Tesis

Analisis Perbedaan Tingkat Metilasi Serta Ekspresi Mrna Gen Reseptor Androgen Dan Reseptor Insulin Pada Sel Granulosa Subjek Sindrom Ovarium Polikistik (SOPK) = Analysis of Methylation and mRNA Expressions Levels of Androgen Receptor and Insulin Receptor Genes in Granulosa Cells of Polycystic Ovary Syndrome (PCOS).

Latar belakang: Sindrom ovarium polikistik (SOPK) merupakan salah satu kelainan endokrin paling umum pada 7-15% wanita usia reproduksi yang menyebabkan infertilitas anovulatori. SOPK sering ditemukan pada wanita gemuk, sekitar 50-75% tetapi sindrom ini juga dapat ditemukan pada wanita kurus sebesar 5,5%. Penyebab dan patogenesis SOPK sampai sekarang masih diperdebatkan tapi hasil penelitian memperlihatkan hiperandrogen dan resistensi insulin terlibat dalam perkembangan perjalanan penyakit dan fenotip SOPK. Efek androgen dimediasi oleh reseptor androgen (AR) sedangkan efek insulin dimediasi oleh reseptor insulin (INSR). Ekspresi dan aksi dari kedua reseptor ini terutama pada sel granulosa ovarium dapat dipengaruhi oleh mekanisme epigenetik yang diduga terlibat dalam perkembangan penyakit SOPK ini. Tujuan: Mengetahui tingkat metilasi DNA pada gen reseptor androgen (AR) dan reseptor insulin (INSR) serta ekspresi mRNAnya pada sel granulosa subjek SOPK dan nir-SPOK. Metode: Penelitian ini merupakan penelitian deskriptif analitik dengan rancangan cross sectional. Sampel berupa sel granulosa dari folikel ovarium didapatkan dari wanita yang melakukan ovum pick up (OPU) di klinik Yasmin RSUPN Ciptomangunkusumo yang kemudian dilakukan isolasi DNA dan RNA. Pada isolat DNA dilakukan konversi bisulfit, Methyl Specifik PCR (MSP), elektroforesis dan analisis ketebalan pita dengan perangkat lunak ImageJ untuk mendapatkan data tingkat metilasi DNA. Pada isolat RNA dilakukan qPCR untuk mendapatkan ekspresi relatif mRNA gen AR dan INSR. Hasil: Analisis data dari 21 subjek SOPK dan 21 subjek nir SOPK menunjukkan terdapat perbedaan bermakna (p=0,00) tingkat metilasi DNA gen AR pada pasien SOPK (70,74 %±20,25) dibandingkan nir-SOPK (95,15%±6,41). Analisis gen INSR, menunjukkan tidak terdapat perbedaan bermakna (p>0,5) tingkat unmetilasi SOPK (97,96%±1,87) dibandingkan nir-SOPK (95,09%±10,15). Dari penelitian juga didapatkan terjadinya peningkatan ekspresi relatif mRNA AR sebesar 2,459 kali dan 1,791 kali pada mRNA INSR. Tidak terdapat korelasi antara tingkat metilasi gen AR dan INSR dengan ekspresi mRNAnya. Kesimpulan: Penurunan tingkat metilasi DNA (hipometilasi) pada gen AR dan INSR dapat meningkatkan tingkat ekspresi mRNA AR dan INSR, yang kemudian berkontribusi terhadap kejadian hiperandrogen dan resistensi insulin pada fenotip subjek SOPK.
Kata kunci: DNA metilasi, ekspresi mRNA, gen AR, gen INSR, SOPK


Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in 7-15% of women of reproductive age who cause anovulatory infertility. PCOS is often found in obese women, around 50-75% but this syndrome can also be found in thin women at 5.5%. The causes and pathogenesis of PCOS is still debated but the results of the study show that hyperandrogen and insulin resistance are involved in the development of the disease course and the PCOS phenotype. The androgen effect is mediated by the androgen receptor (AR) while the effect of insulin is mediated by the insulin receptor (INSR). The expression and action of these two receptors, especially in ovarian granulosa cells can be affected by epigenetic mechanisms that are thought to be involved in the development of PCOS. Objective: To determine the level of DNA methylation in the androgen receptor gene (AR) and insulin receptor (INSR) and its mRNA expression on the subject of PCOS and non-SPOK granulosa cells. Method: This study is a descriptive analytic study with a cross sectional design. Samples in the form of granulosa cells from ovarian follicles were obtained from women who performed ovum pick up (OPU) at the Yasmin clinic Ciptomangunkusumo Hospital which was then isolated from DNA and RNA. DNA isolates were carried out bisulfite conversion, Methyl Specific PCR (MSP), electrophoresis and tape thickness analysis with ImageJ software to obtain DNA methylation level data. qPCR was performed on RNA isolates to obtain the relative expression of the AR and INSR mRNA genes. Results: Analysis of data from 21 SOPK subjects and 21 non-PCOS subjects showed significant differences (p = 0.00) of AR gene DNA methylation rates in PCOS patients (70.74% ± 20.25) compared to non-PCOS (95.15 % ± 6.41). INSR gene analysis showed no significant difference (p> 0.5) for the level of unmetylation of PCOS (97.96% ± 1.87) compared to non-PCOS (95.09% ± 10.15). From the research also found an increase in the relative expression of AR mRNA of 2,459 times and 1,791 times in INSR mRNA. There is no correlation between the rate of methylation of the AR and INSR genes with their mRNA expression. Conclusions: Decreasing levels of DNA methylation (hypomethylation) in AR and INSR genes can increase the level of expression of mRNA AR and INSR, which then contributes to the incidence of hyperandrogen and insulin resistance in the phenotype of the PCOS subject.
Keywords: DNA methylation, mRNA expression, AR gene, INSR gene, PCOS