甲状旁腺激素相关蛋白、单羧酸转运泵家族1信号通路对肺腺癌细胞衰老的调节作用及其机制

摘要

目的 探讨调节甲状旁腺激素相关蛋白(PTHrP)、单羧酸转运泵家族1(MCT1)信号通路对Lewis 肺腺癌细胞株LLC细胞衰老的影响及其机制.方法 将LLC细胞分为A、B、C、D组,每组10孔.分别加入PTHrP信号通路激活剂PTHrP(1~34)、PTHrP受体竞争抑制物PTHrP(7~34)、MCT1信号通路抑制剂 AZD3965、PBS培养72 h后收集细胞.用β-半乳糖苷酶(βGal)染色法观察各组细胞衰老程度,结果以细胞中βGal阳性染色面积占细胞总面积的百分比表示.用实时荧光定量PCR方法检测各组细胞中PTHrP mRNA 、MCT1 mRNA 表达.建立细胞pHi与细胞内BCECF荧光强度关系的标准曲线,细胞pHi结果以495 nm/440 nm延荧光强度比值表示.按照试剂盒说明书操作检测各组细胞外乳酸水平.结果 A、B、C、D组细胞中βGal阳性染色面积占总细胞面积百分比分别为8.47%±0.15%、12.56%±0.20%、16.66%±0.20%、9.52%±0.30%.B、C组细胞中βGal阳性染色面积占总细胞面积百分比与D组相比,P均<0.05.A、B、C、D组细胞中PTHrP mRNA表达量分别为11.49±0.42、7.68±0.10、8.56±0.13、9.35±0.13,MCT1 mRNA 表达量分别为7.84±0.19、4.38±0.09、3.53±0.10、6.58±0.21.A组细胞中PTHrP mRNA 表达量高于B、C、D组(P均<0.05);B组细胞中PTHrP mRNA 表达量低于C、D组(P均<0.05);C组细胞中PTHrP mRNA 表达量低于D组(P<0.05);A组细胞中MCT1 mRNA 表达量高于B、C、D组(P均<0.05);B组细胞中MCT1 mRNA 表达量高于C组(P<0.05),但低于D组(P<0.05);C组细胞中MCT1 mRNA 表达量低于D组(P<0.05).A、B、C、D组细胞pHi分别为3.39±0.12、6.05±0.12、7.08±0.09、4.66±0.08,细胞外乳酸水平分别为(8.34±0.12)、(5.13±0.15)、(3.39±0.10)、(7.33±0.11)U.A组细胞pHi低于B、C、D组(P均<0.05);B组细胞pHi低于C组(P<0.05),但是高于D组(P<0.05);C组细胞pHi高于D组(P<0.05);A组细胞外乳酸水平高于B、C、D组(P均<0.05);B组细胞外乳酸水平高于C组(P<0.05),但是低于D组(P<0.05);C组细胞外乳酸化水平低于D组(P<0.05).Pearson相关性分析结果显示,各组细胞衰老程度与PTHrP mRNA表达量、MCT1 mRNA表达量、pHi、细胞外乳酸水平均相关,r分别为-0.613、-0.880、0.906、-0.961,P均<0.05.结论 调节PTHrP、MCT1信号通路可以影响肺腺癌细胞衰老.其机制可能是由于PTHrP信号通路和MCT1信号通路相互影响,共同调节肺腺癌细胞的乳酸转运.%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.