Ctedoverexpressed A549 cells. In contrast, the expression of LDHA was obviously > 자유게시판

Ctedoverexpressed A549 cells. In contrast, the expression Camptothecin of LDHA was obviously decreased in ENO1-suppressed SPCA-1 cells. To further confirm our results, we examined the level of lactate production in ENO1-overexpressed A549 cells and ENO1-suppressed SPCA-1 cells. Consistent with the results of the Western blot, ENO1-overexpressed A549 cells produced a more amount of lactate compared to its control cells and untreated cells. Conversely, the production of lactate was significantly less in ENO1-suppressed SPCA-1 cells than in its control cells and untreated cells, suggesting the involvement of ENO1 in inducing the glycolysis of NSCLC (Figure 2D).ENO1 promotes cell proliferation, clone formation in vitro, and tumorigenicity in vivoSince ENO1 expression is higher in SPCA-1 than in A549 (Figure 1D), we firstly used lentivirus-mediated fulllength ENO1-GFP (ENO1) to constitutively overexpress ENO1 in A549 cells in order to assess its role in NSCLC. The result showed that ENO1 expression was obviously upregulated in A549-ENO1 cells compared to its control PLV-Ctr cells, and the expression of MBP-1 was not observed (Figure 2A). Further, three lentiviral short hairpin RNA (shRNA) vectors were used to specifically and stably knock down the expression of ENO1 in the SPCA-1 cell line, and the expression levels of ENO1 and MBP-1 were determined by qRT-PCR and Western blot. The result indicated that ENO1 expression was obviously downregulated in shENO1-B and shENO1-C cells compared to their respective control PLV-scrambled control shRNA (shCtr) cells (Figure 2B). Similarly, the expression of MBP1 was not observed in SPCA-1 cells (Figure 2B). To further evaluate the functional significance of ENO1 on NSCLC, small-interfering RNA (siRNA) was used to transiently silence ENO1 in A549 and SPCA-1 cells, and the expression of ENO1 were validated by qRT-PCR and Western blot (Figure 2C).ENO1 regulates the glycolysis in NSCLC cellsNext we assessed the effect of ENO1 expression on A549 cell growth in vitro. The growth curves determined by 3(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that overexpressed ENO1 significantly elevated cell viability compared to its control cells and untreated cells. MTT assays also showed that transiently suppressed ENO1 significantly decreased cell viability in A549 cells (Figure 3A). Colony formation assays showed that overexpressed ENO1 significantly increased cell proliferation compared to its control cells and untreated cells (Figure 3C). On the contrary, suppressed ENO1 expression in SPCA-1 cells significantly inhibited cell viability (Figure PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/16989806 3B) and clone formation (Figure 3D). To confirm the growth effect of ENO1 in vivo, we performed an in vivo tumorigenesis study by inoculating A549 with or without ENO1 overexpression and SPCA-1 cells with or without ENO1 knockdown into nude mice. Mice were sacrificed 15 days after inoculation, with average tumor weights of 0.059 ?0.016 vs 0.73 ?0.12 g in PLV-Ctr vs A549-ENO1 group and 0.95 ?0.13 vs 0.435 ?0.051 g in PLV-shCtr vs shENO1-B group, respectively (P < 0.01) (Figure 3E). These results suggest that ENO1 significantly promotes cell growth in vitro and in vivo.ENO1 promotes cell migration and invasionTo assess the glycolysis changes triggered by ENO1, we used Western blot to detect the expression of lactate dehydrogenase A (LDHA) in ENO1-overexpressed A549 cells and ENO1-suppressed SPCA-1 cells. We found that the protein level of LDHA was markedly.