Research Article Research of the Methylation Status of miR ...downloads.hindawi.com/journals/jir/2013/524204.pdfResearch of the Methylation Status of miR-124a Gene Promoter among Rheumatoid

Hindawi Publishing CorporationClinical and Developmental ImmunologyVolume 2013 Article ID 524204 4 pageshttpdxdoiorg1011552013524204

Research ArticleResearch of the Methylation Status of miR-124a Gene Promoteramong Rheumatoid Arthritis Patients

Qiao Zhou1 Li Long1 Guixiu Shi2 Jing Zhang1 Tong Wu1 and Bin Zhou1

1 Department of Rheumatology Sichuan Academy of Medical Science amp Sichuan Provincial Peoplersquos Hospital No 32 West Section 2Yihuan Road Chengdu Sichuan 610072 China

2Department of Rheumatology The First Affiliated Hospital of Xiamen University Xiamen Fujian 361003 China

Correspondence should be addressed to Bin Zhou bingb2917126com

Received 1 May 2013 Accepted 5 September 2013

Academic Editor Jianying Zhang

Copyright copy 2013 Qiao Zhou et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To analyze the methylation status of miR-124a loci in synovial tissues of rheumatoid arthritis (RA) patients usingmethylation-specific polymerase chain reaction (MSP) Materials and Methods DNA obtained from the frozen tissue of 7 RAsamples 6 osteoarthritis (OA) samples and 3 healthy controls were undergoing bisulfite conversion and then analyzed for miR-124a promoter methylation usingMSP assay Results miR-124-a1 and miR-124-a2 promoter methylation were both seen in 714 ofRA samples compared to 167 of OA samples miR-124-a3 promoter methylation was seen in 571 of RA samples and 0 of OAsamples All the three loci were unmethylated in 3 healthy controls Conclusion The methylation status of miR-124a seen in thisstudy concurs with that reported in tumor cells indicating epigenetic dysregulation constituents a mechanism in the developmentof rheumatoid arthritis

1 Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory sym-metrical polyarticular autoimmune disease affecting sim1of the population worldwide [1 2] The main characteris-tic features of RA are persistent inflammation synoviumhyperplasia lymphocyte infiltration and abnormal prolifer-ation of fibroblast-like synoviocytes (FLS) which eventuallylead to progressive cartilage erosion and bone destruction[3] Although the pathogenesis of RA is not clear muchevidence demonstrates that microRNAs (miRNAs) displayimportant roles in immune response [4 5] miRNAs areendogenous short (about 19ndash25 nucleotides long) single-stranded and noncoding RNAs that can influence the targetmRNA processing at the posttranscriptional level [6] andplay important roles in cell processes such as proliferationapoptosis differentiation or even in tumorigenesis [7] Oneof these important miRNAs is miR-124a Accumulatingevidence shows that miR-124a is downregulated in synovialtissues of RA patients compared with that of osteoarthritis(OA) patients [8] For example Nakamachi et al found thatits level in RA FLS was less than one-sixth of that seen in OA

FLS [9] However the mechanism of the aberrant expressionofmiR-124a inRA synovial tissues is still unknown It is foundthat miR-124a has three genomic loci (miR-124a-1 (8p231)miR-124a-2 (8q123) and miR-124a-3 (20q1333)) that encodefor the same mature miRNA Interestingly the miR-124a-1 and miR-124a-3 genes are located within CpG islandswhereas miR-124a-2 is 760 bp downstream of a CpG islandStudies in several cancer cells demonstrated that all the threeloci are silenced by the hypermethylation of its promoter[10 11] Agirre et al pointed out that the correspondingCpG islands of miR-124a-1 and miR-124a-3 are frequentlymethylated in acute myeloid leukemia [12] Since reporteddata showed that the expression of miR-124a was suppressedin RA synovial tissues it would be of interest to investigatewhether epigeneticmechanism especially DNAmethylationhas played a role in it

2 Materials and Methods

21 Synovial Samples Synovial tissues were obtained fromRA patients OA patients and joint trauma patients (healthycontrol specimens) undergoing total knee arthroplasty from

2 Clinical and Developmental Immunology

Table 1 Clinical characteristics of the patientslowast

Patient Age Sex Disease duration (years) Presurgical CRP (mgdL) Presurgical ESR (mmh) DAS 28 Medications

RA1 72 F 17 19 17 242 MTX 10mg qwLEF 20mg qd


RA3 63 M 21 27 13 187 Pred 75mg qdMTX 125mg qw

RA4 65 F 15 lt1 2 121 Pred 5mg qdLEF 20mg qd

RA5 59 M 6 32 5 149 Pred 5mg qodMTX 10mg qw

RA6 66 F 8 lt1 9 123 LEF 20mg qdRA7 61 F 13 53 28 262 MTX 15mg qwOA1 72 F mdash lt1 3 mdash mdashOA2 75 M mdash lt1 2 mdash mdashOA3 69 M mdash lt1 4 mdash mdashOA4 74 M mdash 33 9 mdash mdashOA5 68 F mdash lt1 3 mdash mdashOA6 70 M mdash 28 11 mdash mdashC1 39 M mdashC2 45 M mdashC3 43 F mdashlowastRA rheumatoid arthritis OA osteoarthritis CRP C-reactive protein ESR erythrocyte sedimentation rate DAS disease activity score MTX methotrexatePred prednisone LEF leflunomides C control

October 2012 to April 2013 in Sichuan Provincial PeoplersquosHospital Tissue was snap-frozen and stored at minus80∘C RAand OA were diagnosed according to the criteria of theAmerican College of Rheumatology [13 14] The clinicalcharacteristics of the patients are shown in Table 1 Sampleswere obtained in accordance with the Declaration of HelsinkiEthical Principles for Medical Research Involving HumanSubjects as approved by the World Medical Association Allpatients signed informed consent forms and the study wasapproved by the Ethics Committees of Sichuan ProvincialPeoplersquos Hospital

22 DNA Extraction and Bisulfite Conversion Genome DNAwas extracted from 25mg of frozen synovial tissue using thePureLink Genomic DNA mini kit (Invitrogen USA) DNAwas quantitated using the Nanodrop (Nanodrop technolo-gies USA) 2120583g DNA was used for bisulfite conversion asdescribed [15] Modified DNA was purified using the QIAEXII Gel Extraction kit (QIAGEN Germany) according to themanufacturer

23 Methylation-Specific PCR The DNA methylation statuswas analyzed by methylation-specific PCR (MSP) usingprimers specific for either the methylated or bisulfate modi-fied unmethylated DNA 15120583L bisulfite-converted DNA wasamplified using 015120583L primers (Sangon Biotech Shanghai)045 120583LMgCl

2(25mM) 18 120583L dNTP (25mM each) 675120583L

2x GC buffer (I) (Takara Japan) and 16 120583L Takara LA taq

polymerase (Takara Japan) Each step included methy-lated (RKO ATCC CRL-2577) and unmethylated (MGC-803 ATCC) controls along with nontemplate control Theamplified products were run on a 2 agarose gel with anexpected size of 164 185 and 150 bp (three loci each) for amethylated product and 165 180 and 155 bp (three loci each)for an unmethylated product

24 Statistical Analysis Statistical comparisons betweengroups were carried out by Chi square test or Fisherrsquos exacttest 119875 values less than 005 were considered significant

3 Results

31 General Clinical Features The mean age of the 7 RApatients was 63 (range 58ndash72) years with a female to maleratio of 25 1 The mean presurgical CRP and ESR were24 plusmn 15mgdL and 14 plusmn 9mmh respectively The meanDAS28 was 186 plusmn 057 The three healthy control patientsunderwent surgery because of car accident falling off andfighting respectively

32 Methylation Status of miR-124a The methylation statusof miR-124a was evaluated in 7 RA 6 OA and 3 controlsynovial tissues (Figure 1) All of the RA tissues were hyper-methylated 5 for miR-124a-1 (57) 5 for miR-124a-2 (57)and 4 for miR-124a-3 (47) Among them 4 tissues (47)showed promoter methylation of all three gene loci and

Clinical and Developmental Immunology 3

M

U

M

U

M

U

R1 R2 R3 R4 R5 R6 R7 O1 O2 O3 O4 O5 O6 C1 C2 C3 803 RKO H2O200 bp100 bp

200 bp100 bp

200 bp100 bp

200 bp100 bp

200 bp100 bp

200 bp100 bp

miR-124-a1

miR-124-a2

miR-124-a3

Figure 1 Gel electrophoresis of the MSP products for miR-124-a1 miR-124-a2 and miR-124-a3 in RA OA patients and healthy controls Rrheumatoid arthritis O osteoarthritis C healthy control 803 is unmethylated positive control RKO is methylated positive control H

2O is

blank control

Table 2 Methylation status of miR-124a in synovial tissues of RAand OAlowast

Gene Methylation frequency 119875 valueRA OA C RA versus OA RA versus C

miR-124-a1 714 167 0 007 008miR-124-a2 714 167 0 007 008miR-124-a3 571 0 0 005 017All three loci 571 0 0 005 017lowastRA rheumatoid arthritis OA osteoarthritis C healthy control

the frequency was 571 higher than that of OA (0 06)and control (0 03) (Table 2) The methylation frequencyof the three genes in RA synovial tissues has no statisticalsignificance compared to each other

4 Discussion

Rheumatoid arthritis is one of the common autoimmunediseases and the molecular mechanism of its pathogene-sis is unclear now miRNAs have been implicated in thepathogenesis of malignant and nonmalignant disease ThemicroRNAmiR-124a was initially identified as a crucial regu-lator involved in neurogenesis [16] In neuronal tissues miR-124a contributes to the differentiation of neural progenitorsinto mature neurons [17] Pierson et al reported that the 31015840-UTR of CDK-6 mRNA is a direct target of miR-124 and thatCDK-6 expression is suppressed by miR-124 overexpressionin medulloblastoma cell lines [18] Nakamachi et al con-firmed that the expression of CDK-6 protein was higher inRA FLS than in OA FLS and that CDK-6 expression wassuppressed when pre-miR-124a was transfected into RA FLSthus the cell cycle was arrested at the G1 phase [9] SinceCDK-6 is known as a G1S phase regulator it is speculated

that miR-124a is an important regulator of the G1S transitionin synovial tissue as well as in tumors

We decided to focus on the methylation status of miR-124a in RA synovial tissues for several reasons (a) Theexpression of miR-124a is downregulated in RA synovialtissues (b) Hypermethylation of gene promoters is a frequentmechanism of gene silencing (c) The three loci of miR-124a are either located within a CpG island or somewheredownstream a CpG island (d) It is reported that miR-124ahas been silenced by gene promoter hypermethylation inseveral cancer cell lines The results of our study supportthe approach showing that the gene promoter of miR-124a is hypermethylated and this might associate with itsdownregulation in RA synovial tissues

The methylation frequencies of the three gene lociobserved among RA synovial tissues (571sim714) weremuch higher than that observed among OA (0sim167) andcontrol synovial tissues (0) However the 119875 values were notso significant considering the small sample size

We obtained good-quality DNA from all frozen tissuesyet therewere someRA samples that showed bothmethylatedand unmethylated band We considered the following rea-sons (a) although we carefully removed all connective tissueand fat the area chosenmight still have very little nonsynovialtissue to confound the results It would be pertinent topoint out that small amounts of contaminating nonsynovialtissues could become a source of contamination and couldprovide false negative results This issue emphasizes the needto carefully choose samples for detection of methylationstatus therefore avoiding potential confounders (b) Regionswithin the synovial tissue might be variably methylated evenwithin the individual clones of the same tissue sample andthe synovial tissue could be a mosaic of variable degreesof methylation Thus we speculate mosaicism could havecontributed to our findings here This issue could be solvedby bisulfite-sequencing


This study highlights the methylation pattern of RA syn-ovial tissues compared with that of OA and healthy controlAlthough it has some limitations such as the number of sam-ples analyzed the study still provides useful information onpromoter methylation pattern of miR-124a This epigeneticdysregulation of miR-124a in RA synovial tissue constitutesan emerging mechanism implicated in the development ofRA and will provide us with new excellent targets for DNAdemethylating agents

Conflict of Interests

None of the authors had any conflict of interests

Acknowledgment

This study was funded by the National Natural ScienceFoundation of China (81102272)

References

[1] A Ruyssen-Witrand A Constantin A Cambon-Thomsenand M Thomsen ldquoNew insights into the genetics of immuneresponses in rheumatoid arthritisrdquo Tissue Antigens vol 80 no2 pp 105ndash118 2012

[2] A Gramling and J R OrsquoDell ldquoInitial management of rheuma-toid arthritisrdquo Rheumatic Diseases Clinics of North America vol38 no 2 pp 311ndash325 2012

[3] F AH Cooles and J D Isaacs ldquoPathophysiology of rheumatoidarthritisrdquo Current Opinion in Rheumatology vol 23 no 3 pp233ndash240 2011

[4] S P Persengiev ldquomiRNAs at the crossroad between hematopoi-etic malignancies and autoimmune pathogenesisrdquo DiscoveryMedicine vol 13 no 70 pp 211ndash221 2012

[5] M de Santis and C Selmi ldquoThe therapeutic potential ofepigenetics in autoimmune diseasesrdquoClinical Reviews in Allergyamp Immunology vol 42 no 1 pp 92ndash101 2012

[6] C L Bartels andG J Tsongalis ldquoMini-reviewsmicrornas novelbiomarkers for human cancerrdquoClinical Chemistry vol 55 no 4pp 623ndash631 2009

[7] C Z Chen ldquoMicroRNAs as oncogenes and tumor suppressorsrdquoThe New England Journal of Medicine vol 353 no 17 pp 1768ndash1771 2005

[8] C-G Miao and Y-Y Yang ldquoNew advances of microRNAsin the pathogenesis of rheumatoid arthritis with a focus onthe crosstalk between DNA methylation and the microRNAmachineryrdquoCellular Signalling vol 25 no 5 pp 1118ndash1125 2013

[9] Y Nakamachi S Kawano M Takenokuchi et al ldquoMicroRNA-124a is a key regulator of proliferation and monocyte chemoat-tractant protein 1 secretion in fibroblast-like synoviocytes frompatients with rheumatoid arthritisrdquo Arthritis amp Rheumatismvol 60 no 5 pp 1294ndash1304 2009

[10] A Lujambio S Ropero E Ballestar et al ldquoGenetic unmaskingof an epigenetically silenced microRNA in human cancer cellsrdquoCancer Research vol 67 no 4 pp 1424ndash1429 2007

[11] A Lujambio and M Esteller ldquoCpG island hypermethylation oftumor suppressor microRNAs in human cancerrdquo Cell Cycle vol6 no 12 pp 1455ndash1459 2007

[12] X Agirre A Vilas-Zornoza A Jimenez-Velasco et al ldquoEpige-netic silencing of the tumor suppressor microRNA Hsa-miR-124a regulates CDK6 expression and confers a poor prognosisin acute lymphoblastic leukemiardquo Cancer Research vol 69 no10 pp 4443ndash4453 2009

[13] F C Arnett S M Edworthy D A Bloch et al ldquoThe AmericanRheumatism Association 1987 revised criteria for the classifica-tion of rheumatoid arthritisrdquo Arthritis amp Rheumatism vol 31no 3 pp 315ndash324 1988

[14] R Altman E Asch D Bloch et al ldquoDevelopment of criteria forthe classification and reporting of osteoarthritis classificationof osteoarthritis of the kneerdquo Arthritis amp Rheumatism vol 29no 8 pp 1039ndash1049 1986

[15] J G Herman J R Graff S Myohanen B D Nelkin and SB Baylin ldquoMethylation-specific PCR a novel PCR assay formethylation status of CpG islandsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no18 pp 9821ndash9826 1996

[16] W P Kloosterman and R H A Plasterk ldquoThe diverse functionsof microRNAs in animal development and diseaserdquo Develop-mental Cell vol 11 no 4 pp 441ndash450 2006

[17] C Conaco S Otto J J Han and G Mandel ldquoReciprocalactions of REST and a microRNA promote neuronal identityrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 103 no 7 pp 2422ndash2427 2006

[18] J Pierson B Hostager R Fan and R Vibhakar ldquoRegulation ofcyclin dependent kinase 6 by microRNA 124 in medulloblas-tomardquo Journal of Neuro-Oncology vol 90 no 1 pp 1ndash7 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Clinical characteristics of the patientslowast

Patient Age Sex Disease duration (years) Presurgical CRP (mgdL) Presurgical ESR (mmh) DAS 28 Medications



RA3 63 M 21 27 13 187 Pred 75mg qdMTX 125mg qw

RA4 65 F 15 lt1 2 121 Pred 5mg qdLEF 20mg qd

RA5 59 M 6 32 5 149 Pred 5mg qodMTX 10mg qw

RA6 66 F 8 lt1 9 123 LEF 20mg qdRA7 61 F 13 53 28 262 MTX 15mg qwOA1 72 F mdash lt1 3 mdash mdashOA2 75 M mdash lt1 2 mdash mdashOA3 69 M mdash lt1 4 mdash mdashOA4 74 M mdash 33 9 mdash mdashOA5 68 F mdash lt1 3 mdash mdashOA6 70 M mdash 28 11 mdash mdashC1 39 M mdashC2 45 M mdashC3 43 F mdashlowastRA rheumatoid arthritis OA osteoarthritis CRP C-reactive protein ESR erythrocyte sedimentation rate DAS disease activity score MTX methotrexatePred prednisone LEF leflunomides C control

October 2012 to April 2013 in Sichuan Provincial PeoplersquosHospital Tissue was snap-frozen and stored at minus80∘C RAand OA were diagnosed according to the criteria of theAmerican College of Rheumatology [13 14] The clinicalcharacteristics of the patients are shown in Table 1 Sampleswere obtained in accordance with the Declaration of HelsinkiEthical Principles for Medical Research Involving HumanSubjects as approved by the World Medical Association Allpatients signed informed consent forms and the study wasapproved by the Ethics Committees of Sichuan ProvincialPeoplersquos Hospital

22 DNA Extraction and Bisulfite Conversion Genome DNAwas extracted from 25mg of frozen synovial tissue using thePureLink Genomic DNA mini kit (Invitrogen USA) DNAwas quantitated using the Nanodrop (Nanodrop technolo-gies USA) 2120583g DNA was used for bisulfite conversion asdescribed [15] Modified DNA was purified using the QIAEXII Gel Extraction kit (QIAGEN Germany) according to themanufacturer

23 Methylation-Specific PCR The DNA methylation statuswas analyzed by methylation-specific PCR (MSP) usingprimers specific for either the methylated or bisulfate modi-fied unmethylated DNA 15120583L bisulfite-converted DNA wasamplified using 015120583L primers (Sangon Biotech Shanghai)045 120583LMgCl

2(25mM) 18 120583L dNTP (25mM each) 675120583L

2x GC buffer (I) (Takara Japan) and 16 120583L Takara LA taq

polymerase (Takara Japan) Each step included methy-lated (RKO ATCC CRL-2577) and unmethylated (MGC-803 ATCC) controls along with nontemplate control Theamplified products were run on a 2 agarose gel with anexpected size of 164 185 and 150 bp (three loci each) for amethylated product and 165 180 and 155 bp (three loci each)for an unmethylated product

24 Statistical Analysis Statistical comparisons betweengroups were carried out by Chi square test or Fisherrsquos exacttest 119875 values less than 005 were considered significant

3 Results

31 General Clinical Features The mean age of the 7 RApatients was 63 (range 58ndash72) years with a female to maleratio of 25 1 The mean presurgical CRP and ESR were24 plusmn 15mgdL and 14 plusmn 9mmh respectively The meanDAS28 was 186 plusmn 057 The three healthy control patientsunderwent surgery because of car accident falling off andfighting respectively

32 Methylation Status of miR-124a The methylation statusof miR-124a was evaluated in 7 RA 6 OA and 3 controlsynovial tissues (Figure 1) All of the RA tissues were hyper-methylated 5 for miR-124a-1 (57) 5 for miR-124a-2 (57)and 4 for miR-124a-3 (47) Among them 4 tissues (47)showed promoter methylation of all three gene loci and


M

U

M

U

M

U


200 bp100 bp

200 bp100 bp

200 bp100 bp

200 bp100 bp

200 bp100 bp

miR-124-a1

miR-124-a2

miR-124-a3


2O is

blank control





4 Discussion










Acknowledgment


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















M

U

M

U

M

U


200 bp100 bp

200 bp100 bp

200 bp100 bp

200 bp100 bp

200 bp100 bp

miR-124-a1

miR-124-a2

miR-124-a3


2O is

blank control





4 Discussion










Acknowledgment


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgment


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Research of the Methylation Status of miR ...downloads.hindawi.com/journals/jir/2013/524204.pdfResearch of the Methylation Status of miR-124a Gene Promoter among Rheumatoid