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AKT/mTOR and C-Jun N-terminal Kinase(JNK) Signaling Pathways Are Required for Chrysotile Asbestos-Induced Autophagy

AKT/mTOR and C-Jun N-terminal Kinase(JNK) Signaling Pathways...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： Ziying Ziying Lin Yahong Wang Gang Liu 广东医学院附属医院临床医学研究中心

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseasessuch as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms inwhich chrysotile asbestos fibers induce pulmonary disease have been identified, the role ofautophagy in human lung epithelial cells has not been examined. In the present study, weevaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelialcells, and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos-induced autophagy in A549 cells based on a series of biochemical and microscopicautophagy markers. We observed that asbestos increased A549 cell microtubule-associated protein 2 light chains 3(LC3-II) expression, an autophagy marker, in conjunctionwith dephosphorylation of phospho-AKT, phosphomTOR, and phospho-P70s6 ***, AKT1/AKT2 double knockout(AKT DKO) murine embryonic fibroblasts(MEFs) hadnegligible asbestos-induced LC3-II expression supporting a crucial role for AKT *** asbestos also led to the phosphorylation/activation of Jun N-terminal kinase(JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibitedthe protein expression of LC3-II. Moreover, JNK2-/- MEFs but not JNK1-/- MEFsblocked LC3-II levels induced by chrysotile asbestos. In addition, NAC, an antioxidant, attenuatedchrysotile asbestos-induced dephosphorylation of P-AKT and completely abolishedphosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitors 3-methyladenine(3-MA) or ATG5(autophagy-related gene 5) siRNA, indicating thatchrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findingsdemonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotileasbestosinduced autophagy. These data provide a mechanistic basis for possible futureclinical applications targeting these signaling path

关键词： chrysotile asbestos autophagy LC3-Ⅱ pulmonary disease A549 cells

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Character and pathway of proapoptotic PEDF functional peptides responsible for tumor suppression

Character and pathway of proapoptotic PEDF functional peptid...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： Qing Gong Shuai Li Yachao Yao Di Che Juan Feng Weibin Cai Xia Yang Guoquan Gao Zhonghan Yang Department of Biochemistry Zhongshan School of Medicine Sun Yatsen University

Background: PEDF inhibits prostate cancer growth via anti-angiogenic activity,however,the direct effect of PEDF on prostate cancer cells and its functional epitope as well as theunderlying mechanism involved pathway from extracellular receptors to nuclear transcriptionalfactors have not been fully elucidated. This study investigates the functional epitopesof PEDF, clarifies their characters including secondary structure, hydropathicity andthe interaction with potential receptors, and further explores the mechanism to suppressprostate ***: PEDF and its epitope peptides, PEDF34 and PEDF44, were obtained by recombinanttechnology. Molecular modeling and secondary structure prediction, as well as the localizationof alkaline amino acids in their epitopes, were annotated using Cn3 D and Raswinsoftware. Circular dichroism(CD) assay was used for analyzing the conformation of PEDF34 and PEDF44 peptides in the solution. Hydropathicity of PEDF34 and PEDF44 was analyzedby ProtScale. Apoptosis of cells was analyzed using TUNEL, Hoechst33258 and AnnexinV/PI staining. Protein expression were examined by Western-blot. Targeted genes wereknockdown by RNA interference(RNAi). The activity of transcriptional factor binding to itspromoter was determined by the luciferase reporter gene ***: CD spectra shows that α-helix is the main structural feature for PEDF34, but β-sheet and random structure for PEDF44, which is consistent with the features predicted byCn3 D. We predicted hydropathicity of the two peptides in the solution and found thatGrand average of hydropathicity(GRAVY) of PEDF34 was-0.271, while GRAVY of PEDF44 is0.423, which means the affinity to water of PEDF34 is higher than that of PEDF44. Wefound that Lys28-Lys33 motif or basic electrostatic surface potential presented as alkalineamino acid, Lys and Arg, within α-helix in PEDF34 has more LamRbinding motif than thatin PEDF44, which means that PEDF34 interacts with Laminin receptor(LamR) more thanPEDF44 doe

关键词： PEDF epitope apoptosis

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MicroRNA-34a functions as a tumor suppressor in non-small celllung cancer H1299 cell line by targeting TGFγR2

MicroRNA-34a functions as a tumor suppressor in non-small ce...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： zhongliang ma pinpin hou yanli li detao wang jiali wei botao zhao jiatao lou xintaizhao yan jin youxin jin School of life sciences Shanghai University School of Life Sciences Shanghai University Department of Laboratory Medicine Shanghai Chest Hospital affiliated to Shanghai Jiaotong University

MicroRNAs(MiRNAs) are small non-coding RNA molecules which act as important regulatorsof post-transcriptional gene expression by binding 3′-untranslated region(3′-UTR) oftarget mRNA. In this study, we analyzed miRNA-34a(miR-34a) as a tumor suppressor innon-small cell lung cancer(NSCLC) H1299 cell line. The expression level of miR-34 a in fourdifferent NSCLC cell lines, H1299, A549, SPCA-1 and HCC827 was significantly lower thanthat in the normal bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34 a expression level was also down-regulated in pT2-4 compared with the correspondingnon-tumor tissues. Moreover, miR-34 a mimics could inhibit the proliferation and triggeredapoptosis in H1299 cells. Luciferase assays revealed that miR-34 a inhibited TGFβR2 expressionby targeting one binding site in the 3′-UTR of TGFβR2 mRNA. Quantitative real-timePCR(qRT-PCR) and western blot assays verified that miR-34 a reduced TGFβR2 expressionat both mRNA and protein levels. The expression level of miR-34 a and its target geneTGFβR2 was inverted corresponding in human NSCLC tissues and NSCLC cell lines. Furthermore,down-regulation TGFβR2 by siRNA showed the same effects on the proliferation andapoptosis as miR-34 a mimics in H1299 cells. Our results demonstrated that miR-34 a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the down-regulation of its target gene TGFβR2.

关键词： miR-34a non-small cell lung cancer apoptosis TGFβR2

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Juglanthraquinone C induces intracellular ROS increase andapoptosis by activating Akt/FoxO signal pathway in HepG2 cells

Juglanthraquinone C induces intracellular ROS increase andap...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： Ya-Qin Hou Yong-Li Bao Zhen-Bo Song Lu-Guo Sun Chun-Lei Yu Yan-Xin Huang Yu-Xin Li National Engineering Laboratory for Druggable gene and Protein Screening Northeast Normal University Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education Northeast Normal University Institute of Geneticsa nd Cytology Northeast Normal University

Cancer is a major public health problem in the world. Natural products have become an increasinglyimportant source of anticancer drugs. Juglans mandshurica Maxim has beenused in folk medicine for many years, however little is known about which compoundsfrom it involved in its anticancer roles. Our previous study revealed that JuglanthraquinoneC(JC) was a naturally occurring anthraquinone extracted from Juglans mandshurica Maximand can induce apoptosis of HepG2 in vitro. To investigate the mechanisms by which JC inducedthe apoptosis of HepG2 cells, Affymetrix HG-U133 Plus 2.0 arrays were used to analyzethe mRNA expression in HepG2 cells exposed to JC or DMSO. We found that JC caninduce apoptosis of HepG2 which is consistent with our previous results. Surprisedly, wealso found that the mRNA levels of several genes which can positive regulate Akt signalingwere up-regulated, after treated with JC for 4 hours. Western blot analysis revealed thatAkt, a well-known survival protein was activated. To determine the effect of Akt activationon JC-induced apoptosis, siRNAs or shRNAs was used to downregulate cellular expressionof Akt, the result shown that Akt deficiency obviously inhibits JCinduced apoptosis, whileoverexpression of Akt constitutive activation mutant accelerated JC-induced apoptosis. Wenext analyzed the expression of FOXO3 a which is a downstream target of Akt. After treatedwith JC, FOXO3 a transcriptional activity was inhibited, which led to a decrease of ROS *** addition, we found that JC treatment led to an increase of intracellular ROS levelswhich induced apoptosis of HepG2 cells. Collectively, our study demonstrated that JC inducedAkt/Foxo signal pathway activation, which induced high levels of ROS productionand eventually led to apoptosis of HepG2 cells.

关键词： anthraquinone compound Akt activation ROS apoptosis

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the Effect of Taurine on Human hepatoma HepG2 cells Proliferation and Apoptosis

the Effect of Taurine on Human hepatoma HepG2 cells Prolifer...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： shuo tu fusheng wan Basic Medical College of Nanchang University Medical College of Nanchang University

Taurine(Tau) is a sulfur amino acid with simple chemical structur, more and more researcheshave shown that there are some kind of connection between taurine and *** vitro experiment mainly focus on the anti-tumor activities of taurine derivatives on tumorcells alone or in combination with chemotherapy drugs, but few researches focus ontaurine itself. This study mainly focus on Tau's anti-cancer ***:Human hepatoma HepG2 cells were treated with different Tau concentration(0mM,20 mM,40mM, 80 mM and 160mM),were analysed by MTS,Flow cytometry, Hochest33342 nuclear staining method, RT-qPCR and Western blot methods. MTS assay to analyse cellactive change after transfected PUMA into human hepatoma HepG2 cells or co-treatedwith Tau. PUMA specificity siRNA(PUMA-siRNA, si-PUMA) was transfected to human hepatomaHepG2 cells by liposome,were analysed by flow cytometer,RT-qPCR and Westernblot ***:With the increase of Tau concentration, MTS showed the inhibition rate appeared graduallyincreasing in time-dose dependent manner, Flow cytometry data showed that the apoptosisrate of HepG2 cells increased gradually(P<0.05), Morphology detection viaHochest33342 nuclear staining showed that the number of apoptosis cells were raised(P<0.05), RT-qPCR and Western Blot results showed that PUMA, Bax mRNA and proteinexpression level was up regulated, but Bcl-2 mRNA and protein expression level was downregulated(P<0.05). The recombinant plasmid pCEP4-(HA)2-PUMA was transfected into humanhepatoma HepG2 cells in vitro by liposome DNA transfection reagent. MTS showedthat the proliferation inhibition rate of pCEP4-(HA)2-PUMA, and pCEP4-(HA)2-PUMA+Taugroup cells was significantly higher than control group. After siRNA specific interferencePUMA gene expression on HepG2 cells, Flow cytometer analysis showed the apoptosis rateof si-PUMA group were significantly lower than Control group, the apoptosis rate of si-PUMA+Tau group appeared to riser compared with si-PUMA group,(P0.

关键词： taurine p53 up-regulated modulator of apoptosis(PUMA) apoptosis RNAi nterference

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Expression and bioinformatics analysis of SRPK2 identified by LCESI-MS during mouse spermatogenesis

Expression and bioinformatics analysis of SRPK2 identified b...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： Hong Yang Xiaobin Li Lijuan Yang Hailong Wang Rui Guo Department of Biochemistry and Molecular Biology Shanxi Medical University Department of Parasitology Shanxi Medical University

Objective: To analyze the expression characteristics of mouse spermatogenesis-related gene SRPK2 and to reveal its molecular roles during ***: Various bioinformatics tools were used to analyze the structure and functional characteristics of SRPK2 gene which was recently identified by automatic 2D-Nano-LC-ESIMS/MS analysis. Semi-quantitative RT-PCR was used here to demonstrate the tissue-specific expression pattern of SRPK2 gene in mice. Real-time quantitative PCR was applied to identify the stage-specific expression pattern of SRPK2 during mouse *** protein samples from different tissues like heart, liver, brain, kidney,spleen, lung and testis of mice were conducted with Western blotting. SRPK2 protein distributionsin mice testicular seminiferous tubules was detected by immunohistochemicalstaining and SRPK2 protein subcellular localization in spermatic cells was examined by multiple indirect immunofluorescence ***: The full length cDNA of 6435 bp of mouse SRPK2 gene encoded a putative protein of 682 amino acids with a molecular weight of 76.884 kDa. Cellular mapping prediction ofSRPK2 indicated that its protein would be expresssed in nuclear. Protein domain predicted that the protein encoded by SRPK2 contained a STYKc domain which may be associated with cells of the organism. SRPK2 belong to SRPK family that can regulate the cell cycle,cell proliferation and apoptosis. RT-PCR analysis revealed that SRPK2 mRNA was highly expressed in mice testis. Real-time quantitative PCR analysis demonstrated that SRPK2 mRNA was expressed abundantly at later stage during mouse spermatogenesis and probably participated in spermiogenesis. Western blotting revealed the tissue-specific expression of SRPK2 in testis, brain and kidney. Immunohistochemical staining showed thatSRPK2 protein located around the elongating spermatids in mouse testicular seminiferoustubules. Multiple indirect immunofluorescence staining revealed that SRPK2

关键词： SRPK2 spermatogenesis mice bioinformatic

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Really interesting new gene finger protein 121(RNF121) is anovel Golgi-localized membrane protein that regulates apoptosis

Really interesting new gene finger protein 121(RNF121) is an...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： Yuanbo Zhao Dubeiqi Hong Dalong Ma Yingyu Chen Key Laboratory of Medical Immunology Ministry of Health Peking University Health Science Center Peking University Center for Human Disease Genomics Peking University

Really interesting new gene(RING)finger protein is a large protein family in the *** have very important functions in physiological activities and cancer *** biological functions of many RING finger proteins remain *** thisstudy,we describe the biological activity of a novel,human Golgi-localized RING fingerprotein 121(RNF121),the function of which is,thus far,*** RNF121 proteincontains five conserved TM domains and that the RING finger domain faces the *** endoplasmic reticulum-localized RNF121 in Caenorhabditis elegans,human RNF121was ***121 predominantly localized to the Golgi apparatus in ***121 mRNA is expressed in a variety of normal human ***121 knockdown inhibited cell growth and induced apoptosis,which was accompaniedby caspase-3 activation and the inactivation of poly(adenosine diphosphate-ribose)polymerase.Z-VAD-FMK,a pan-caspases inhibitor,inhibited the RNF121knockdown–*** of wild-type RNF121 decreased RNF121 knockdown-inducedapoptosis but the RING domain mutants of RNF121did not have this *** theRING-domain mutated(RNF121 C229A C259A)or truncated RNF121(RNF121 1-225)couldn't alleviate the cell death induced by knockdown of ***121 C229A C259Aand RNF121 1-225 also showed no anti-apoptosis effect when *** indicatesthat the RING domain is required for RNF121-regulated ***,RNF121 knockdown enhanced etoposide-induced *** RNF121has anti-apoptosis effect in cancer cells,and sheds new light to the relation betweenRNF121 and *** finger proteins have been proved to play crucial roles in *** is possible that RNF121 regulates apoptosis through targeting apoptosis-related molecules for *** is also possible that RNF121 regulates apoptosisthrough regulation of Golgi-related transport *** study instigates further investigation to the exact mechanism that RNF121 regula

关键词： RING finger protein 121 RNF121 apoptosis RING domain Golgi apparatus

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GAS2-Calpain axis modulates the growth of leukemic cells

GAS2-Calpain axis modulates the growth of leukemic cells

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： Lili Sun Haixia Zhou Yue Ge Wei-Wei Qu Hong Liu Depei Wu Yun Zhao Soochow University The First Affiliated Hospital of Soochow University

Growth arrest specific 2(GAS2)is a microfilament-associated protein,whose expression isincreased during growth arrest in mammalian ***2 is involved in a variety of cellularfunctions,including cell cycle,apoptosis,and the regulation of calcium-activated enzyme(m-calpain)activity.N-terminus of GAS2 is able to interact with m-calpain and C-terminusof GAS2 inhibits the activity of ***’s reported that GAS2/calpain/p53 axis preventsmalignant transformation;in contrast,GAS2/calpain/beta-catenin pathway promotesin the growth of tumor ***,the expression of GAS2 and its role in leukemic cellsremain *** this study,we first assessed the expression of GAS2 in human acute myeloid leukemia(AML,n=57),chronic myeloid leukemia(CML,n=38),acute lymphoid leukemia(ALL,n=19)and chronic lymphoid leukemia cells(CLL,n=5)in comparison to those from healthydonors(n=7).GAS2 had significantly higher expression in all types of leukemic cells versusnormal *** calpain activity of cells from AML patients(n=22)was significantly lowerthan that from healthy donors(n=7).The interaction between GAS2 and m-calpain wasvalidated in THP-1(AML cell line)and Jurkat cells(T-ALL cell line).Next,we validated twoindependent shRNA sequences to silence GAS2,and delivered them to various leukemiccells including THP-1,K562(CML cell line),Jurkat and Namalwa(B-ALL cell line)***2silence enhanced the calpain activity and inhibited the proliferation in liquid culture;in addition,GAS2 silence suppressed the colony-formingcell(CFC)capacities of these cells rangingfrom 32-58%.Moreover,GAS2 silence strongly inhibited the tumor formation of THP-1 cells in nude mice(control group,7/8;shRNA#1 group,1/8;shRNA#2 group,0/8).Conversely,the re-expression of GAS2 in leukemic cells was able to restore GAS2 silence inducedgrowth *** delivered GAS2Δ171-313 to leukemic cells as well,and thecalpain activity was elevated and the growth was inhibited as *** address the importanceof cal

关键词： GAS2 Calpain Leukemia

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The Tetrahymena cyclin B3, Tcy2p, is essential for meiosis

The Tetrahymena cyclin B3, Tcy2p, is essential for meiosis

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： Jing Xu Xiaoqi Ren Bo Chen Wei Wang Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education Institute of Biotechnology Shanxi University

Cyclin and their cofactor, CDKs(cyclin-dependent kinases), co-regulate the cell cycle. Thenumber of B-type cyclins varies between different organisms but all known eukaryotescontain at least one cyclin B3 gene. Lost or over-expression of cyclin B3 arrest meiosis orform abnormal gamete in mammalian. Tetrahymena thermophila is a unicellular model organismwith two different nuclei: a diploid germline micronucleus and a somatic polyploidymacronucleus. In vegetative cells, the micronucleus divides mitotically, the macronucleusdivides amitotically. During the sexual process of conjugation, the micronucleusundergoes meiosis to form two haploid pronuclei. We identified 16 cyclins in T. thermophilaand found cyclin Tcy2 is a cyclin B3 homolog. The qRT-PCR indicated that TCY2 specificallyexpress during conjugation stage and the expression level reach maximum after 2h mating. HA-Tcy2 localized into cytoplasm during conjugation sage. Overexpression ofTCY2 has no effect for the sexual development. When TCY2 was deleted, the conjugationprocess blocked at the prophase of meiosis. The "crescent" structure fail to form and thehomologous recombination also abort. When TCY2 knockout cell and wild type cell weremated, the meiotic development could be rescued. There was no γ-H2 AX signal occur atthe prophase of meiosis for TCY2 knockout cells, in contrast, strongγ-H2 AX signal appearfor the "crescent" micronuclei of wild type. The meiotic double-strand break regulatoryfactor SPO11 was seriously decline in TCY2 knock out cells. Furthermore, the arrested pairswere treated with cis-platinum or MMS, "crescent" structure still can not form for TCY2 knock out cells. When wild type cells were treated with paclitaxel, 23.4% pairs form"crescent" or enter metakinesis. Furthermore, the TCY2 knock out cells were treated withpaclitaxel, the development of the micronuclei were disrupted more severely. Taken together,the results showed that Tcy2 not only involved in micronuclear chromo some broken and

关键词： Tetrahymena cyclin meiosis nuclear development

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Effects of Kidney-Tonifying Recipe on Neural Cell Apoptosis and Spatial Learning & Memory of Aging Rats

Effects of Kidney-Tonifying Recipe on Neural Cell Apoptosis ...

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中国生物化学与分子生物学会第十一次会员代表大会暨2014年全国学术会议

作者： xiangping kang chao liang guoqin jin xueli zhang zhangbin gong weiwei dai Shanghai University of TCM Longhua Hospital Shanghai University of TCM

Objective:To observe the effects of kidney-tonifying recipe——Zuogui Wan(左归丸)and Radix Rehmanniae Praeparata(熟地黄)on learning & memory ability and themRNA expressions of proBDNF,p75 NTR,sortilin,caspase-3,bcl-2 and bax of ageingrats. Methods:The rats were randomly divided into five groups: young group,ageingmodel group,ageing rats + Zuogui Wan group,ageing rats + high dose Radix RehmanniaePraeparata group(8g/kg) and ageing rats + low dose Radix Rehmanniae Praeparatagroup(4g/kg). The rats were injected D- galactose(500mg/kg) to preparing ageingmodels. The spatial learning & memory abilities were observed by Morris water maze *** contents of β-galactose glucoside enzyme in the serum were examined by using theenzyme-linked immunosorbent assay(ELISA). The apoptosis of neural cell in the hippocampuswere observed by transmission electron microscope(TEM). The mRNA expressionsof proBDNF,p75 NTR,sortilin,caspase-3,bcl-2 and bax in the hippocampus weretested by real-time quantitative PCR. The regulating effects of Zuogui Wan and RadixRehmanniae Praeparata on above indices were also studied. Results:Compared withyoung group, the spatial learning & memory abilities of ageing rats were lower(P<0.05),the contents of β-galactose glucoside enzyme in the serum were higher(P<0.05) and theneural cells apoptosis of ageing rats were obvious. The mRNA expressions of proBDNF,p75 NTR,sortilin,caspase-3 and bax in the hippocampus were obviously increased(P<0.05) and the same of bcl-2 was decreased of ageing rats(P<0.05). ZuoguiWan, high dose Radix Rehmanniae Praeparata(8g/kg)and low dose Radix RehmanniaePraeparata(4g/kg) could improve the spatial learning & memory abilities of ageingrats(P<0.05),lower the contents of β- galactose glucoside enzyme in the serum(P<0.05),downregulate the mRNA expressions of proBDNF and sotilin in the hippocampus(compared with the ageing rats,P<0.05). Apart from that,Zuogui Wan andlow dose Radix Rehmanniae Praeparata(4g/kg) could also down-regulate the mRNA e

关键词： Ageing Rats Apoptosis Learning and Memory D-galactose

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