Disertasi

Analisis Ekspresi mRNA dan Metilasi DNA Promoter Gen TNF-α, Sitokin CXCL16 dan P53 pada Darah Menstruasi Penderita Endometriosis dengan Nyeri dan Nir-Endometriosis = Analysis of mRNA Expression and Promoter DNA Methylation of TNF- α, CXCL16 and P53 Genes in Menstrual Blood of Endometriosis Patients with Pain and Non-Endometriosis.

Latar Belakang: TNF-α dan CXCL16 terlibat dalam patofisiologi endometriosis melalui regulasi respon inflamasi dan pengkode nyeri endometriosis. Peningkatan TNF-α berperan dalam jalur pensinyalan P53 untuk apoptosis. Darah menstruasi sebagai pelepasan jaringan endometrium dapat digunakan dalam mengidentifikasi biomarker untuk diagnosis penyakit endometriosis tanpa memerlukan biopsi. Penelitian ini bertujuan untuk menganalisis tingkat ekspresi mRNA dan metilasi DNA gen TNF-α, CXCL16 dan P53 pada darah menstruasi dan jaringan endometrium pasien endometriosis dengan nyeri untuk mengembangkan metode diagnosis endometriosis noninvasif. Metode: 40 wanita dengan dan tanpa endometriosis terlibat dalam penelitian ini. Sampel darah menstruasi subjek dikumpulkan dengan menggunakan pembalut kertas saring dan jaringan endometrium dikumpulkan dengan melakukan biopsi, yang kemudian diekstraksi DNA dan RNAnya. Tingkat metilasi DNA diukur dengan menggunakan metode pyrosequencing setelah perlakuan konversi bisulfit. Sementara itu, tingkat ekspresi mRNA diukur dengan menggunakan metode quantitative polymerase chain reaction (qPCR) dan dianalisis dengan metode Livak Hasil: Ekspresi mRNA gen TNF-α pada darah menstruasi pasien endometriosis meningkat signifikan 3,73 kali lipat dibandingkan ekspresi pada kontrol (p=0,005). Terdapat 1,21 kali signifikan ekspresi jaringan endometrium dibandingkan darah menstruasi pasien endometriosis (p=0,012). Gen TNF-α mengalami hipermetilasi dan berbeda bermakna dalam darah menstruasi pasien endometriosis dibandingkan kontrol (p=0,008). Sedangkan ekspresi mRNA gen CXCL16 pada darah menstruasi pasien endometriosis meningkat 2,42 kali (p=0,030) dibandingkan ekspresi mRNA darah menstruasi pada kontrol, namun tidak terdapat perbedaan signifikan ekspresi mRNA darah menstruasi dibandingkan jaringan endometrium pada pasien endometriosis (p=0,173). Gen CXCL16 mengalami hipometilasi, tingkat metilasinya dalam darah menstruasi pasien endometriosis berbeda bermakna dibandingkan kontrol (p=0,004). Pada P53 terjadi terjadi peningkatan ekspresi gen P53 yaitu 1,52 kali terhadap darah menstruasi kontrol. Ekspresi mRNA gen TNF-α dan CXCL16 pada subjek nyeri berat lebih tinggi dibandingkan subjek nyeri sedang, dan terdapat korelasi positive antara ekspresi gen TNF-α dan CXCL16 dengan intensitas nyeri. Kesimpulan: Penelitian ini menunjukkan bahwa peningkatan ekspresi mRNA TNF-α dan CXCL16 dalam darah menstruasi pasien endometriosis dapat menjadi penanda langsung untuk mendiagnosis endometriosis. Namun, untuk memvalidasi lebih lanjut temuan ini dan mengeksplorasi potensi sebagai alat diagnostik, penelitian tambahan yang melibatkan kelompok pasien yang lebih besar diperlukan.
Kata Kunci: CXCL16, Darah Menstruasi, Ekspresi mRNA, Jaringan Endometrium, Metilasi DNA, P53, TNF-α.


Background: TNF-α and CXCL16 are implicated in the pathophysiology of endometriosis through the regulation of inflammatory response and the coding of endometriosis pain. Elevated TNF-α is implicated in the P53 signaling pathway for apoptosis. Menstrual blood, as a discharge of endometrial tissue, presents an opportunity for identifying biomarkers for the diagnosis of endometriosis without resorting to biopsy. This study aims to analyze the mRNA expression level and DNA methylation of TNF-α, CXCL16, and P53 genes in menstrual blood and endometrial tissue of endometriosis patients with pain to develop a noninvasive diagnostic method for endometriosis. Method: Forty women, both with and without endometriosis, participated in this study. Menstrual blood samples were collected using filter paper pads, and endometrial tissues were obtained via biopsy, from which DNA and RNA were extracted. DNA methylation levels were assessed using the pyrosequencing method after bisulfite conversion treatment. Meanwhile, mRNA expression levels were measured using the quantitative polymerase chain reaction (qPCR) method and analyzed using the Livak method. Results: The mRNA expression of the TNF-α gene in menstrual blood of endometriosis patients increased significantly by 3.73 times compared to controls (p=0.005). There was a significant 1.21 -fold increase in expression in endometrial tissue compared to menstrual blood of endometriosis patients (p=0.012). The TNF-α gene exhibited hypermethylation, significantly differing in menstrual blood of endometriosis patients compared to controls (p=0.008). The mRNA expression of the CXCL16 gene in menstrual blood of endometriosis patients increased by 2.42 times (p=0.030) compared to controls, although there was no significant difference in expression between menstrual blood and endometrial tissue in endometriosis patients (p=0.173). The CXCL16 gene displayed hypomethylation, with methylation levels in menstrual blood of endometriosis patients significantly differing from controls (p=0.004). There was an increase in P53 gene expression, which was 1.52 times higher than in control menstrual blood. The mRNA expression of TNF-α and CXCL16 genes in subjects experiencing severe pain was higher than in those with moderate pain, and there was a positive correlation between TNF-α and CXCL16 gene expression and pain intensity. Conclusion: This study suggests that increased mRNA expression of TNF-α and CXCL16 in menstrual blood of endometriosis patients may serve as direct markers for diagnosing endometriosis. However, further validation of these findings and exploration of their potential as diagnostic tools requires additional studies involving larger patient cohorts.
Keywords: CXCL16, Menstrual Blood, mRNA Expression, Endometrial Tissue, DNA Methylation, P53, TNF-α.