EKSPRESI MicroRNA-21 (hsa-miR-21) DAN PHOSPHATASE TENSIN HOMOLOG (PTEN) PADA SEL LINI MCF-7 RESISTEN DOXORUBICIN (MCF-7/DOX)

GUSTI M. SOFYANNOOR, Prof. dr. Sofia Mubarika H., M.Med.Sc., Ph.D.; Dr. Med. dr. Indwiani Astuti

2015 | Tesis | S2 Bioteknologi

Kanker payudara merupakan tumor ganas epitelial dengan insidensi dan mortalitas tertinggi pada wanita di seluruh dunia maupun di Indonesia. Pasien biasanya datang pada stadium lanjut, sehingga pengobatan dengan kemoterapi tidak memberikan hasil yang diharapkan karena adanya potensi resistensi terhadap obat antitumor. Pada tingkat molekuler, microRNA-21 (hsa-miR-21) berperan sebagai post transcriptional regulator dalam mekanisme regulasi pasca transkripsi sehingga menekan atau menghambat translasi mRNA gen tumor supresor Phosphatase Tensin Homolog (PTEN). Penurunan atau hilangnya ekspresi PTEN berdampak meningkatnya proliferasi sel dan inhibisi jalur apoptosis yang akhirnya memicu tumorigenesis dan resistensi kanker payudara. Untuk memahami mekanisme tersebut, dilakukan eksperimen in vitro pada kultur sel lini MCF-7 resisten Doxorubicin guna mengetahui ekspresi hsa-miR-21 dan PTEN. Dilakukan analisis in silico untuk memprediksi interaksi hsa-miR-21 dan mRNA PTEN berdasarkan basis data bioinformatik. Eksperimen in vitro dilakukan dengan metode paparan Doxorubicin secara bertingkat pada sel MCF-7 sehingga diperoleh varian sel resisten Doxorubicin (MCF-7/Dox) berdasarkan karakteristik peningkatan nilai IC50 melalui MTT assay. Ekspresi protein P-gp dan PTEN dideteksi secara imunositokimia dengan antibodi monoklonal. Adapun analisis ekspresi hsa-miR-21 dilakukan menggunakan qRT-PCR dengan U6SnRNA sebagai reference gene dan UniSp6 sebagai kontrol positif. Hasil menunjukkan bahwa terdapat peningkatan nilai IC50 pada sel MCF-7/Dox (5,78 μg/ml) dibandingkan MCF-7 parental (0,68 μg/ml) dengan indeks resistensi sebesar 8,5. Analisis qRT-PCR membuktikan bahwa hsa-miR-21 mengalami overekspresi pada sel lini MCF-7/Dox dengan fold change expression 7,94. Meningkatnya regulasi hsa-miR-21 seiring dengan penurunan ekspresi protein PTEN pada sel MCF-7/Dox. Secara imunositokimia terdeteksi ekspresi P-gp, namun protein PTEN tidak terekspresi pada sel MCF-7/Dox. Penelitian ini berhasil membuktikan bahwa hsa-miR-21 mengalami overekspresi pada sel MCF-7/Dox sebagai model resistensi kanker payudara. Overekspresi hsa-miR-21 diduga kuat berperan dalam represi atau inhibisi translasi protein melalui mekanisme regulasi pascatranskripsi mRNA gen PTEN sehingga protein PTEN tidak terekspresi pada sel MCF-7/Dox.

Breast cancer (BC) is epithelial carcinoma with high incidency and mortality worldwide, including in Indonesia. In clinical case, most of the patient having chemotherapy on the late stadium of BC which is costly ineffective and contribute to resistance. At molecular level, it is proposed that microRNA-21 (hsa-miR-21) plays important role in post transcriptional regulation by repressing or inhibiting translation of mRNA tumor suppressor gene Phosphatase Tensin Homolog (PTEN). Decreasing or loss of PTEN expression, which is code by PTEN gene, has impact on increased cell proliferation and aberrant apoptosis pathway which lead to tumorigenesis and chemotherapy resistance. To understand these molecular mechanism, in vitro experiment conducted in Doxorubicinresistant MCF-7 cell line to investigate the expression of hsa-miR-21 and PTEN. Interaction between hsa-miR-21 and mRNA PTEN were predicted by in silico analysis based on bioinformatics database. Parental MCF-7 cell culture has periodically Doxorubicin exposured to obtain resistant MCF-7 cell line with IC50 value determined by MTT assay. P-gp and PTEN protein expression were analyzed by immunocytochemistry using monoclonal antibody. qRT-PCR was performed to detect the expression of hsa-miR-21 as well as U6SnRNA served as reference gene and UniSp6 as positive control. Results showed that Doxorubicin resistant MCF-7 cell line had increased IC50 value (5,78 μg/ml) compared to parental MCF-7 (0,68 μg/ml) with resistance index value 8,5. Based on qRT-PCR analysis, hsa-miR-21 was overexpressed in MCF-7/Dox cell line with fold change expression 7,94. The upregulation of hsa-miR-21 in MCF-7/Dox cell line was concurrent with downregulation of PTEN protein. It was shown that P-gp expression was detected while PTEN found underexpression on MCF-7/Dox cell line. This study revealed that hsa-miR-21 was overexpressed in MCF-7/Dox cell line as Doxorubicin-resistance model in breast cancer. This finding suggest that overexpresion of hsa-miR-21 play important role in post transcriptional regulation by repressing or inhibiting translation of PTEN mRNA which affect the underexpression of PTEN protein in MCF-7/Dox breast cancer cell line.

Kata Kunci : Resistensi, Doxorubicin, MCF-7/Dox, miR-21, PTEN