"SUPEREXPRESSION OF THE PIPKIIALPHA ENZYME GENE IN CULTURES OF NORMAL ERYTHROID CELLS AND ERYTHROID CELLS FROM PATIENTS WITH HEMOGLOBIN H DISEASE"

• Grant number: 12/07969-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: August 01, 2012
• End date: July 31, 2015
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Maria de Fatima Sonati
• Grantee: Daniela Maria Ribeiro
• Host Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Phosphatidylinositol-phosphate kinases (PIPKs) belong to a family of lipid kinase enzymes that generate various lipid messengers, including an important second messenger known as phosphatidylinositol-4,5-biphosphate, which regulates a number of cell activities, such as modulation of the actin cytoskeleton, vesicle transport, formation of focal adhesions and a variety of nuclear events. The PIPK subfamily is divided into types I (alpha, beta and gamma), II (alpha, beta and gamma) and III according to signaling specificity. In a study carried out in our laboratory, the PIPKIIalpha gene was differentially expressed in reticulocytes from two siblings with hemoglobin H disease. PIPKIIalpha and beta-globin gene expression were elevated in the patient with the higher hemoglobin H level, suggesting a possible relationship between PIPKIIalpha and globin production. In addition, analyses carried out using CD34+ cells from cultures undergoing erythroid differentiation showed that PIPK gene expression increased as the cells became more differentiated and was similar to that of the globins. However, there has been little research into the role of PIPK in the hematopoietic process. The aim of this project is to overexpress the PIPKIIalpha gene in cultures of normal erythroid cells and erythroid cells from patients with Hb H disease using RNA-induced gene activation technology (RNAa). To achieve this, CD34+ progenitor cells will be transduced with shRNA molecules targeting the promoter region of the PIPKIIalpha gene with the aid of lentiviral vectors. Expression of the genes of this enzyme and of the globins will be assessed using quantitative real-time PCR (qRT-PCR) and Western Blot techniques in an attempt to elucidate the functions of PIPKIIalpha and determine its role in the (direct or indirect) regulation of globin genes.