Analisis Hubungan Anemia terhadap Hipoksia Jaringan serta Hubungannya dengan Regulasi Transkripsi Gen Eritropoietin (Epo)

Dewi, FirliRahmahPrimula (2013) Analisis Hubungan Anemia terhadap Hipoksia Jaringan serta Hubungannya dengan Regulasi Transkripsi Gen Eritropoietin (Epo). Magister thesis, Universitas Brawijaya.

Abstract

Anemia merupakan kondisi dimana level Hb seseorang kurang dari 12 g/dL. Kondisi anemia pada pasien kanker ditambah dengan tingginya tingkat proliferasi sel kanker diduga merupakan faktor utama terjadinya kondisi hipoksia pada jaringan kanker. Kondisi hipoksia merupakan kondisi dimana level pO2 jaringan sangat rendah. Pada kondisi hipoksia, sel kanker mampu mensintesis protein Eritropoietin (EPO) dibawah kontrol oleh faktor transkripsi hypoxia inducible factor-1 alpha (HIF-1 α ). EPO merupakan protein yang berfungsi untuk meningkatkan pertahanan diri sel. Persentase metilasi yang berbeda-beda pada region promotor dan enhancer gen EPO diduga berpengaruh terhadap aktivitas HIF1 α . Penelitian ini dilakukan dalam 2 tahap penelitian. Penelitian tahap pertama dilakukan dengan pewarnaan HE dan imunofluoresen untuk mengamati proliferasi sel, serta ekspresi EPO dan reseptornya (EPOR) pada jaringan kanker payudara pasien anemia dan non-anemia. Penelitian tahap kedua dilakukan secara in silico untuk mengetahui pengaruh metilasi pada region regulator gen EPO terhadap aktivitas HIF1. Persentase metilasi yang dianalisis adalah 0% (kondisi hipoksia), 46% (sel kanker kondisi normoksia), dan 8% (sel normal). Hasil penelitian ini menunjukkan bahwa pada kelompok anemia, ekspresi EPO dan EPOR lebih tinggi dibandingkan pada kelompok non-anemia, sehingga diduga bahwa pada kelompok anemia, kondisi jaringan kanker adalah lebih hipoksik dibandingkan dengan kelompok non-anemia. Hasil penelitian secara in silico menunjukkan bahwa energi paling kecil yang dibutuhkan untuk proses interaksi dengan HIF1 adalah pada enhancer gen EPO yang tidak termetilasi (kondisi hipoksia). Kecilnya energi yang dibutuhkan untuk proses interaksi menyebabkan ikatan yang terbentuk adalah lebih stabil, sehingga kemungkinan HIF1 dalam mentranskripsi gen EPO adalah lebih besar. Hasil tersebut mendukung hasil penelitian tahap pertama, dimana kondisi hipoksia akan menyebabkan HIF1 lebih aktif dalam mentranskripsi gen EPO, sehingga ekspresi EPO menjadi lebih tinggi pada jaringan yang mengalami kondisi hipoksia.

English Abstract

Anemia is a frequent complication of malignant diseases and cancer rapy. In cancer patients, anemia has been hypo sized to lead to tumor hypoxia. Tumor hypoxia might lead to tumor growth and resistance to rapy, because it leads to angiogenesis, genetic mutations, and resistance of apoptosis. Erythropoietin (Epo) is a glycoprotein hormone which - in normoxic condition - is produced in adult kidney and fetal liver that regulates growth, differentiation, and survival of erythroid progenitors. Epo circulates in blood vessels and binds to Epo receptors (EpoR) to accelerate viability, proliferation and differentiation, resulting in an increased number of erythrocytes. promoter region of Epo becomes methylated in normoxic (non-hypoxia) condition, but not in hypoxic condition. Methylation of Epo enhancer region declines transcription activity of Epo gene. However, mechanism of this regulation needs more proper examination. This research was conducted in two steps. First step was to investigate Epo and EpoR expression in anemic and non anemic group of breast cancer tissue by using immunofluorescence methods, and second was to investigate effect of different percentage of methylation on HIF1 activity by using in silico analysis. From a preliminary study, it`s known that in normal cells at non-hypoxic conditions, promoter and enhancer region of EPO gene become methylated up to 8% (referring to number of cytosine being able to be methylated), whereas in cancer cells methylation can reach up to 46%. But at hypoxic condition, promoter and enhancer region of EPO gene are not methylated. Epo and EpoR analyzed by immunofluorescence showed a higher expression, especially in anemia group compared to non-anemia. Comparing different signal intensities, we found that Epo and EpoR expression was significantly higher in anemia than in non anemia. re was a negative correlation between cancer cell proliferation with Epo and EpoR expression in anemic group. In contrast to situation with anemic group, we found a positive correlation between cancer cell proliferation with Epo and EpoR expression in non-anemic group. In silico analysis show that binding energy in 46% methylated DNA was higher than in unmethylated DNA and 8% methylated DNA. So, we presume that a silencing mechanism of Epo gene by methylation is correlated with binding energy, which is required for interaction. A higher methylation percentage correlates with a higher binding energy which can cause an unstable interaction between DNA and transcription factor. In conclution, methylation of promoter and enhancer region of Epo gene leads to silencing.

Item Type:	Thesis (Magister)
Identification Number:	TES/616.152/DEW/a/041307533
Subjects:	600 Technology (Applied sciences) > 616 Diseases > 616.1 Diseases of cardiovascular system
Divisions:	S2/S3 > Magister Biologi, Fakultas MIPA
Depositing User:	Endro Setyobudi
Date Deposited:	04 Feb 2014 14:53
Last Modified:	14 Mar 2024 00:58
URI:	http://repository.ub.ac.id/id/eprint/158296

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