Biogenesis of MiRNA-195 and its role in biogenesis, the cell cycle, and apoptosis (1)

J BIOCHEM MOLECULAR TOXICOLOGYVolume25,Number6,2011 Biogenesis of MiRNA-195and Its Role in Biogenesis,the Cell Cycle,and ApoptosisJun-Feng He1Yu-Mei Luo,1Xin-Hong Wan,1Deqian Jiang21Department of Cardiology,Shenzhen Longgang District People’s Hospital,Guang Dong Medicine College,Shenzhen,518172,People’s Republic of China;E-mail:hjf5529406@2Department of Cardiology,the Second Xiangya Hospital,Central South University,Changsha,410011,Hunan,People’s Republic of China Received24February2011;revised30March2011;accepted16April2011ABSTRACT:microRNA-195(miR-195)is an important member of the micro-15/16/195/424/497family,and which is activated in multiple diseases,such as can-cers,heart failure,and schizophrenia.Mir-195regu-lates a plethora of target proteins,which are involved in the cell cycle,apoptosis,proliferation.WEE1,CDK6, and Bcl-2are confirmed target genes of miR-195that are involved in miR-195-mediated cell-cycle and apop-tosis effects.However,the mechanism of miR-195ac-tion is not completely understood.This review summa-rizes recent the research progress regarding the roles of miR-195in the cell cycle and in apoptosis.C 2011 Wiley Periodicals,Inc.J Biochem Mol Toxicol25:404–408, 2011;View this article online at .DOI10:1002/jbt.20396KEYWORDS:microRNA-195,biogenesis,cell cycle, apoptosisMiRNA BIOGENESIS AND MECHANISMMicroRNAs(miRNAs),which are approximately 21nucleotides in length,are a recently discovered class of endogenous,small,noncoding RNAs that regulate about30%of the encoding genes of the human genome. MiRNAs occurs naturally and downregulate pro-tein levels by posttranscriptional regulation of target mRNAs.MiRNA genes exist in the introns of protein-coding genes,in introns and exons of noncoding genes,or even in the3 -UTR of protein-coding genes.MiRNAs are transcribed by RNA pol II or RNA pol III as mono-or polycistronic primary miRNAs(pri-miRNA)tran-scripts,often containing thousands of nucleotides that form hairpins(stem-loops)[1],pri-miRNAs are pro-Correspondence to:Xin-Hong Wan.c 2011Wiley Periodicals,Inc.cessed in the nucleus by the Drosha-DGCR8complex (in human)or Drosha-Pasha(in Drosophila and C.ele-gans)to produce∼70nt-long precursor miRNAs(pre-miRNAs).Pre-miRNAs are then transported to the cy-toplasm by exportin-5and Ran-GTP.In the cytoplasm,pre-miRNAs and mirtrons share the same processing pathway.Another RNAase III-like endonuclease Dicer processes∼70nt pre-miRNAs into mature∼22nt miRNA duplexes,which are des-ignated miRNA/miRNA*.The next step is loading of one of the two strands of this duplex onto the RISC (RNA-induced silencing complex),to form miRISC. Only the miRNA is successfully incorporated into the miRISC(miRNP),whereas the miRNA*is eliminated and degraded.Residues2–8at the5 end of the miRNA play a pivotal role in the specificity of the interaction miRNA with mRNA.This region is called the“seed.”The miRISC complex binds via the seed region to the 3 untranslated region(3 -UTR)of target mRNAs,and this imperfect match leads to translational inhibition without influencing mRNA integrity.Biogenesis of miRNA-195The miRNA-195sequence was predicted based on homology to a verified miRNA from mouse[2] and was later verified in human[3].The hsa-miR-195gene is located from6881953bp to6862065bp on the chromosome17p13.1[4](Figure1),with in-tron7and on the opposite strand of an mRNA gene AK098506(/ cgi-bin/miRGen/v3),which may encode a hypothet-ical protein LOC284112(/ cgi-bin/source/sourceResult).The function of this pro-tein is currently unknown.MiR-195is aberrantly expressed in multiple types of diseases including human breast cancer[5],glioblas-toma multiforme[6],gastric cancer[7],human hepa-tocellular carcinoma[8],adrenocortical adenomas[9],404Volume25,Number6,2011BIOGENESIS AND ROLES OF miRNA-195405FIGURE1.Location of Hsa-miR-195on chromosome17p13.1.chronic lymphocytic leukemia[10],bladder cancer[11], schizophrenia[12–15],and cardiac hypertrophy[16,17] (Table1).The expression of miR-195varies with differ-ent stages of diseases[16–19].MicroRNAs binded to the3 -UTR region of the target mRNA and downregulate protein levels by translational repression or by mRNA degradation mechanisms.In Table2,we summarize some important miR-195targets and the respective regulatory mecha-nisms.Multiple studies have shown that miR-195pre-vents cell proliferation,promotes apoptosis in diverse cancers and in cardiovascular diseases[8,20,21].The roles of miR-195targets in cell cycle regulation and their aberrant expression in various cancers suggest that they belong to a novel class of tumor suppressor gene depending on the targets they regulate.The Roles of miRNA-195in the Cell CycleMany studies have indicated that miRNA-195has a regulatory function in the cell-cycle,especially with re-spect to cell proliferation.Mitotic cell cycle progression is accomplished through a set sequence of events,DNA replication(S phase)and mitosis(M phase)are sepa-rated temporally by gaps known as G1and G2phases. The expression and activation of cyclin-dependent ki-nases(CDKs)and of cyclin determine whether cells proceed through G1into S phase,and from G2into M phase,This sequence of events is regulated by mecha-nisms that are conserved in more complex eukaryotes. Many transcription factors,such as WEE1and E2F3,af-fect the regulation of the cell cycle.Joglekar et ed an antisense miR-195-specific inhibitor in regenerating pancreatic cells and found that miR-195reduced the number of hormone-producing cells during pancreas development[19].Ichimi et al.showed that significant decreases in cell growth occurred in miR-195-trans-fectants[11].Van Rooij et al.reported that6week-old miR-195transgenic mice(Tg mice)displayed thinning of left ventricular walls[17].The Role of miR-195in the G2/M Transition WEE1is well characterized as a cell-cycle check-point kinase that regulates the entry into mitosis in dividing cells.Three inhibitory kinases,WEE1,WEE2, and MYT1regulate G2CycB/CDK complex activity in mammalian cells.However,WEE1is the only one of these kinases that negatively regulates CycB/CDK [24,25].Qi et al.found that miR-195overexpression in hESCs directly or indirectly acts on targets that nor-mally inhibit the rate of the cell cycle at the G2/M transition[20].They identified potential targets usingJ Biochem Molecular Toxicology DOI10:1002/jbt406HE ET AL.Volume25,Number6,2011TABLE1.Disordered Expression of mi-195in Different DiseasesDisease Type Reference Upregulation/Downregulation Breast cancer[5]UpregulationMCF-7cells[22]UpregulationU251R[6]UpregulationGastric cancer[7]Downregulation Chronic lymphocytic leukemia[10]Upregulation Adrenocortical adenomas[9]Upregulation Human hepatocellular carcinoma cells[8]Downregulation Bladder cancer[11]Downregulation Placentae with severe pre-eclampsia[23]Upregulation Schizophrenia[12–15]Upregulation Cardiac hypertrophy[16,17]Upregulation Different stages of pancreatic regeneration and development[19]Upregulation Irreversible growth arrest states in WI-38humanfibroblasts[18]UpregulationTargetscan and found that WEE1contains two pre-dicted miR-195binding sites in its3 UTR.They used a luciferase assay in an HCT116Dicer hypomorphic cell line and found that overexpression of miR-195results in a reduction of luciferase activity in the WEE13 UTR re-porter construct and that mutations in the WEE13 UTR relieve the repression of luciferase activity mediated by miR-195.In addition,they showed that miR-195regu-lates the hESC cell cycle by repressing WEE1levels. Reduction of miR-195levels in hESCs results in atten-uated cell division.The Role of miR-195in the G1/S TransitionXu et al.showed that miR-195may block the G1/S transition in hepatocellular carcinoma cells(HCCs)by repressing Rb-E2F signaling through targeting multi-ple molecules,including cyclin D1,CDK6,and E2F3[8]. Cyclin D1,CDK6,and E2F3are part of the G1/S transi-tion pathway.They promote the G1into S phase transi-tion via the Rb-E2F signaling pathway.Silencing these factors induces cell cycle arrest in G1phase.Xu et al. constructed expression vectors pc3-miR-195,pc3-gab-CCND1,pc3-gab-CDK6,and pc3-gab-E2F3and tested the affects between miR-195and the expressed factors. MiR-195directly inhibits the expression of the cyclin D1,CDK6,and E2F3through their3 UTRs,but miR-195overexpression caused a reduction only in the level of CDK6mRNA but not in the levels of cyclin D1or E2F3mRNA.Together these data indicate that inhibi-tion of miR-195promotes G1/S transition.The Role of miRNA-195in ApoptosisLiu et al.showed that microRNA-195promotes apoptosis and suppresses tumorigenicity of human col-orectal cancer cells[21].The process of programmed cell death is generally characterized by distinct mor-phological characteristics and energy-dependent bio-chemical mechanisms.Inappropriate apoptosis,either too little or too much,is a factor in many human condi-tions including neurodegenerative diseases,ischemic damage,autoimmune disorders and many types of cancer[28].Liu et al.constructed an miR-195expression plasmid and a reporter plasmid of luciferase inserted into the3 UTR of Bcl-2.When both were transfected into the human CRC cell lines HT29and LoVo,miR-195suppressed the activity of the luciferase reporter. This result further proved that the apoptosis promot-ing effect of miR-195occurs mainly through inhibition of Bcl-2expression.CONCLUSIONS AND PERSPECTIVESHere,we have summarized our current under-standing of miR-195,which is a member of the micro-15/16/195/424/497family of miRNAs.Numerous studies have suggested that miR-195promotes cell di-vision and apoptosis while inhibiting cell proliferation. The main pathophysiological changes in hypertension and cancer are cell proliferation.In transgenic mice,a 25-fold expression of miR-195induced cardiac growth with disorganization of cardiomyocytes and heart fail-ure[17].The mechanism of miR-195in cardiovascu-lar diseases is not completely understood.To explore whether miR-195had potential therapeutic effect in cardiovascular diseases and at which level of miR-195 would inhibit the incorrect proliferation.Different lev-els of mimics of miR-195was given to spontaneously hypertensive rats treated with or without captopril(80 mg/kg/day),the degree of myocardial hypertrophy and vascular proliferation and its molecular mecha-nism would be observed.From this experiment,we could know whether miR-195had potential therapeu-tic effect in cardiovascular diseases and at which level of miR195would inhibit the incorrect proliferation.J Biochem Molecular Toxicology DOI10:1002/jbtVolume25,Number6,2011BIOGENESIS AND ROLES OF miRNA-195407TABLE2.miR-195TargetsmRNA Validated by Function ReferenceWEE1Luciferase reporter assay,real-time PCR,western blot,immunostaining Protein-tyrosine kinase activity,proteinserine/threonine kinase activity,transferase activity,nucleotidebinding,ATP binding,proteinbinding,mitosis,protein amino acid phosphorylation,nucleus,cell cycle, regulation of progression throughcell cycle,cell division[20]CDK6Luciferase reporter assay,real-time PCR,western blot,immunohistochemistryPositive regulation of G1/S[8]Bcl-2Luciferase reporter assay,real-time PCR,western blot Mitochondrion,negative regulation ofcell proliferation,endoplasmicreticulum,apoptosis,regulation of apoptosis,release of cytochrome cfrom mitochondria,cytosol,mitochondrial outer membrane,protein binding,integral tomembrane,humoral immuneresponse,nucleus,membrane,identical protein binding,regulationof progression through cell cycle, antiapoptosis[21]BNDF Luciferase reporter assay,miRNAs microarray,solutionhybridization,ISH withLNA-modifiedoligonucleotides,immunoassay,chromatinimmunoprecipitation,neuronal transduction Axon target recognition,negativeregulation of neuroblastproliferation,growth factor activity, regulation of metabolism, mechanoreceptor differentiation,dendrite morphogenesis,positiveregulation of neuron differentiation,protein binding,extracellular space, cytoplasmic membrane-boundvesicle,regulation of retinalprogrammed cell death,neuron recognition,feeding behavior,nervous system development,antiapoptosis[15]ADP ribosylation factor-like2(Arl2)Luciferin-luciferase ATP assay,luciferase reporter assay,real-time PCR,western blotModulates cellular ATP levels[26]FMS-like tyrosine kinase3(FLT3)Luciferase reporter assay Extracellular space,protein binding,protein serine/threonine kinaseactivity,integral to membrane,lymphocyte differentiation[2]Further research is required to understand the reg-ulation of miR-195expression to detect gene networks that are regulated by this microRNA and to develop potent miR-195-based therapeutics.REFERENCES1.Choudhuri S.Small noncoding RNAs:biogenesis,func-tion,and emerging significance in toxicology.J Biochem Mol Toxicol2010;24(3):195–216.J Biochem Molecular Toxicology DOI10:1002/jbt408HE ET AL.Volume25,Number6,2011gos-Quintana M,Rauhut R,Meyer J,Borkhardt A,Tuschl T.New microRNAs from mouse and human.RNA 2003;9(2):175–179.ndgraf P,Rusu M,Sheridan R,Sewer A,Iovino N,Aravin A,Pfeffer S,Rice A,Kamphorst AO,Landthaler M,Lin C,Socci ND,Hermida L,Fulci V,Chiaretti S,Fo`a R, Schliwka J,Fuchs U,Novosel A,M¨uller RU,Schermer B, Bissels U,Inman J,Phan Q,Chien M,Weir DB,Choksi R, De Vita G,Frezzetti D,Trompeter HI,Hornung V,Teng G, Hartmann G,Palkovits M,Di Lauro R,Wernet P,Macino G,Rogler CE,Nagle JW,Ju J,Papavasiliou FN,Benzing T, Lichter P,Tam W,Brownstein MJ,Bosio A,Borkhardt A, Russo JJ,Sander C,Zavolan M,Tuschl T.A mammalian microRNA expression atlas based on small RNA library sequencing.Cell2007;129(7):1401–14.4.Flavin RJ,Smyth PC,Laios A,O’Toole SA,Barrett C,Finn SP,Russell S,Ring M,Denning KM,Li J,Aherne ST, Sammarae DA,Aziz NA,Alhadi A,Sheppard BL,Lao K, Sheils OM,O’Leary JJ.Potentially important microRNA cluster on chromosome17p13.1in primary peritoneal carcinoma.Mod Pathol2009;22(2):197–205.5.Heneghan HM,Miller N,Lowery AJ,Sweeney KJ,Newell J,Kerin MJ.Circulating microRNAs as novel minimally invasive biomarkers for breast cancer.Ann Surg2010;251(3):499–505.6.Ujifuku K,Mitsutake N,Takakura S,Matsuse M,SaenkoV,Suzuki K,Hayashi K,Matsuo T,Kamada K,Nagata I, Yamashita S.miR-195,miR-455–3p and miR-10a*are im-plicated in acquired temozolomide resistance in glioblas-toma multiforme cells.Cancer Lett2010;296(2):241–248.7.Guo J,Miao Y,Xiao B,Huan R,Jiang Z,Meng D,WangY.Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues.J Gastroen-terol Hepatol2009;24(4):652–657.8.Xu T,Zhu Y,Xiong Y,Ge YY,Yun JP,Zhuang SM.MicroRNA-195suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells.Hepatology2009;50(1):113–121.9.Soon PSH,Tacon LJ,Gill AJ.Bambach CP,Sywak MS,Campbell PR,Yeh MW,Wong SG,Clifton-Bligh RJ, Robinson BG,Sidhu SB.miR-195and miR-483–5p iden-tified as predictors of poor prognosis in adrenocortical cancer.Clin Cancer Res2009;15(24):7684–7692.10.Zanette DL,Rivadavia F,Molfetta GA,Barbuzano FG,Proto-Siqueira R,Falc˜a o RP,Zago MA,Silva-Jr.WA.miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukaemia.Braz J Med Biol Res 2007;40(11):1435–1440.11.Ichimi T,Enokida H,Okuno Y,Kunimoto R,ChiyomaruT,Kawamoto K,Kawahara K,Toki K,Kawakami K, Nishiyama K,Tsujimoto G,Nakagawa M,Seki N.Identi-fication of novel microRNA targets based on microRNA signatures in bladder cancer.Int J Cancer2009;125(2):345–352.12.Guo AY,Sun J,Jia P,Zhao Z.A novel microRNAand transcription factor mediated regulatory network in schizophrenia.BMC Syst Biol2010;4:10.13.Dinan TG.MicroRNAs as a target for novel antipsy-chotics:a systematic review of an emergingfield.Int J Neuropsychopharmacol2010;13(3):395–404.14.Mellions N,Huang HS,Baker SP,Galdzicka M,GinnsE,Akbarian S.Molecular determinants of dysregu-lated GABAergic gene expression in the prefrontal cortex of subjects with schizophrenia.Biol Psychiatry 2009;65(12):1006–1014.15.Mellios N,Huang HS,Grigorenko A,Rogaev E,AkbarianS.A set of differentially expressed miRNAs,including miR-30a-5p,act as post-transcriptional inhibitors BDNF in prefrontal cortex.Hum Mol Genet2008;17(19);3030–3042.16.Busk PK,Cirera S.MicroRNA profiling in early hyper-trophic growth of the left ventricle in rats.Biochem Bio-phys Res Commun2010;396(4):989–993.17.van Rooij E,Sutherland LB,Liu N,Williams AH,McAnally J,Gerard RD,Richardson JA,Olson EN.A sig-nature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure.Proc Natl Acad Sci U S A2006;103(48):18255–18260.18.Maes OC,Sarojini H,Wang E.Stepwise up-regulationof microRNA expression levels from replicating to re-versible and irreversible growth arrest states in WI-38 humanfibroblasts.J Cell Physiol2009;221(1):109–119. 19.Joglekar MV,Parekh VS,Mehta S,Bhonde RR,HardikarAA.MicroRNA profiling of developing and regenerating pancreas reveal post-transcriptional regulation of neuro-genin3.Dev Biol2007;311(2):603–612.20.Qi J,Yu J-Y,Shcherbata HR,Mathieu J,Wang AJ,SealS,Zhou W,Stadler BM,Bourgin D,Wang L,Nelson A, Ware C,Raymond C,Lim LP,Magnus J,Ivanovska I, Diaz R,Ball A,Cleary MA,Ruohola-Baker H.microRNAs regulate human embryonic stem cell division.Cell Cycle 2009;8(22):3729–3741.21.Liu L,Chen L,Xu Y,Li R,Du X.microRNA-195pro-motes apoptosis and suppresses tumorigenicity of hu-man colorectal cancer cells.Biochem Biophys Res Com-mun2010;400(2):236–240.22.Zhang H,Su SB,Zhou QM,Lu YY.Differential expressionprofiles of microRNAs between breast cancer cells and mammary epithelial cells.Ai Zheng2009;28(5):493–499.23.Hu Y,Li P,Hao S,Liu L,Zhao J,Hou Y.Differentialexpression of microRNAs in the placenta of Chinese pa-tients with severe pre-eclampsia.Clin Chem Lab Med 2009;47(8):923–929.24.Tominaga Y,Li C,Wang RH,Deng CX.Murine Wee1plays a critical role in cell cycle regulation and pre-implantation stages of embryonic development.Int J Biol Sci2006;2:161–170.25.Price DM,Jin Z,Rabinovitch S,Campbell SD.Ectopicexpression of the Drosophila Cdk1inhibitory kinases, Wee1and Myt1,interferes with the second mitotic wave and disrupts pattern formation during eye development.Genetics2002;161:721–731.26.Nishi H,Ono K,Iwanaga Y,Horie T,Nagao K,TakemuraG,Kinoshita M,Kuwabara Y,Mori RT,Hasegawa K, Kita T,Kimura T.MicroRNA-15b modulates cellular ATP levels and degenerates mitochondria via Arl2in neona-tal rat cardiac myocytes.J Biol Chem2010;285(7):4920–4930.27.Gao XN,Lin J,Li YH,Wang LL,Yu L.Construction ofFLT33 -UTR-luciferase reporter vector and evaluation of its activity.Zhongguo Shi Yan Xue Za Zhi2010;18(3):694–697.28.Elmore S.Apoptosis:a review of programmed cell death.Toxicol Pathol.2007;35(4):495–516.J Biochem Molecular Toxicology DOI10:1002/jbt。