoted cell death in gallbladder cancer cells in the presence of cisplatin. In contrast, knockdown of miR-125b-5p reduced cell death in gallbladder cancer cells treated with cisplatin. Up-regulation of miR-125b-5p significantly decreased tumor growth in combination with cisplatin in a mouse model. We identified Bcl2 as a direct target of miR-125b-5p which mediates the function of miR-125b-5p in gallbladder cancer. In clinical samples, miR-125b-5p was down-regulated in gallbladder cancer whereas Bcl2 was up-regulated and their expression was inversely correlated. Moreover, low miR-125b-5p expression or high expression of Bcl2 is correlated with poor prognosis in gallbladder cancer. Taken together, our findings indicate that miR-125b-5p is a potent chemotherapy sensitizer and may function as a new biomarker for the prognosis of gallbladder cancer patients.
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http://dx.doi.org/10.1038/srep43109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5335654PMC
March 2017

Combination of metformin and sorafenib suppresses proliferation and induces autophagy of hepatocellular carcinoma via targeting the mTOR pathway.

Int J Oncol 2017 Jan 9;50(1):297-309. Epub 2016 Dec 9.

Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning 116044, P.R. China.

The multi‑kinase inhibitor sorafenib is the only drug for which randomized control trials have shown improved patient survival in advanced hepatocellular carcinoma (HCC). However, life expectancy is extended in these cases by only a few months. The anti‑type II diabetes agent metformin was used in this study in an effort to find a more efficient approach to HCC treatment. Sorafenib effectively reversed the activation status of mTORC2 induced by metformin and enhanced the suppression of the mTORC1 and MAPK pathway by metformin in HCC cells, which may be responsible for reduced proliferation upon combined treatment. The metformin and sorafenib combination led to increased impaired proliferation and tumor inhibition of HCC in vitro and in vivo compared to single agent, which was partially bridged by disrupting the mTORC1/mTORC2 feedback loop. Metformin and sorafenib cooperated to promote apoptosis and autophagy in HCC cells. Pharmacological inhibition of autophagy sensitized HCC cells to metformin and sorefenib‑induced apoptotic cell death. Therefore, the anti‑autophagy treatment should be considered in metformin and sorafenib-based treatments in HCC cells.
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http://dx.doi.org/10.3892/ijo.2016.3799DOI Listing
January 2017

Alternative Splicing of EZH2 pre-mRNA by SF3B3 Contributes to the Tumorigenic Potential of Renal Cancer.

Clin Cancer Res 2017 07 22;23(13):3428-3441. Epub 2016 Nov 22.

Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China.

Deregulation or mutation of the gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). Although several splice variants of EZH2 have been identified, little is known about how EZH2 splicing is regulated or the contribution of alternative splicing to its protumorigenic functions. We conducted RT-PCR, Western blot analysis, and IHC techniques to examine EZH2 and its alternative splicing transcript expression in renal cancer tissue and renal cancer cell lines. Proliferation, migration, clonogenicity, and tumorigenicity of renal cancer cells either exhibiting knockdown of EZH2 or its splicing factor SF3B3 were assessed by CCK8, Transwell assay, and murine xenograft experiments. We found that the inclusion of alternative EZH2 exon 14 was significantly increased in ccRCC samples and renal cancer cell lines. In ccRCC lines, enforced expression of EZH2Δ14 inhibited, and EZH2 promoted, cell growth, migration, proliferation, and tumorigenicity in a xenograft model. Mechanistic studies demonstrated that EZH2Δ14 isoform functions as a dominant-negative inhibitor of full-length EZH2. Coexpression of EZH2Δ14 variant with full-length EZH2 not only abrogated DAB2IP and HOXA9 suppression but also inhibited EZH2-driven tumorigenesis. Strikingly, the splicing factor SF3B3 stimulates inclusion of exon14 and has pro-proliferative activity. Importantly, the upregulation of SF3B3 expression observed in clinical ccRCC samples parallels the increased inclusion of EZH2 exon14, and the SF3B3 level is associated with higher tumor stage and poor overall survival. These results suggest SF3B3 as a key regulator of EZH2 pre-mRNA splicing and SF3B3 may represent a novel prognostic factor and potential therapeutic target in ccRCC. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440213PMC
July 2017

Time trend analysis of primary liver cancer incidence in Sihui county of Guangdong Province, China (1987-2011).

BMC Cancer 2016 10 12;16(1):796. Epub 2016 Oct 12.

Department of Cancer Prevention Research, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China; Collaborative Innovation Center Medicine, 21 Qingcaigang, Jianshe 6 road, Guangzhou, Guangdong, China.

Background: Southern China is an endemic area for primary liver cancer (PLC), but it is unclear if rates have changed in recent decades. We evaluated PLC incidence and estimated the effects of age, period of diagnosis, and birth cohort in Sihui City, Guangdong Province, China.

Methods: Age-standardized rates (ASRs) of PLC were examined for both males and females from 1987 to 2011. Joinpoint regression analysis was conducted to estimate the annual percent changes in PLC incidence. The age-period-cohort (APC) model was used to investigate the effects of age, diagnosis period, and birth cohort on the relative risk (RR) of PLC.

Results: A total of 2988 PLC cases were identified in this period, with average ASRs of 51.1/100,000 for males and 11.7/100,000 for females. Joinpoint regression analysis revealed increasing PLC incidence throughout the entire period in both males (average annual change of 1.65 %) and females (0.20 %). RRs increased gradually in both sexes from the youngest age group (30-34 years) to the oldest (80-84 years). In males, the RR decreased during diagnosis period from 1987-1991 to 1997-2001 and remained stable thereafter. In females, RRs fluctuated with diagnosis period throughout the entire period. Incidence tended to increase with birth cohort from 1905-1909 to 1975-1979 in both males and females; however, female incidence plateaued in the youngest cohorts born between 1955 and 1974, while incidence in males increased sharply in the cohorts born between 1965 and 1974. According to APC analysis, the full age-period-cohort (APC) model fit the data best, and the period-cohort (PC) model would be enough to explain variability of rates in females.

Conclusion: The PLC incidence rate in males of Sihui City has increased more significantly than female over the last 25 years. Despite the age effect in male, this trend mainly reflects the effects of risk factors that are present in early life (birth cohort) and period change in both genders.
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http://dx.doi.org/10.1186/s12885-016-2817-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062930PMC
October 2016

The changing 50% inhibitory concentration (IC50) of cisplatin: a pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer.

Oncotarget 2016 Oct;7(43):70803-70821

Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.

Inconsistencies in the half-maximal (50%) inhibitory concentration (IC50) data for anticancer chemotherapeutic agents have yielded irreproducible experimental results and thus reciprocally contradictory theories in modern cancer research. The MTT assay is currently the most extensively used method for IC50 measurements. Here, we dissected the critical reasons behind MTT-dependent IC50 inconsistencies. We showed that IC50 errors caused by the technical deficiencies of the MTT assay are large and not adjustable (range: 300-11,000%). To overcome severe MTT artifacts, we developed an unbiased direct IC50 measurement method, the limiting dilution assay. This detection technique led us to the discovery of the inherent density-dependent chemoresistance variation of cancer cells, which is manifold and unpredictable in its forms. The subsequent intracellular signaling pathway analysis indicated that pAkt and p62 expression levels correlated with alterations in the IC50 values for cisplatin in ovarian cancer, providing an explainable mechanism for this property. An in situ pAkt-and-p62-based immunohistochemical (IHCpAkt+p62) scoring system was thereby established. Both the limiting dilution assay and the IHCpAkt+p62 scoring system accurately predicted the primary chemoresistance against cisplatin in ovarian cancer patients. Furthermore, two distinct chemoresistant recurrence patterns were uncovered using these novel detection tools, which were linked to two different forms of density-chemoresistance relationships (positively vs. negatively correlated), respectively. An interpretation was given based on the cancer evolution theory. We concluded that the density-related IC50 uncertainty is a natural property of the cancer cells and that the precise measurement of the density-dependent IC50 spectrum can benefit both basic and clinical cancer research fields.
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http://dx.doi.org/10.18632/oncotarget.12223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342590PMC
October 2016

Suppression of MicroRNA 200 Family Expression by Oncogenic KRAS Activation Promotes Cell Survival and Epithelial-Mesenchymal Transition in KRAS-Driven Cancer.

Mol Cell Biol 2016 Nov 13;36(21):2742-2754. Epub 2016 Oct 13.

Center for Research on Reproduction & Women's Health, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA Department of Obstetrics and Gynecology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Oncogenic KRAS contributes to malignant transformation, antiapoptosis, and metastasis in multiple human cancers, such as lung, colon, and pancreatic cancers and melanoma. MicroRNAs (miRNAs) are endogenous 18- to 25-nucleotide noncoding small RNAs that regulate gene expression in a sequence-specific manner via the degradation of target mRNAs or inhibition of protein translation. In the present study, using array-based miRNA profiling in IMR90 and MCF10A cells expressing oncogenic KRAS, we identified that the expression of the microRNA 200 (mir-200) family was suppressed by KRAS activation and that this suppression was mediated by the transcription factors JUN and SP1 in addition to ZEB1. Restoration of mir-200 expression compromised KRAS-induced cellular transformation in vitro and tumor formation in vivo In addition, we found that enforced expression of mir-200 abrogated KRAS-induced resistance to apoptosis by directly targeting the antiapoptotic gene BCL2 Finally, mir-200 was able to antagonize the epithelial-mesenchymal transition (EMT) driven by mutant KRAS. Collectively, our results suggest that repression of endogenous mir-200 expression is one of the important cellular responses to KRAS activation during tumor initiation and progression.
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http://dx.doi.org/10.1128/MCB.00079-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064220PMC
November 2016