Tesis

Analisis Metilasi DNA dan Ekspresi mRNA Nerve Growth Factor dan Purinergic Receptor P2x Ligand-Gated Ion Channel 3 pada Darah Menstruasi Subjek Endometriosis dengan Nyeri = Analysis of DNA Methylation and mRNA Expression of Nerve Growth Factor and Purinergic Receptor P2x Ligand-Gated Ion Channel 3 in Menstrual Blood of Endometriosis Subjects with Pain.

Latar Belakang: Endometriosis merupakan penyakit ginekologi yang mengakibatkan nyeri panggul pada wanita usia reproduksi. Namun, penyakit ini memiliki etiologi yang kompleks, yang melibatkan faktor epigenetik. Metilasi DNA merupakan mekanisme yang meregulasi perubahan ekspresi gen dan sering dihubungkan dengan patologis endometriosis. Diketahui gen NGF dan P2RX3 mengalami peningkatan ekspresi pada susukan peritoneal endometriosis dan mengakibatkan nyeri endometriosis. Tujuan penelitian ini adalah untuk memeriksa metilasi DNA, ekspresi mRNA dan analisis korelasinya terhadap gen NGF dan P2RX3 pada darah menstruasi subjek endometriosis dan tanpa endometriosis. Metode: Pengukuran metilasi DNA gen NGF dan P2RX3 menggunakan metode Methylated Specific PCR (MSP) dan pyrosequencing. DNA genom setiap jaringan diekstraksi, dan dimodifikasi bisulfit. Persen metilasi sampel dihitung berdasarkan nilai rata-rata semua lokasi CpG yang dianalisis. Pengukuran ekspresi mRNA menggunakan RT-qPCR dan dianalisis dengan metode Livak. Hasil: Metilasi DNA gen NGF mengalami hipometilasi (39,3%) pada darah menstruasi endometriosis dengan nilai signifikansi p=0,025. Terdapat peningkatan ekspresi mRNA NGF secara bermakna sebesar 4,29 kali pada darah menstruasi subjek endometriosis dibandingkan terhadap subjek tanpa endometriosis (p=0,021). Sedangkan metilasi gen P2RX3 sebesar 81,1% pada darah menstruasi endometriosis dan 80,6% pada kelompok tanpa endometriosis, tidak berbeda bermakna (p=0,799). Terdapat perbedaan ekspresi mRNA P2RX3 secara bermakna lebih tinggi 2,23 kali (p=0,004). Penelitian ini tidak menunjukkan korelasi yang bermakna antara metilasi DNA dan ekspresi mRNA baik gen NGF maupun P2RX3. (Gen NGF nilai p=0,702; r=-0,179, dan gen P2RX3 nilai p=0,461; r=0,264). Kesimpulan: Pada sampel darah menstruasi endometriosis terjadi hipometilasi pada gen NGF, namun tidak pada gen P2RX3. Terjadi peningkatan ekspresi mRNA NGF dan P2RX3 pada darah menstruasi subjek dengan endometriosis dibandingkan dengan tanpa endometriosis. Hasil riset ini dapat dimanfaatkan untuk pengembangan biomarker dalam penegakkan diagnosis endometriosis non-invasif.
Kata kunci: metilasi DNA, ekspresi mRNA, darah menstruasi, endometriosis.


Background: Endometriosis is a gynecological disease that causes pelvic pain in women of reproductive age. However, the disease has a complex etiology, involving epigenetic factors. DNA methylation is a mechanism that regulates changes in gene expression and is often associated with pathological endometriosis. It is known that the NGF and P2RX3 genes have increased expression in the peritoneal implants of endometriosis and cause endometriosis pain. The purpose of this study was to examine the level of DNA methylation, mRNA expression and analysis of its correlation to the NGF and P2RX3 genes in the menstrual blood of subjects with endometriosis and without endometriosis. Methods: Measurement of DNA methylation of NGF and P2RX3 genes using Methylated Specific PCR (MSP) and pyrosequencing methods. The genomic DNA of each tissue was extracted, and bisulfite modified. Percent methylation of the sample was calculated based on the average value of all analyzed CpG sites. Measurement of mRNA expression using RT-qPCR and analyzed by the Livak. Results: The level of DNA methylation of the NGF gene was hypomethylated (39.3%) in endometriosis menstrual blood with a significance value of p=0.025. There was a significant increase in NGF gene mRNA expression of 4.29 times in menstrual blood of endometriosis subjects compared to subjects without endometriosis (p=0.021). Meanwhile, the methylation level of the P2RX3 gene was 81.1% in endometriosis menstrual blood and 80.6% in the group without endometriosis, not significantly different (p=0.799). There was a difference in the mRNA expression of the P2RX3 gene which was significantly higher by 2.23 times (p=0.004). This study did not show a significant correlation between the level of DNA methylation and mRNA expression of either the NGF or P2RX3 genes. (NGF gene p value = 0.702; r = -0.179, and P2RX3 gene p value = 0.461; r = 0.264). Conclusion: In endometriosis menstrual blood samples, hypomethylation occurred in the NGF gene, but not in the P2RX3 gene. There was an increase in the expression of NGF and P2RX3 mRNA in the menstrual blood of subjects with endometriosis compared to those without endometriosis. The results of this research can be used for the development of biomarkers in the non-invasive diagnosis of endometriosis.
Keywords: DNA methylation, mRNA expression, menstrual blood, endometriosis.