PTHrP、MCT1信号通路对非小细胞肺癌细胞衰老的调节机制研究

Regulatory Mechanism of PTHrP and MCT1 Signaling Pathway on Senescence of Non-small Cell Lung Cancer Cells

目的研究探讨甲状旁腺激素相关蛋白(PTHrP)、单羧酸转运泵家族1(MCT1)信号通路对非小细胞肺癌衰老的调节机制。方法将2014年5月-2018年5月在该医院培养的80孔非小细胞肺癌A549细胞分为A、B、C、D共4组,每组20孔。其中A组细胞中加入PTHrP信号通路抑制剂、B组细胞加入MCT1信号通路抑制剂、C组细胞加入PTHrP信号通路激活剂、D组细胞加入等量的PBS培养液,4组细胞经联系72 h培养。采用β-半乳糖苷酶染色法(β-Gal)观察4组细胞的衰老程度。对4组细胞进行PCR扩增,以实时荧光定量PCR方法检测PTHrP mRNA、MCT1mRNA的表达量。并对各组细胞中pHi染色小体的和细胞外乳酸水平进行检测比较。并采用Pearson检验对各指标间进行相关性分析。结果①4组细胞间β-Gal染色阳性率比较差异有统计学意义(P<0.05),其中A组、B组阳性率(13.98±2.53)%、(14.23±3.21)%均高于D组(10.09±2.98)%,C组阳性率(8.29±2.10)%则低于D组,各组间比较差异有统计学意义(P<0.05)。②4组细胞的PTHrP mRNA、MCT1 mRNA表达量比较差异有统计学意义(P<0.05),其中A组、B组PTHrP mRNA、MCT1 mRNA表达量[(7.09±1.53)、(5.29±1.22);(7.26±1.61)、(4.98±1.17)]低于D组[(9.13±1.98)、(7.19±1.68)],而C组表达率[(12.32±2.09)、(9.57±1.98)]则高于D组,各组间比较差异有统计学意义(P<0.05)。③4组细胞的p Hi染色小体的和细胞外乳酸水平比较有统计学差异,A组、B组pHi染色小体水平高于D组,C组则低于D组,A组、B组细胞外乳酸水平低于D组,C组则高于D组,各组间比较差异有统计学意义(P<0.05)。④Pearson相关性检验显示,各组细胞的β-Gal染色阳性率(即衰老程度)与PTHrP mRNA、MCT1 mRNA表达量呈负相关性,与pHi染色小体水平呈正相关性,与细胞外乳酸水平呈负相关性(P<0.05)。结论 PTHrP、MCT1信号通路状态可以对非小细胞肺癌细胞的衰老起到调节作用,PTHrP、MCT1间相互影响,共同调节肺癌细胞的乳酸转运过程。

Objective To investigate the regulation mechanism of parathyroid hormone-related protein(PTHrP) and monocarboxylate transport pump family 1(MCT1) signaling pathway on the senescence of non-small cell lung cancer. Methods The A549 cells of 80-well non-small cell lung cancer cultured in People’s Hospital from May 2014 to May 2018 were divided into 4 groups: A, B, C, and D, each with 20 holes. In group A, PTHrP signaling pathway inhibitor was added, group B cells were added with MCT1 signaling pathway inhibitor, group C cells were added with PTHrP signaling pathway activator,group D cells were added with equal amount of PBS medium, and four groups of cells were cultured for 72 h. The degree of senescence of the four groups of cells was observed by β-galactosidase staining(β-Gal). Four groups of cells were subjected to PCR amplification, and the expression levels of PTHrP mRNA and MCT1 mRNA were detected by real-time fluorescent quantitative PCR. The pHi staining and extracellular lactate levels in each group of cells were detected and compared.Correlation analysis was carried out between the indicators using the Pearson test. Results 1.There was a statistically significant difference(P<0.05) in the positive rate of β-Gal staining between the four groups. The positive rate of group A and group B(13.98±2.53)% and(14.23±3.21)% were higher than those of group D(10.09±2.98)%. The positive rate of group C(8.29±2.10)% was lower than that of group D, and there was significant significant difference between the groups(P<0.05).2.The expression levels of PTHrP mRNA and MCT1 mRNA in the four groups of cells were statistically significant(P<0.05). The expression levels of PTHrP mRNA and MCT1 mRNA in group A and group B were(7.09±1.53),(5.29±1.22);(7.26±1.61)(4.98±1.17)], lower than group D [(9.13±1.98),(7.19±1.68)], and the expression rate of group C [(12.32±2.09),(9.57±1.98)], higher than group D, and there were significant significant differences between the groups(P<0.05). 3.There were significant significant differences in p Hi staining and extracellular lactate levels between the three groups of cells. The p Hi staining level of group A and group B was higher than that of group D, and that of group C was lower than that of group D, group A and group B. The level of external lactate was lower than that of group D, and that of group C was higher than that of group D. There was statistically significant difference between groups(P<0.05). 4.Pearson correlation test showed that the positive rate of β-Gal staining(ie, aging) of each group was negatively correlated with the expression of PTHrP mRNA and MCT1 mRNA, and positively correlated with the level of p Hi staining, and the level of extracellular lactate of negative correlation(P<0.05). Conclusion The state of PTHrP and MCT1 signaling pathway can regulate the senescence of non-small cell lung cancer cells. PTHrP and MCT1 interact with each other to regulate the lactic acid transport process of lung cancer cells.