The Functional Identification Of Human Novel Gene TMEM98Associated With Inflammation

In2008, Chinese National Human Genome Center analysed the GenomeDatabase in order to obtain novel cytokines.They selected many candidates andverified many kinds of proteins.TMEM98(Transmembrane protein98) was one ofthem. So far, the functions and the functional mechanisms of TMEM family waspoorly understanded.There was no functional report about the human TMEM98gene.It was asequence known but function unknown gene which expressed in a lot of tissues andorgans like spleen,thymus,lymphonodus and bone marrow. Besides,it appeared anup-regulation under the stimulation of inflammation. It is suggested that there shouldbe some relationships between TMEM98and the development of inflammation. SoTMEM98could be of great value in the treatment of inflammation.Objectives:The fuction of TMEM98in innate immunity particularly in anti-infectionimmunity by the research of inflammation related cells in vitro and the model of acutepneumonia mice in vivo.Method:1.In vivoPathogenic Strains:Single colony of Klebsiella pneumonia was transferred intoLB chutivation after cultured on the plate of LB.After7~11h`s culture,ODvalue(OD=0.7) was tested to make the Kp at a concentration of107cfu/ml.AnimalModel:20μl of Kp was injected into the mouse trachea to induce pneumonia.AnimalTreatment:306~8weeks female C57BL/6mice were randomly divided into Control group(6mice),Model group(12mice),Experiment group(12mice).Each mouse ofExperiment group was injected with TMEM98protein(100ng/mouse) through thevena caudalis,while each mouse of Model group and control group was injected withequal volume of saline by the same way.1h later,every mouse was proceed a neckoperate,each mouse of the Model group and Experiment group was injected with20μlKp,while the Control group was injected with equal valume of saline.Detectivemethod:Mice were put to death after24h when Kp was injected. Lung tissues werecollected to proceed lung HE staining,lung MPO assay,RT-PCR and WesternBlotting.2.In vitroCell culture,grouping and treatment:Human umbilical vein endothelial cells(HUVECs) and human monocyte lines THP-1cells were incubated in vitro, then theywere treated with Tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS). Cellswere divide into different groups, including PBS group, TNF-α/LPS group,TMEM98group, TNF-α/LPS+TMEM98group. Detective method: The effect ofTMEM98on cell proliferation and apoptosis were measured by MTT assay. Real-timePCR were performed to check the mRNA transcription of inflammatorycytokines(IL-6,IL-8,MCP-1,CXCL-2). Western blotting was used to detect theexpression of VCAM-1, signal transduction pathway proteins (MAPK8and P65).Result:1.In vivoHE staining:The lung marking of Experiment group was more clear than theModel group,there is only some infiltration of inflammation cells in Experimentgroup.Kp induced pneumonia inflammation was reduced in Experiment group. LungMPO assay:The activity of MPO was significant reduced in the Experiment groupcompared with Model group(P＜0.05).RT-PCR test:The mRNA level of cytokinessuch as IL-1β、IL-6、TNF-α were significant reduced in the Experiment groupcompared with Model group(P＜0.05).Western blotting assay:The protein level ofNF-κB was significant reduced in the Experiment group compared with Model group(P＜0.05).2.In vitroMTT assay: The assay indicated that TMEM98can inhibit HUVECs`growthsignificantly in TNF+TMEM98group compared with TNF group(P＜0.05). Real-timeqPCR assay: The mRNA levels of IL-6,CXCL-2in HUVECs cells were reducedsignificantly in TNF+TMEM98group compared with TNF group(P＜0.05),whilethere is no significantly difference between TMEM98group and PBS group(P＞0.05).Besides, the mRNA levels of IL-8,CXCL-2in THP-1cells were reducedsignificantly in TNF+TMEM98group compared with TNF group(P＜0.05),whilethere is no significantly difference between TMEM98group and PBS group(P＞0.05).Otherwise, the mRNA level of MCP-1in HUVECs cells was elevatedsignificantly in TNF+TMEM98group compared with TNF group(P＜0.05).Westernblotting assay: The protein level of VCAM-1, NF-Κb,MAPK8was reducedsignificantly in TNF+TMEM98group compared with TNF group(P＜0.05),while thesame appearance was occurred between TMEM98group and PBS group(P＜0.05).Conclusions:1.The TMEM98was predicted to be a protein secreted by an unclassical way2.The protein TMEM98can significantly reduce the acute inflammatory reactioncaused by Kp by reducing the secretion of a lot of cytokines,besides, the inhibition ofNF-κB pathway may play an important role.3.The protein TMEM98can significantly inhibit the proliferation and reduce thesecretion of many cytokines of HUVECs in vitro while cells were stimulated byTNF-α,besides, this protein can also significantly educe the secretion of manycytokines of THP-1in vitro while cells were stimulated by LPS.This inhibition maybe closely associated with the down-regulate of NF-κB、 MAPK8pathway.