Expression and functional analysis of novel gene AngRem104 associated with glomerular sclerosis / 中国病理生理杂志

AIM: To study the expression and function of novel gene AngRem104. METHODS: Northern blot was performed to detect the distribution of AngRem104 in human multiple normal tissues as well as the effect of AngⅡ and AT1R antagonist (losartan) on AngRem104 expression. The sense and antisense eukaryotic expression vectors of AngRem104 were constructed and transfected into human mesangial cells. RT-PCR was used to detect the expression of FN when AngRem104 was over-expressed. Primary sequence and motif analysis of AngRem104 protein were performed by on-line ExPasy predictive tools. RESULTS: AngRem104 was predicted to localize at the cellular nucleus. It was widely expressed in human heart, placenta, liver, muscle, kidney and pancreas. Moreover, the up-regulated expression of AngRem104 induced by AngⅡ was inhibited by losartan in a dose-dependent manner. CONCLUSION: AngRem104 is a novel nuclear protein related to the expression of fibronectin and could be up-regulated by AngⅡ in human MC.

Screening for genes related to novel gene AngRem104 by gene chip / 中华肾脏病杂志

Objective To screen the genes related to novel gene AngReml04. Methods Gene chip was employed to detect the genes related to AngRem104 by its over-expressed constructs, which was produced by transfection of the sense- and antisense-AngReml04 into human mesangial cells, and then RT-PCR was used to confirm the up-regulated expression of related genes. Results There were 94 genes up-regulated and 2 genes down-regulated when AngRem104 was over-expression. The different expression genes were cataloged as extracellular matrix and receptor protein, DNA-binding and transcription-related protein, immune-related protein, cytoskeletal and dynamic protein, synthesis and metabolism related protein. Fibronectin (FN) and integrin beta 1 (fibronectin receptor) were dramatically over-expressed in the top of all up-regulated genes. Furthermore, the correlative expression between AngReml04 and FN was detected by RT-PCR. Conclusion Gene chip not only demonstrates the clues for further investigation of novel gene AngRem 104, but also reveals the co-expression of AngRem104 and fibronectin.

Interaction of novel gene AngRem104 and Bardet-Biedl syndrome 2 protein in mammalian cells / 中华肾脏病杂志

Objective To screen for proteins interacting with novel gene AngRem104 and to identify the putative interaction of novel gene AngRem104 and Bardet-Bied1 syndrome 2 (BBS2) protein in mammalian cells. Methods The yeast strain AH109 was transformed with AngRem104pGBKT7/c-myc and yeast-mating was utilized to screen for interacting proteins with AngRem104 in pretransformed human kidney cDNA library. The human embryonic kidney (HEK 293T) cells were transformed with two recombined plasmids,AngRem104-pcDNA3.1/V5-His and BBS2-pCMV/c-myc. Mouse anti-human V5 monoclonal antibody and mouse anti-human c-myc monoclonal antibody were used in immunoprecipitation and immunoblot analysis, respectively. Results Seven proteins that interact with AngRem104, including BBS2 were identified. The AngRem104-V5 and the BBS2-c-myc fusion protein were detected respectively in the immunoprecipitation by anti-c-myc and anti-V5 antibody. Conclusion The novel gene AngRem104 may interact with BBS2 protein in mammalian cells,which provides insights as to the function exploration of novel gene AngRem104 and the pathogenesis investigation of Bardet-Biedl syndrome.

AngRem104 potentially regulates the transcription of fibronectin through the activation of promoter / 中国病理生理杂志

AIM: To investigate the mechanism of AngRem104-mediated regulation of fibronectin gene in human mesangial cells. METHODS: A series of deleted FN promoter sequences was constructed, which fuse to a luciferase reporter gene, and then the potential active regions that respond to AngRem104 in the upstream regulatory sequence of human FN gene was screened by detecting the luciferase activity of promoter-reporter gene. RESULTS: The detection of relative luciferase activity revealed that there was no significant differences when the HMC were transfected with FN122-reporter gene together with sense AngRem104 construct, but the luciferase activity significantly increased when transfection of FN507-reporter gene construct together with sense AngRem104 construct. However, no increase in luciferase activity was observed when transfection of FN1280-reporter gene construct together with sense AngRem104 construct. The potential regulatory region responds to AngRem104 is in the upstream sequence (-122 to -507) of human FN gene. CONCLUSION: Our preliminary study provided the evidence that AngRem104 may mediate the transcription of the FN gene via the activation of its promoter.