001) Neither cell line had significant changes in the G2 populat

001). Neither cell line had significant changes in the G2 population (Figure 4C)(Table 1). Table 1 Overexpressed ECRG4 retarded cell cycle progression from G1 to S phase Cells G1 S G2

pEGFP-ECRG4-5 64.93 ± 1.54 16.37 ± 1.12 18.7 ± 0.44 pEGFP-ECRG4-7 5.77 ± 1.34 15.23 ± 1.30 19.0 ± 0.44 Ctr-Vector 54.67 ± 1.27 26.13 ± 0.91 19.2 ± 2.05 U251 54.73 ± 0.86 25.87 ± 1.27 19.4 ± 1.77 ECRG4 inhibited the expression of NF-Kb We were further interested in exploring the molecular mechanism of ECRG4 tumor-suppression in glioma. We found that restoration of ECRG4 expression in https://www.selleckchem.com/products/BafilomycinA1.html glioma U251 cells inhibited expression of transcription factor NF-κB (Figure 5A). This suggested that ECRG4 may be involved in NF-κB pathway in glioma. Figure 5 Overexpresed ECRG4 expression suppressed the expression of NF-kB protein. A. Protein expression of NF-kB was decreased in pEGFP-ECRG4-5 and -7 cells Microtubule Associated inhibitor compared to Control-vector cells. Data were presented as mean ± SD. *P < 0.05. Discussion Malignant glioma is a highly invasive and clinically challenging tumor of the central nervous system, and its molecular basis remains poorly understood. We became interested in ECRG4 because it JNJ-26481585 is

normally expressed in the brain yet was found to be downregulated in gliomas. Northern blot assays revealed that ECRG4 is also expressed in other tissues including heart, placenta, lung, liver, skeletal muscle, kidney and pancreas [14]. Further, ECRG4 promoter hypermethylation has been attributed to decreased expression in esophageal, prostatic, and colorectal cancers. Together these results suggest that ECRG4 might play a suppressive role in tumor pathogenesis. ECRG4 contains a 772-bp full-length cDNA fragment, and its open reading frame is 444bp

encoding a 148-amino acid polypeptide with molecular weight of 17 kDa. ECRG4 gene is located at chromosome 2q12.2 and contains 4 exons spanning about 12,500 bp. In order to assess the role of ECRG4 in glioma, we first performed real-time PCR to measure the expression of ECRG4 mRNA transcripts in 10 paired gliomas and their adjacent brain tissues. Similar to observations by Götze et al [12], we found that ECRG4 expression was Alanine-glyoxylate transaminase significantly downregulated in 9 glioma tissues compared to their matched normal tissues. To examine whether ECRG4 plays a suppressive role in glioma pathogenesis, we applied a gain-of-function approach by introducing ECRG4 into cells to investigate its biological function. To this end, we chose the U251 glioma cell line which exhibits relatively low expression level of endogenous ECRG4 (data not shown) and provides a biologically relevant model for our study. U251 cells were transfected with ECRG4-GFP-expressing eukaryotic vector followed by selection with G418. We successfully established lines stably expressing ECRG4 protein at dramatically elevated levels compared to control cells.

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