== Construction of pcDNA3 HA HGF vector and HGF-expression. compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5’untranslated region of thePf-Sub2 promoter. Taken together, it is suggested that HGF controls Parecoxib and interacts with the promoter region of thePf-Sub2 gene. Parecoxib Keywords:Plasmodium falciparum, malaria, subtilisin-like protease 2, hepatocyte growth factor, 5’untranslated reagion, promoter == INTRODUCTION == Malaria, which is caused by infection with the hemoprotozoan parasitePlasmodium, afflicts approximately 500 million people each year, leading to 2.7 million deaths annually worldwide [1]. Malaria results in diverse clinical outcomes presumably because of dynamic relationships between parasite-expressed virulence factors and individual host responses to these determinants [2]. The last few years have seen a rapid growth in the number of reported genetic associations with susceptibility and resistance to malaria in the host; they are mainly variants in erythrocyte, cytoadherence factor, immune system, and inflammatory genes [3,5]. However, molecular basis of malaria infection remains incompletely defined. Identification of genetic factors in both the host and the parasite involved in malaria susceptibility and resistance would lead not only to a greater understanding of this complex disease, but potentially also to the development of effective medical Parecoxib interventions [2,6]. Interactions betweenPlasmodiumand its host hepatocyte factors are of great research interest [7-9]. On one hand, it is well established that Sirt6 malaria sporozoites exploit hepatocyte growth factor (HGF) and its receptor MET signaling during host invasion [10]. On the other hand,Plasmodiumis a life cycle-specific disease that includes liver injury at the erythrocyte stage of the parasite [11]. Indeed, both humans and mice suffer from liver injury after infection with the fatal strainsPlasmodium falciparum(Pf) andP. berghei(Pb) [12,13]. BecausePbmerozoites do not parasitize liver parenchymal cells in mice, it was proposed that the mechanism ofPb-induced liver injury is likely to be caused by the local production of cytokines that activate lymphocytes residing in the liver [14]. Pathologically, malaria-induced mouse liver injury is characterized by dense infiltration of lymphocytes, and the presence of apoptotic and necrotic hepatocytes [15]. Plasmodiumsubtilisin-like protease 2 (subtilase, Sub2) is an essential integral membrane serine protease in bothPfandPb. It functions in the shedding of the ectodomain components of 2 surface proteins, membrane surface protein 1 (MSP1), Parecoxib and apical membrane antigen 1 (AMA1), upon host invasion [16-18]. The importance of the Sub2 gene is evidenced by the demonstration that gene-knock out parasites cannot be recovered [19]. The correlation of HGF and malaria infection is not well understood. In this Parecoxib study, to understand the relationship between the malaria infection-related proteinPf-Sub2 and HGF, thePf-Sub2 5’untranslated region (UTR) was analyzed and its promoter activity was estimated. In addition, the influence of HGF on its promoter activity was analyzed. == MATERIALS AND METHODS == == Parasite manipulations == Pf3D7 strain was obtained from Malaria Research and Reference Reagent Resource Center (MR4; Manassas, Virginia, USA;http://www.mr4.org). ThePf3D7 culture condition was set according to the MR4 standard protocol. Parasitemia was calculated by counting at least 1,000 cells following staining with Diff Quick (Sysmex, Kobe, Japan). == Cell culture == Cell culture media and components, including fetal bovine serum (FBS), were obtained from Invitrogen (Carlsbad, California, USA). All cells were purchased from the American Type Culture Collection (ATCC, Rockville, Maryland, USA) and were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS. HeLa, Huh-7, HepG2, and 293T cells were used for the experiments. == DNA isolation and plasmid vector construction == All plasmids were purified using JETstar2.0 mini and maxiprep Kit (GenoMed, St. Louis, Missouri, USA) according to the manufacturer’s instructions.Pf3D7 small scale gDNA isolation followed.