Publication: Büyüme hormonunun SMN2 gen ekspresyonu üzerindeki etkisinin araştırılması
Abstract
Büyüme hormonunun SMN2 gen ekspresyonu üzerindeki etkisinin araştırılması Amaç: Spinal Müsküler Atrofi (SMA), survival motor nöron (SMN) proteininin yokluğu veya yetersizliğinden kaynaklanan nörodejeneratif hastalıktır. SMN proteini, %100 fonksiyonel üreten SMN1 ve yalnızca %10 fonksiyonel üreten SMN2 genlerinden sentezlenir. SMN1 genindeki delesyon veya mutasyon yetersiz protein üretimi ile sonuçlanır ve hastalık kendini gösterir. SMN2 kopya sayısının hastalığın klinik şiddeti üzerinde etkisi olduğu bilinmektedir. SMN2 gen ekspresyon seviyelerinin artırılması SMA için potansiyel bir tedavi olarak ortaya çıkmıştır. Bu çalışmada, Çin hamster yumurtalık (CHO) hücrelerinde raportör kullanarak büyüme hormonunun (GH) SMN2 promotör aktivitesi üzerindeki etkisi incelenmiştir. Gereç ve Yöntem: Bu çalışmada, GH’ye yanıt veren raportör yapının gen ifadesine etkisini incelemek için SMN2 geninin üç promotör bölgesi (588 bp, 1,036 bp ve 1,705 bp uzaklıkta) kullanılmıştır. Bu bölgeler spesifik SMN2 primerleri ile Polimeraz Zincir Reaksiyonu (PCR) ile çoğaltılmıştır. SMN2 promotör PCR ürünleri T4 DNA ligaz ile promotörsüz Promega Lusiferaz Raportör Vektör 3 (pGL3)’e klonlanmış ve DH5α hücrelerine transforme edilmiştir. Pozitif koloniler koloni PCR ile ve plazmidlerdeki SMN2 promotör insertleri SMN2 spesifik primerlerle PCR ile doğrulanmıştır. Plazmidler CHO hücrelerine transfekte edilmiştir. Lusiferaz genini uyaran SMN2 promotörünü ifade eden CHO hücreleri 24 saat boyunca büyüme hormonu ile muamele edilmiş ve ardından CHO hücrelerinin lusiferaz aktivitesi ölçülmüştür. Bulgular: SMN2 promotörleri içeren tüm raportör yapılar büyüme hormonuna yanıt vermiştir. Büyüme hormonu, 1,036 bp SMN2 promotörüne sahip raportör yapı içeren lusiferaz genini indüklemiştir ve bu bölgede yüksek düzeyde gen ekspresyonu gerçekleşmiştir. Ancak, büyüme hormonu ile muamele edilen ve insert içermeyen plazmid ile karşılaştırıldığında, büyüme hormonunun 1,705 bp SMN2 promotörüne sahip lusiferaz geninin gen ekpreyonunu baskıladığı görülmüştür. Sonuç: Bu bulgular, büyüme hormonunun hSMN2 ifadesini hSMN2 promotörüne bağlı bir şekilde modüle edebileceğini önermektedir. GH, hSMN2 ifadesini artırarak SMA tedavisi için potansiyel bir hormon adayı olabilir.
Determination of the effect of GH on SMN2 gene expression Objective: Spinal Muscular Atrophy (SMA) is a neurodegenerative disease caused by the absence or deficiency of the survival motor neuron (SMN) protein. SMN protein is synthesized from two genes: SMN1, which produces 100% functional protein, and SMN2, which produces only about 10% functional protein. Deletions or mutations in the SMN1 gene result in insufficient protein production and disease onset. The copy number of SMN2 is known to influence the clinical severity of SMA. Increasing SMN2 gene expression has emerged as a potential therapeutic approach for SMA. In this study, the effect of growth hormone (GH) on SMN2 promoter activity was investigated using a reporter system in Chinese hamster ovary (CHO) cells. Materials and Methods: To examine the effect of a GH-responsive reporter construct on gene expression, three promoter regions of the SMN2 gene (located 588 bp, 1.036 bp, and 1.705 bp upstream) were used. These regions were amplified by Polymerase Chain Reaction (PCR) with specific SMN2 primers. The PCR products of SMN2 promoters were cloned into the promoterless Promega Luciferase Reporter Vector 3 (pGL3) using T4 DNA ligase and transformed into DH5α cells. Positive colonies were identified by colony PCR, and SMN2 promoter inserts in the plasmids were confirmed by PCR with SMN2-specific primers. The plasmids were then transfected into CHO cells. CHO cells expressing the SMN2 promoter driving luciferase were treated with GH for 24 hours, after which luciferase activity was measured. Results: All reporter constructs containing SMN2 promoters responded to GH. GH induced the highest level of gene expression luciferase gene for the reporter construct containing the 1.036 bp SMN2 promoter, however, GH suppressed gene expression of luciferase gene for 1.705 bp SMN2 promoter vector when compared to the gene expression of reporter plazmid alone with GH. Conclusion: These findings suggest that growth hormone (GH) can modulate hSMN2 expression in hSMN2 promoter dependent manner. GH may be candidate hormone for SMA treatment by enhancing hSMN2 expression.
Determination of the effect of GH on SMN2 gene expression Objective: Spinal Muscular Atrophy (SMA) is a neurodegenerative disease caused by the absence or deficiency of the survival motor neuron (SMN) protein. SMN protein is synthesized from two genes: SMN1, which produces 100% functional protein, and SMN2, which produces only about 10% functional protein. Deletions or mutations in the SMN1 gene result in insufficient protein production and disease onset. The copy number of SMN2 is known to influence the clinical severity of SMA. Increasing SMN2 gene expression has emerged as a potential therapeutic approach for SMA. In this study, the effect of growth hormone (GH) on SMN2 promoter activity was investigated using a reporter system in Chinese hamster ovary (CHO) cells. Materials and Methods: To examine the effect of a GH-responsive reporter construct on gene expression, three promoter regions of the SMN2 gene (located 588 bp, 1.036 bp, and 1.705 bp upstream) were used. These regions were amplified by Polymerase Chain Reaction (PCR) with specific SMN2 primers. The PCR products of SMN2 promoters were cloned into the promoterless Promega Luciferase Reporter Vector 3 (pGL3) using T4 DNA ligase and transformed into DH5α cells. Positive colonies were identified by colony PCR, and SMN2 promoter inserts in the plasmids were confirmed by PCR with SMN2-specific primers. The plasmids were then transfected into CHO cells. CHO cells expressing the SMN2 promoter driving luciferase were treated with GH for 24 hours, after which luciferase activity was measured. Results: All reporter constructs containing SMN2 promoters responded to GH. GH induced the highest level of gene expression luciferase gene for the reporter construct containing the 1.036 bp SMN2 promoter, however, GH suppressed gene expression of luciferase gene for 1.705 bp SMN2 promoter vector when compared to the gene expression of reporter plazmid alone with GH. Conclusion: These findings suggest that growth hormone (GH) can modulate hSMN2 expression in hSMN2 promoter dependent manner. GH may be candidate hormone for SMA treatment by enhancing hSMN2 expression.