Abstract
Objective To investigate the influence of parathyroid hormone-related protein (PTHrP) and monocarboxylic acid transporter 1 (MCT1) signaling pathway on the aging of Lewis lung adenocarcinoma cell line LLC and its mechanism.Methods LLC cells were divided into groups A, B, C, and D with 10 holes in each group.PTHrP cell pathway activators PTHrP (1-34), PTHrP receptor competition inhibitor PTHrP (7-34), MCT1 signaling pathway inhibitor AZD3965, and PBS were added to the culture cells of the above four groups for 72 h, respectively.The degree of cell senescence in each group was observed by β-Gal (βGal) staining, which was shown as the percentage of positive staining area of βGal in total cell area.The expression of PTHrP mRNA and MCT1 mRNA in each group was detected by real-time fluorescent quantification PCR, and the results were expressed by Ct (threshold, cycle).The standard curve of relationship between cell pH value (pHi) and intracellular BCECF fluorescence intensity was established, and the results of cell pHi were expressed by 495 nm/440nm ratio.The level of extracellular lactic acid in each group was detected according to the kit instruction.Results The percentages of βGal positive staining area in groups A, B, C, and D were 8.47%±0.15%, 12.56%±0.20%, 16.66%±0.20%, and 9.52%±0.30%, respectively.In groups B and C, the percentage of positive staining area of βGal in total cell area was higher than that in group D (P<0.05).The expression of PTHrP mRNA in the groups A, B, C, and D was 11.49±0.42, 7.68±0.10, 8.56±0.13, and 9.35±0.13, respectively.The expression of MCT1 mRNA was 7.84±0.19, 4.38±0.09, 3.53±0.10, and 6.58±0.21, respectively.The expression of PTHrP mRNA in the group A was higher than that in the groups B, C and D (all P<0.05).The expression of PTHrP mRNA in the group B was lower than that in the groups C and D (P<0.05).The expression of PTHrP mRNA in the group C was lower than that in the group D (P<0.05).The expression of MCTl mRNA in group A was higher than that in the groups B, C and D (all P<0.05).The expression of MCTl mRNA in the group B was higher than that in the group C (P<0.05), but lower than that in the group D (P<0.05).The expression of MCTl mRNA in the group C was lower than that in the group D (P<0.05).pHi cells in the groups A, B, C, and D were 3.39±0.12, 6.05±0.12, 7.08±0.09, and 4.66±0.08;the extracellular lactate levels were (8.34±0.12), (5.13±0.15), (3.39±0.10), and (7.33±0.11)unit (A.U.).The pHi in the group A was lower than that in the groups B, C, and D (all P<0.05).The pHi in the group B was lower than that in the group C (P<0.05), but was higher than that in the group D (P<0.05).The pHi in the group C was higher than that in the group D (P<0.05).The level of extracellular lactic acid in the group A was higher than those in the groups B, C and D (all P<0.05).The level of extracellular lactic acid in the group B was higher than that in the group C (P<0.05), but was lower than that in the group D (P<0.05).The level of extracellular lactic acid in the group C was lower than that in the group D (P<0.05).Pearson correlation analysis showed that the aging of cells in each group was related to the PTHrP mRNA, MCT1 mRNA, pHi, and extracellular lactate level (r=-0.613,-0.880, 0.906, and-0.961, respectively, all P<0.05).Conclusions The regulation of PTHrP and MCT1 signaling pathway may affect the aging of lung adenocarcinoma cells.The mechanism may be that the PTHrP signaling pathway and the MCT1 signaling pathway interact with each other to modulate the lactate transport in lung adenocarcinoma cells.关键词
肺腺癌/甲状旁腺激素相关蛋白/单羧酸转运泵家族1/细胞衰老/β-半乳糖苷酶/细胞内pH值/乳酸Key words
lung adenocarcinoma/parathyroid hormone-related proteins/monocarboxylic acid transport pump family 1/cell senescence/β-galactosidase/intracellular pH value/lactic acid分类
医药卫生
