Genome-Wide DNA Methylation Indicates Silencing of

Tumor Suppressor Genes in Uterine Leiomyoma

Antonia Navarro1, Ping Yin1, Diana Monsivais1, Simon M. Lin2, Pan Du2, Jian-Jun Wei3, Serdar E. Bulun1*

Manuela Gómez Londoño. Estefania Jaramillo Jaramillo.

INTRODUCTIONUterine leiomyomas or fibroids are benign smooth

muscle tumors of myometrial that rapidly proliferate.

A cause of this disease is a dysregulated epigenetics such as DNA metilation, histone modification and non-coding RNAs

INTRODUCTIONin this study is looking

for the molecular causes of leiomyoma to look for a treatment because this is one of the most common tumors in USA.

LEIOMYOMA.Uterine leiomyomas are the most

common tumor in women and can occur in 75% in reproductive age.

Leiomyomas are benign tumor, its origin can be muscular and vascular.

It can be asymptomatic or may present with bleeding, abdominal pain, irregular cycles and dysuria.

DNA METILATIONIs a epigenetic proccess that is involved in regulation

of gene expression in two ways, directly by preventing the binding of transcription factors and indirectly promoting structure closed chromatin.

It consist in add a metil group by DNA methyltransferases (DNMTs).

GENE SILENCING

Is when a gene is inactivated and is not expressed, this is for to control transcription, this causes heterochromatin changes which may be inherited

Is caused by several molecules that act at the level of the DNA or mRNA

RELATIONSHIPLeiomyoma is a benign myometrium tumor caused

by inhibition of the expression of tumor suppressor genes myometrial, it means gene silencing in which transcription is altered because they are methylated.

MAIN OBJECTIVEDetermine the relationship between differential DNA

methylation and mRNA expression in uterine leiomyoma by performing a genome-wide analysis. Comparing leiomyoma vs normal myometrial tissue under epigenetic control.

Their findings will be a contribution of pathogenesis of leiomyoma

MATERIALES YMETODOS

ETICA Para obtener tejidos humanos, se siguió el protocolo aprobado por la Junta de Revisión Institucional para la Investigación en Seres Humanos de las universidades de Nueva York y del Noroeste, escribiendo un consentimiento informado.

ADQUISICION DE TEJIDOS 1. Se obtuvieron miometrios con leiomioma 23

afroamericanas y 14 caucasicas por histerectomia.

Estos fueron congelados en nitrogeno, antes del aislamiento con DNA y RNA

2. Se usaron 4 de cada uno para la prueba de secuenciacion y 18 afroamericanas y 10 de raza blanca para la TR-PCR

AISLAMIENTO CELULARLas celulas pueden ser aisladas

de los tejidos por ejemplo mediante hidrólisis enzimatica

Las células que se cultivan directamente desde un sujeto se conocen como células primarias.

Las células primarias para evitar cambios en el fenotipo y la expresión génica

METILACION DEL ADN

El ADN presenta regiones de 1000-1500 pb ricas en dinucleótidos CpG ("islas CpG"), que son reconocidas por las enzimas ADN-metiltransferasas, las cuales, durante la replicación del ADN metilan el carbono 5 de las citosinas de la cadena recién sintetizada, manteniéndose así la memoria del estado metilado en la molécula hija de ADN.

La metilación del ADN es un marcador epigenético del

silenciamiento de genes.

SECUENCIACION DEL ADN

Se realiza para obtener la secuencia de nucleótidos de un segmento de ADN, se necesita:

1. El ADN molde o segmento de ADN que se desea secuenciar.

2. Un enzima que replique el ADN, normalmente la ADN Polimerasa I del bacteriofago T4

SECUENCIACION DEL ADN3. Un cebador o "primer" (oligonucleótido de 20PB).

4. Los cuatro nucleótidos trifosfato (dATP, dCTP, dGTP y dTTP).

5.  nucleótidos didesoxi (ddATP, ddTTP, ddCTP y ddGTP).

RT-PCRConsiste en la amplificacion

directa de un gen o un fragmento de DNA o indrecta de un RNA.

Amplifica y simultaneamente cuantifica el producto de la amplificacion del ADN.

Mediante deteccion con fluorescencia se mide durante la amplificacion la cantidad de DNA sintetizado

ANALISIS DE PROTEINASEs una prubea utilizada para

detectar proteínas específicas en una muestra dada de tejido.

Se utilizan anticuerpos para detectar la presencia de una determinada proteina

Finalmente, se detecta la union antigeno-anticuerpo por actividad enzimatica o fluoresencia

RESULTADOS

DISCUSSION

AUTHOR APPROACH ARTICLE RELATION

Li S, Chiang, Richard Davis, Barrett, McLachlan

Genes such as ESR1 had been studied, shown to be hipomethylated in leiomyomas.

This approach is not related to the article because it shows that genes involved in leiomyoma are hypermethylated

Xue Q, Lin Z, Yin P, Milad MP, ChengSkubitz KM, Skubitz

DNA hypermethylation is related to transcriptional suppression

This approach is related to the article because the authors checked DNA hypermethylation causes transcriptional suppression

Ellenrieder V

KLF1 is expressed in a number of human tissues and is repressed in several human cancers

KFL1 is a transcription factor involved in key cellular functions such as apoptosis, proliferation and differentiation. They demonstrated the downregulation of KFL1 in leiomyoma

Daigo Y, Nishiwaki T, Kawasoe T, Tamari M, Tsuchiya E

Hypermethylation of DLEC1 promoter is associated with transcriptionalrepression in a variety cancer like lung, esophagus and ovary

The hypermetilation of CpG in promoter region is responsible for the repression of DLEC1 expression in uterine leiomyoma

CONCLUSIONUnderstanding the mechanism underlying the pathogenesis of uterine leiomyoma will be critical for developing new preventive and therapeutic approaches to the disease.

Epigenetic profile that could inform the development of diagnostic biomarkers for uterine leiomyoma as well as identify potential therapeutic targets.