| Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a phosphoprotein that not only plays an important role in the synthesis of rRNA and the biosynthesis of ribosomes, but also participates in related signaling pathways control on cell inflammatory response, cell growth, cell apoptosis and tumorigenesis. Previous studies suggest that NOLC1 is crucial for normal cell growth and plays a role in the regulation of tumorigenesis of nasopharyngeal carcinoma (NPC) and liver cancer. Yet, the effect of NOLC1 in lung cancer remains unknown.First, pENTR/D-TOPO-NOLC1was successfully constructed by TOPO cloning technology. pAd-CMV-V5-NOLC1 expression vector was successfully constructed by pathways cloning system technology. After adenovirus vector was successfully packaged into adenovirus, NOLC1 mRNA and protein expression was assessed in normal human embryo lung cells (HEL) and lung cancer cells (A549) by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The result showed that MOI=100, two kinds of cells NOLC1 has the highest amount of mRNA and protein.Cells infected with Ad-V5-NOLC1 (overexpression) or transfected with shRNA-NOLC1 (knockdown) were subjected to cell viability assay (MTT assay). The result showed that both over-expression and knockdown of NOLC1 induced much losser in cell viability in A549 cells than in HEL cells. In order to determine whether apoptosis induced by overexpression of NOLC1 in HEL and A549 was mediated via the mitochondrial pathway, changes in mitochondrial membrane potential (ΔΨm) in these cells were measured. The results suggested that overexpression of NOLC1 in HEL cells did not really contribute to any increase in the proportion of early stage apoptosis, but overexpression of NOLC1 in A549 would increase the proportion of early stage apoptosis, with the increase being more profound with time. It was illustrated NOLC1 over-expression influence A549 decreased mitochondrial membrane potential, may lead to pro-apoptosis release, caspase activated and eventually make the cell apoptosis. Real-time PCR assays were then used to determine whether apoptotic signaling pathways were changed by NOLC1 over-expression. a total of ten genes, include Caspases families (CASP3, CASP6, CASP7, CASP8), tumor necrosis factor (TNF) ligand and receptor families (TNF, TNF10B), B-cell lymphoma 2 (BCL2) families (BCL2L11, BAX, BAG1, BCL2A1) were detected in A549 cells and HEL cells. The result showed that of the ten pro-apoptotic genes, but fold changes from one to two in HEL cells. This suggested that, there was insufficient activation of relevant pro-apoptotic pathways to support apoptosis compared to control cells. In contrast, in A549 cells, of the eight pro-apoptotic genes were up-regulated from 4.0 to 8.0, while Anti-apoptosis gene BCL2A1 was up-regulated. Collectively, the up-regulation of the ten pro-apoptotic genes combined with down-regulation of one anti-apoptotic gene would suggest that apoptosis would be favored, and important protein CASP8 and Bax were up-regulated markedly, but two important protein were not up-regulated markedly in HEL cells. It was illustrated NOLC1 over-expression induced apoptosis A549 cells apoptosis by mitochondrial pathway and death receptor pathway. This supported the interpretation that over-expression of NOLC1 reduced cell numbers through apoptosis in transformed A549 cells but not in HEL normal cells.This study identified that over-expression NOLC1 accelerated the apoptosis of A549 cells, but it had not effected on HEL cells, which provides theoretical basis for further study of cell apoptosis mechanism and provides experimental basic for study the effect of NOLC1 protein overexpression on animal level. |
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