Skip to content
2000
Volume 14, Issue 2
  • ISSN: 2211-5366
  • E-ISSN: 2211-5374

Abstract

Research on microRNAs is constantly expanding and evolving due to their role in the regulation of gene expression. miR-122, a 22-nucleotide microRNA, was first discovered as a liver-specific miRNA. Subsequently, it was found to be present in a wide range of tissues, such as the breast, testes, ovaries, and heart. The research on miR-122 in the liver has been extensive over the past few decades, leading to several important discoveries. However, its role in extrahepatic tissues is largely incompletely understood. Therefore, in light of the established clinical relevance of miR-122 as a potential biomarker and/or drug target in the liver, available information on miR-122 is compiled as it pertains to health and disease. This review discusses novel information generated in recent years and the corresponding progress in our understanding of the physiology of extrahepatic miR-122.

Loading

Article metrics loading...

/content/journals/mirna/10.2174/0122115366334187250116164121
2025-07-01
2025-12-16
Loading full text...

Full text loading...

References

  1. BhaskaranM. MohanM. MicroRNAs.Vet. Pathol.201451475977410.1177/030098581350282024045890
    [Google Scholar]
  2. O’BrienJ. HayderH. ZayedY. PengC. Overview of MicroRNA biogenesis, mechanisms of actions, and circulation.Front. Endocrinol. (Lausanne)2018940240210.3389/fendo.2018.0040230123182
    [Google Scholar]
  3. TétreaultN. De GuireV. miRNAs: Their discovery, biogenesis and mechanism of action.Clin. Biochem.20134610-1184284510.1016/j.clinbiochem.2013.02.00923454500
    [Google Scholar]
  4. FuG. BrkićJ. HayderH. PengC. MicroRNAs in human placental development and pregnancy complications.Int. J. Mol. Sci.20131435519554410.3390/ijms1403551923528856
    [Google Scholar]
  5. PaulP. ChakrabortyA. SarkarD. Interplay between miRNAs and human diseases.J. Cell. Physiol.201823332007201810.1002/jcp.2585428181241
    [Google Scholar]
  6. AndoW. KikuchiK. UematsuT. Novel breast cancer screening: Combined expression of miR-21 and MMP-1 in urinary exosomes detects 95% of breast cancer without metastasis.Sci. Rep.2019911359510.1038/s41598‑019‑50084‑531537868
    [Google Scholar]
  7. MonteleoneN.J. MooreA.E. IaconaJ.R. LutzC.S. DixonD.A. miR-21-mediated regulation of 15-hydroxyprostaglandin dehydrogenase in colon cancer.Sci. Rep.201991540510.1038/s41598‑019‑41862‑230931980
    [Google Scholar]
  8. CardozoE.R. FosterR. KarmonA.E. MicroRNA 21a-5p overexpression impacts mediators of extracellular matrix formation in uterine leiomyoma.Reprod. Biol. Endocrinol.20181614610.1186/s12958‑018‑0364‑829747655
    [Google Scholar]
  9. Fomison-NurseI. SawE.E.L. GandhiS. Diabetes induces the activation of pro-ageing miR-34a in the heart, but has differential effects on cardiomyocytes and cardiac progenitor cells.Cell Death Differ.20182571336134910.1038/s41418‑017‑0047‑629302057
    [Google Scholar]
  10. KongL. ZhuJ. HanW. Significance of serum microRNAs in pre-diabetes and newly diagnosed type 2 diabetes: A clinical study.Acta Diabetol.2011481616910.1007/s00592‑010‑0226‑020857148
    [Google Scholar]
  11. ShenY. XuH. PanX. miR 34a and miR 125b are upregulated in peripheral blood mononuclear cells from patients with type 2 diabetes mellitus.Exp. Ther. Med.20171465589559610.3892/etm.2017.525429285097
    [Google Scholar]
  12. ZhangL. HeS. GuoS. Down-regulation of miR-34a alleviates mesangial proliferation in-vitro and glomerular hypertrophy in early diabetic nephropathy mice by targeting GAS1.J. Diabetes Complications201428325926410.1016/j.jdiacomp.2014.01.00224560136
    [Google Scholar]
  13. ZhangX. SongS. LuoH. Regulation of podocyte lesions in diabetic nephropathy via miR-34a in the Notch signaling pathway.Medicine (Baltimore)20169544e505010.1097/MD.000000000000505027858840
    [Google Scholar]
  14. LinY. ShenJ. LiD. MiR-34a contributes to diabetes-related cochlear hair cell apoptosis via SIRT1/HIF-1α signaling.Gen. Comp. Endocrinol.2017246637010.1016/j.ygcen.2017.02.01728263817
    [Google Scholar]
  15. YanT. OoiW.F. QamraA. HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis.Nat. Commun.20189110010.1038/s41467‑017‑02601‑129311615
    [Google Scholar]
  16. PuH.Y. XuR. ZhangM.Y. Identification of microRNA-615-3p as a novel tumor suppressor in non-small cell lung cancer.Oncol. Lett.20171342403241010.3892/ol.2017.568428454411
    [Google Scholar]
  17. LiuJ. JiaY. JiaL. LiT. YangL. ZhangG. MicroRNA 615-3p inhibits the tumor growth and metastasis of NSCLC via inhibiting IGF2.Oncol. Res.201927226927910.3727/096504018X1521501922768829562959
    [Google Scholar]
  18. De GuireV. RobitailleR. TétreaultN. Circulating miRNAs as sensitive and specific biomarkers for the diagnosis and monitoring of human diseases: Promises and challenges.Clin. Biochem.20134610-1184686010.1016/j.clinbiochem.2013.03.01523562576
    [Google Scholar]
  19. SugimachiK. MatsumuraT. HirataH. Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation.Br. J. Cancer2015112353253810.1038/bjc.2014.62125584485
    [Google Scholar]
  20. GanepolaG.A.P. RutledgeJ.R. SumanP. YiengpruksawanA. ChangD.H. Novel blood-based microRNA biomarker panel for early diagnosis of pancreatic cancer.World J. Gastrointest. Oncol.201461223310.4251/wjgo.v6.i1.2224578785
    [Google Scholar]
  21. HannaJ. HossainG.S. KocerhaJ. The potential for microRNA therapeutics and clinical research.Front. Genet.20191047810.3389/fgene.2019.0047831156715
    [Google Scholar]
  22. GrantM.J. BoothA. A typology of reviews: An analysis of 14 review types and associated methodologies.Health Info. Libr. J.20092629110810.1111/j.1471‑1842.2009.00848.x19490148
    [Google Scholar]
  23. Lagos-QuintanaM. RauhutR. YalcinA. MeyerJ. LendeckelW. TuschlT. Identification of tissue-specific microRNAs from mouse.Curr. Biol.200212973573910.1016/S0960‑9822(02)00809‑612007417
    [Google Scholar]
  24. LandgrafP. RusuM. SheridanR. A mammalian microRNA expression atlas based on small RNA library sequencing.Cell200712971401141410.1016/j.cell.2007.04.04017604727
    [Google Scholar]
  25. JoplingC. Liver-specific microRNA-122: Biogenesis and function.RNA Biol.20129213714210.4161/rna.1882722258222
    [Google Scholar]
  26. WardJ. KanchagarC. Veksler-LublinskyI. Circulating microRNA profiles in human patients with acetaminophen hepatotoxicity or ischemic hepatitis.Proc. Natl. Acad. Sci. USA201411133121691217410.1073/pnas.141260811125092309
    [Google Scholar]
  27. CoulouarnC. FactorV.M. AndersenJ.B. DurkinM.E. ThorgeirssonS.S. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties.Oncogene200928403526353610.1038/onc.2009.21119617899
    [Google Scholar]
  28. XuJ. WuC. CheX. Circulating MicroRNAs, miR-21, miR-122, and miR-223, in patients with hepatocellular carcinoma or chronic hepatitis.Mol. Carcinog.201150213614210.1002/mc.2071221229610
    [Google Scholar]
  29. ClossE.I. BoisselJ.P. HabermeierA. RotmannA. Structure and function of cationic amino acid transporters (CATs).J. Membr. Biol.20062132677710.1007/s00232‑006‑0875‑717417706
    [Google Scholar]
  30. YanW. CaoM. RuanX. Cancer-cell-secreted miR-122 suppresses O-GlcNAcylation to promote skeletal muscle proteolysis.Nat. Cell Biol.202224579380410.1038/s41556‑022‑00893‑035469018
    [Google Scholar]
  31. ChahalJ. GebertL.F.R. CamargoC. MacRaeI.J. SaganS.M. miR-122–based therapies select for three distinct resistance mechanisms based on alterations in RNA structure.Proc. Natl. Acad. Sci. USA202111833e210367111810.1073/pnas.210367111834385308
    [Google Scholar]
  32. KundenR.D. KhanJ.Q. GhezelbashS. WilsonJ.A. The role of the liver-specific microRNA, miRNA-122 in the HCV replication cycle.Int. J. Mol. Sci.20202116567710.3390/ijms2116567732784807
    [Google Scholar]
  33. WeiX. DingJ. TianW. YuY.C. MicroRNA-122 as a diagnostic biomarker for hepatocellular carcinoma related to hepatitis C virus: A meta-analysis and systematic review.J. Int. Med. Res.2020488030006052094163410.1177/030006052094163432790532
    [Google Scholar]
  34. Faramin LashkarianM. HashemipourN. NiarakiN. MicroRNA-122 in human cancers: From mechanistic to clinical perspectives.Cancer Cell Int.20232312910.1186/s12935‑023‑02868‑z36803831
    [Google Scholar]
  35. HowellL.S. IrelandL. ParkB.K. GoldringC.E. MiR-122 and other microRNAs as potential circulating biomarkers of drug-induced liver injury.Expert Rev. Mol. Diagn.2018181475410.1080/14737159.2018.141514529235390
    [Google Scholar]
  36. MurrayD.D. SuzukiK. LawM. Circulating miR-122 and miR-200a as biomarkers for fatal liver disease in ART-treated, HIV-1-infected individuals.Sci. Rep.2017711093410.1038/s41598‑017‑11405‑828883647
    [Google Scholar]
  37. MoosaM.S. RussomannoG. DorfmanJ.R. Analysis of serum microRNA‐122 in a randomized controlled trial of N‐acetylcysteine for treatment of antituberculosis drug‐induced liver injury.Br. J. Clin. Pharmacol.20238961844185110.1111/bcp.1566136639145
    [Google Scholar]
  38. ChangJ. NicolasE. MarksD. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1.RNA Biol.20041210611310.4161/rna.1.2.106617179747
    [Google Scholar]
  39. DenzlerR. AgarwalV. StefanoJ. BartelD.P. StoffelM. Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.Mol. Cell201454576677610.1016/j.molcel.2014.03.04524793693
    [Google Scholar]
  40. BurchardJ. ZhangC. LiuA.M. microRNA‐122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma.Mol. Syst. Biol.20106140210.1038/msb.2010.5820739924
    [Google Scholar]
  41. ValdmanisP.N. KimH.K. ChuK. miR-122 removal in the liver activates imprinted microRNAs and enables more effective microRNA-mediated gene repression.Nat. Commun.201891532110.1038/s41467‑018‑07786‑730552326
    [Google Scholar]
  42. TsaiW.C. HsuS.D. HsuC.S. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis.J. Clin. Invest.201212282884289710.1172/JCI6345522820290
    [Google Scholar]
  43. EsauC. DavisS. MurrayS.F. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.Cell Metab.200632879810.1016/j.cmet.2006.01.00516459310
    [Google Scholar]
  44. KimN. KimH. JungI. KimY. KimD. HanY.M. Expression profiles of miRNAs in human embryonic stem cells during hepatocyte differentiation.Hepatol. Res.201141217018310.1111/j.1872‑034X.2010.00752.x21269386
    [Google Scholar]
  45. CastoldiM. Vujic SpasicM. AltamuraS. The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice.J. Clin. Invest.201112141386139610.1172/JCI4488321364282
    [Google Scholar]
  46. ElménJ. LindowM. SchützS. LNA-mediated microRNA silencing in non-human primates.Nature2008452718989689910.1038/nature0678318368051
    [Google Scholar]
  47. ElménJ. LindowM. SilahtarogluA. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver.Nucleic Acids Res.20083641153116210.1093/nar/gkm111318158304
    [Google Scholar]
  48. KrützfeldtJ. RajewskyN. BraichR. Silencing of microRNAs in vivo with ‘antagomirs’.Nature2005438706868568910.1038/nature0430316258535
    [Google Scholar]
  49. MitchellP.S. ParkinR.K. KrohE.M. Circulating microRNAs as stable blood-based markers for cancer detection.Proc. Natl. Acad. Sci. USA200810530105131051810.1073/pnas.080454910518663219
    [Google Scholar]
  50. ArroyoJ.D. ChevilletJ.R. KrohE.M. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.Proc. Natl. Acad. Sci. USA2011108125003500810.1073/pnas.101905510821383194
    [Google Scholar]
  51. El-HefnawyT. RajaS. KellyL. Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics.Clin. Chem.200450356457310.1373/clinchem.2003.02850614718398
    [Google Scholar]
  52. VogtJ. SheinsonD. KatavolosP. Variance component analysis of circulating miR-122 in serum from healthy human volunteers.PLoS One2019147e022040610.1371/journal.pone.022040631348817
    [Google Scholar]
  53. KyrmiziI. HatzisP. KatrakiliN. TroncheF. GonzalezF.J. TalianidisI. Plasticity and expanding complexity of the hepatic transcription factor network during liver development.Genes Dev.200620162293230510.1101/gad.39090616912278
    [Google Scholar]
  54. LaudadioI. ManfroidI. AchouriY. A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation.Gastroenterology2012142111912910.1053/j.gastro.2011.09.00121920465
    [Google Scholar]
  55. YamadaH. SuzukiK. IchinoN. Associations between circulating microRNAs (miR-21, miR-34a, miR-122 and miR-451) and non-alcoholic fatty liver.Clin. Chim. Acta20134249910310.1016/j.cca.2013.05.02123727030
    [Google Scholar]
  56. HenkeJ.I. GoergenD. ZhengJ. microRNA-122 stimulates translation of hepatitis C virus RNA.EMBO J.200827243300331010.1038/emboj.2008.24419020517
    [Google Scholar]
  57. KojimaK. TakataA. VadnaisC. MicroRNA122 is a key regulator of α-fetoprotein expression and influences the aggressiveness of hepatocellular carcinoma.Nat. Commun.20112133810.1038/ncomms134521654638
    [Google Scholar]
  58. AntoineD.J. DearJ.W. LewisP.S. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital.Hepatology201358277778710.1002/hep.2629423390034
    [Google Scholar]
  59. WuY. GaoC. CaiS. Circulating miR-122 is a predictor for virological response in chb patients with high viral load treated with nucleos(t)ide analogs.Front. Genet.20191024324310.3389/fgene.2019.0024330967899
    [Google Scholar]
  60. TsaiW.C. HsuP.W.C. LaiT.C. MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma.Hepatology20094951571158210.1002/hep.2280619296470
    [Google Scholar]
  61. KutayH. BaiS. DattaJ. Downregulation of miR‐122 in the rodent and human hepatocellular carcinomas.J. Cell. Biochem.200699367167810.1002/jcb.2098216924677
    [Google Scholar]
  62. BaiS. NasserM.W. WangB. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib.J. Biol. Chem.200928446320153202710.1074/jbc.M109.01677419726678
    [Google Scholar]
  63. PeiZ.J. ZhangZ.G. HuA.X. YangF. GaiY. miR-122-5p inhibits tumor cell proliferation and induces apoptosis by targeting MYC in gastric cancer cells.Pharmazie201772634434729442023
    [Google Scholar]
  64. RaoM ZhuY ZhouY CongX FengL MicroRNA-122 inhibits proliferation and invasion in gastric cancer by targeting CREB1. Am. J. Cancer Res.20177232333328337380
    [Google Scholar]
  65. XuX. GaoF. WangJ. MiR-122-5p inhibits cell migration and invasion in gastric cancer by down-regulating DUSP4.Cancer Biol. Ther.201819542743510.1080/15384047.2018.142392529509059
    [Google Scholar]
  66. DuanY. DongY. DangR. MiR‐122 inhibits epithelial mesenchymal transition by regulating P4HA1 in ovarian cancer cells.Cell Biol. Int.201842111564157410.1002/cbin.1105230136751
    [Google Scholar]
  67. YangY. LiuY. LiuW. miR-122 inhibits the cervical cancer development by targeting the oncogene RAD21.Biochem. Genet.202260130331410.1007/s10528‑021‑10098‑z34191246
    [Google Scholar]
  68. WangS. ZhengW. JiA. ZhangD. ZhouM. Overexpressed miR-122-5p promotes cell >viability, proliferation, migration and glycolysis of renal cancer by negatively regulating PKM2.Cancer Manag. Res.2019119701971310.2147/CMAR.S22574231814765
    [Google Scholar]
  69. FanY. MaX. LiH. miR‐122 promotes metastasis of clear‐cell renal cell carcinoma by downregulating Dicer.Int. J. Cancer2018142354756010.1002/ijc.3105028921581
    [Google Scholar]
  70. JingushiK. KashiwagiY. UedaY. High miR-122 expression promotes malignant phenotypes in ccRCC by targeting occludin.Int. J. Oncol.201751128929710.3892/ijo.2017.401628534944
    [Google Scholar]
  71. HuZ. ShenW.J. CortezY. Hormonal regulation of microRNA expression in steroid producing cells of the ovary, testis and adrenal gland.PLoS One2013810e7804010.1371/journal.pone.007804024205079
    [Google Scholar]
  72. LiuT. HuangY. LiuJ. MicroRNA-122 influences the development of sperm abnormalities from human induced pluripotent stem cells by regulating TNP2 expression.Stem Cells Dev.201322121839185010.1089/scd.2012.065323327642
    [Google Scholar]
  73. MenonB. SindenJ. Franzo-RomainM. BottaR.B. MenonK.M.J. Regulation of LH receptor mRNA binding protein by miR-122 in rat ovaries.Endocrinology2013154124826483410.1210/en.2013‑161924064360
    [Google Scholar]
  74. MenonB. GulappaT. MenonK.M.J. Molecular regulation of LHCGR expression by miR-122 during follicle growth in the rat ovary.Mol. Cell. Endocrinol.2017442818910.1016/j.mce.2016.12.00227940300
    [Google Scholar]
  75. JeonT.I. OsborneT.F. miRNA and cholesterol homeostasis.Biochim. Biophys. Acta Mol. Cell Biol. Lipids201618611212 Pt B2041204610.1016/j.bbalip.2016.01.00526778752
    [Google Scholar]
  76. Toran-AllerandC.D. TinnikovA.A. SinghR.J. NethrapalliI.S. 17α-estradiol: A brain-active estrogen?Endocrinology200514693843385010.1210/en.2004‑161615947006
    [Google Scholar]
  77. MenonB. GuoX. GarciaN. GulappaT. MenonK.M.J. miR-122 regulates LHR expression in rat granulosa cells by targeting Insig1 mRNA.Endocrinology201815952075208210.1210/en.2017‑0327029579170
    [Google Scholar]
  78. MenonB. GulappaT. MenonK.M.J. miR-122 regulates LH receptor expression by activating sterol response nlm binding protein in rat ovaries.Endocrinology201515693370338010.1210/en.2015‑112126125464
    [Google Scholar]
  79. AgarwalV. BellG.W. NamJ.W. BartelD.P. Predicting effective microRNA target sites in mammalian mRNAs.eLife20154e0500510.7554/eLife.0500526267216
    [Google Scholar]
  80. AbouhamedM. GrobeK. Leefa Chong SanI.V. Myosin IXa regulates epithelial differentiation and its deficiency results in hydrocephalus.Mol. Biol. Cell200920245074508510.1091/mbc.e09‑04‑029119828736
    [Google Scholar]
  81. Bozal-BasterraL. Gonzalez-SantamartaM. MuratoreV. LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is a contributing factor in Townes-Brocks Syndrome.eLife20209e5595710.7554/eLife.5595732553112
    [Google Scholar]
  82. SinghK. JayaramM. KaareM. Neural cell adhesion molecule Negr1 deficiency in mouse results in structural brain endophenotypes and behavioral deviations related to psychiatric disorders.Sci. Rep.201991545710.1038/s41598‑019‑41991‑830932003
    [Google Scholar]
  83. Biological insights from 108 schizophrenia-associated genetic loci.Nature2014511751042142710.1038/nature1359525056061
    [Google Scholar]
  84. HydeC.L. NagleM.W. TianC. Identification of 15 genetic loci associated with risk of major depression in individuals of European descent.Nat. Genet.20164891031103610.1038/ng.362327479909
    [Google Scholar]
  85. KaraM. AxtonR.A. JacksonM. A role for MOSPD1 in mesenchymal stem cell proliferation and differentiation.Stem Cells201533103077308610.1002/stem.210226175344
    [Google Scholar]
  86. BonneauE. NeveuB. KostantinE. TsongalisG.J. De GuireV. How close are miRNAs from clinical practice? A perspective on the diagnostic and therapeutic market.EJIFCC201930211412731263388
    [Google Scholar]
  87. LindowM. KauppinenS. Discovering the first microRNA-targeted drug.J. Cell Biol.2012199340741210.1083/jcb.20120808223109665
    [Google Scholar]
  88. BaekJ. KangS. MinH. MicroRNA-targeting therapeutics for hepatitis C.Arch. Pharm. Res.201437329930510.1007/s12272‑013‑0318‑924385319
    [Google Scholar]
  89. LiuY. LiP. LiuL. ZhangY. The diagnostic role of miR-122 in drug-induced liver injury.Medicine (Baltimore)20189749e1347810.1097/MD.000000000001347830544438
    [Google Scholar]
  90. CioneE. Abrego GuandiqueD.M. CaroleoM.C. LucianiF. ColosimoM. CannataroR. Liver damage and microRNAs: An Update.Curr. Issues Mol. Biol.2022451789110.3390/cimb4501000636661492
    [Google Scholar]
  91. FuS. WuD. JiangW. Molecular biomarkers in drug-induced liver injury: Challenges and future perspectives.Front. Pharmacol.202010166710.3389/fphar.2019.0166732082163
    [Google Scholar]
  92. SalaminO. JaggiL. BaumeN. RobinsonN. SaugyM. LeuenbergerN. Circulating microRNA-122 as potential biomarker for detection of testosterone abuse.PLoS One2016115e015524810.1371/journal.pone.015524827171140
    [Google Scholar]
  93. Cortez-DiasN. CostaM.C. Carrilho-FerreiraP. Circulating miR-122-5p/miR-133b ratio is a specific early prognostic biomarker in acute myocardial infarction.Circ. J.201680102183219110.1253/circj.CJ‑16‑056827593229
    [Google Scholar]
  94. MagedA.M. DeebW.S. El AmirA. Diagnostic accuracy of serum miR‐122 and miR‐199a in women with endometriosis.Int. J. Gynaecol. Obstet.20181411141910.1002/ijgo.1239229149541
    [Google Scholar]
/content/journals/mirna/10.2174/0122115366334187250116164121
Loading
/content/journals/mirna/10.2174/0122115366334187250116164121
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test