Cloning of ADAMTS2 Gene and Colony Formation Effect of ADAMTS2 in Saos-2 Cell Line Under Normal and Hypoxic Conditions

Year 2020, Volume: 10 Issue: 2, 413 - 426, 30.12.2020

Sümeyye Aydogan Türkoğlu , Arife Sinem Gültekin Tosun

https://doi.org/10.37094/adyujsci.731430

Abstract

ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motifs 2), an N- propeptidase isoenzyme, is an enzyme involved in collagen biosynthesis by providing the amino ends of procollagen to be cut away. ADAMTS2 has anti-angiogenic activity as well as provides the processing of collagen. With this activity, it has become a target in cancer studies. Hypoxic regulation is a process that affects the expression of a large number of genes at the cellular level. Within the scope of our study, the cloning of the ADAMTS2 gene and its expression in Saos-2 (human bone carcinoma) cell line were performed ectopically. For this purpose, the transient transfection of the expression vector containing ADAMTS2 coding sequence was transfected by the calcium-phosphate precipitation method. Recombinant ADAMTS2 mRNA expression was checked by Real-Time PCR in Saos-2 cells. It was observed that there was a 50-fold increase in ADAMTS2 mRNA expression in the transfected Saos-2 cells compared to the control group. Then, the effect of recombinant ADAMTS2 on the colony-forming potential of Saos-2 cells was investigated in both normal and chemically induced hypoxic conditions. Colony formation was reduced in Saos-2 cells, in which ADAMTS2 was expressed ectopically under normal conditions compared to control group cells. Also, a stronger reducing effect on the colony-formation potential of ectopic expression in hypoxic conditions in Saos-2 cells was determined. In this study, the strong inhibitory effect of ADAMTS2 in hypoxic conditions was determined in Saos-2 cells for the first time.

Keywords

Cloning, ADAMTS-2, Saos-2, Hypoxia, Ectopic expression, Colony formation

Supporting Institution

Balikesir University Scientific Research Projects Units (BAP)

Project Number

2015/42

ADAMTS2 Geninin Klonlanması ve ADAMTS2’nin Saos-2 Hücre Hattında Normal ve Hipoksik Koşullarda Koloni Oluşumuna Etkisi

Abstract

N-propeptidaz izoenzimlerinden ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motifs 2), prokollajenlerin amino uçlarının kesilip uzaklaştırılmasını sağlayarak kollejen biyosentezinde görev alan bir enzimdir. ADAMTS2, prokollajenlerin işlenmesinin yanı sıra anti-anjiyojenik aktiviteye de sahiptir. Bu aktivitesi ile kanser çalışmalarında bir hedef haline gelmiştir. Hipoksik regülasyon hücresel düzeyde çok sayıda genin ifadesini etkileyen bir süreçtir. Çalışmamız kapsamında ADAMTS2 geninin klonlanması ve ektopik olarak Saos-2 (insan kemik karsinomu) hücre hattında ifadesi gerçekleştirilmiştir. Bu amaçla ADAMTS2 kodlayıcı sekans içeren ekspresyon vektörünün, Saos-2 hücrelerine kalsiyum-fosfat presipitasyonu ile geçici transfeksiyonu gerçekleştirilmiştir. Rekombinant ADAMTS2’nin Saos-2 hücrelerinde ektopik olarak üretilip üretilmediği vektör transfekte edilmeyen hücrelerdeki ADAMTS2 mRNA ifadesi ile kontrol edilmiştir. Transfekte edilen Saos-2 hücrelerinde ADAMTS2 mRNA ifadesi kontrol grubuna kıyasla 50 kat artış olduğu gözlenmiştir. Daha sonra rekombinant ADAMTS2’nin Saos- 2 hücrelerinin koloni oluşturma potansiyeli üzerindeki etkisi hem normal hem de kimyasal olarak indüklenmiş hipoksik koşullarda incelenmiştir. Kontrol grubu hücrelerine kıyasla normal koşullarda ektopik ifadenin sağlandığı Saos-2 hücrelerinde koloni oluşumu azalmıştır. Ayrıca Saos-2 hücrelerinde hipoksik koşullarda yapılan ektopik ifadenin hücrelerin koloni oluşturma potansiyellerini üzerinde daha güçlü yönde azaltıcı etkisi tespit edilmiştir. Çalışmamızda, ilk kez ADAMTS2'nin hipoksik koşullarda güçlü inhibe edici etkisi Saos-2 hücrelerinde belirlenmiştir.

Keywords

ADAMTS2, Saos-2, hipoksi, Ektopik ifade, Koloni formasyon

Project Number

2015/42

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