CD44^+/CD24^(-/low)亚群比例和p63的表达强度对甲状腺乳头状癌细胞体外转移侵袭能力的影响

Expression of CD44^+/CD24^(-/low) and p63 are associated with the invasion and metastasis of papillary thyroid carcinoma

目的探讨CD44^+/CD24^(-/low)亚群比例和p63的表达强度对甲状腺乳头状癌细胞(PTC)体外转移侵袭能力的影响。方法选取PTC 4个细胞株TPC-1、GLAG-66、BCPAP、NPA和正常甲状腺细胞株Nthyori 3-1进行体外实验,采用流式细胞术检测CD44^+/CD24^(-/low)亚群比例,Western印迹法检测p63蛋白的表达情况,Transwell侵袭实验检测细胞的转移侵袭能力,细胞计数实验(CCK-8法)观察化学治疗药物多柔比星对不同侵袭能力肿瘤细胞生长的抑制作用,定量反转录(qRT)-PCR检测全长型p63(TAp63)和N端截短型p63(ΔNp63)mRNA的表达。结果 PTC细胞株TPC-1、BCPAP、GLAG-66、NPA和正常甲状腺细胞株Nthy-ori3-1的CD44^+/CD24^(-/low)亚群比例分别为(34.59±1.88)%、(0.28±0.02)%、(44.15±1.42)%、(56.96±1.94)%和(0.25±0.01)%,PTC 4个细胞株间CD44^+/CD24^(-/low)亚群比例的差异均有统计学意义(P值均<0.01),TPC-1、GLAG-66和NPA与Nthy-ori 3-1间CD44^+/CD24^(-/low)亚群比例的差异均有统计学意义(P值均<0.05)。PTC各细胞株中均有p63蛋白表达,正常甲状腺细胞株Nthy-ori 3-1中几乎无p63蛋白表达。NPA、GLAG-66、TPC-1、BCPAP穿过人工基底膜的细胞数分别为(126.00±2.31)、(71.00±1.95)、(36.00±3.05)和(12.00±2.40)个/高倍镜视野,4组间差异有统计学意义(P值均<0.05);正常甲状腺细胞株Nthy-ori 3-1未见穿过人工基底膜的细胞。不同浓度的多柔比星溶液对PTC细胞株NPA、GLAG-66、TPC-1、BCPAP和正常甲状腺细胞株Nthy-ori3-1均有不同程度的抑制作用,且在同一浓度下(除浓度为0.04\mmol/L外),对各细胞株的生长抑制率由低至高依次为NPA、GLAG-66、TPC-1、BCPAP、Nthy-ori 3-1。PTC细胞株TPC-1、BCPAP、GLAG-66、NPA中ΔNp63 mRNA的表达水平分别为0.000 570±0.000 160、0.000 057±0.000 007、0.000 800±0.000 085、0.001 300±0.000 091,4组间的差异有统计学意义(P值均<0.05);PTC细胞株TPC-1、BCPAP、GLAG-66、NPA中TAp63mRNA几乎无表达。结论 PTC细胞株的转移、侵袭能力可能与CD44^+/CD24^(-/low)亚群比例和p63表达强度有关,高CD44^+/CD24^(-/low)亚群比例和p63表达强度的细胞株对化学治疗药物有较强的耐药性,CD44和p63对于未来靶向治疗的靶点选择具有一定参考意义。

Objective To explore the effects of the propotion of CD44＋/CD24-/low and p63 levels on the invasion and metastasis of papillary lhyroid carcinoma （PTC） cell lines. Methods Four PTC celt strains （TPC-1, BCPAP, GLAG-66 and NPA） were used for in vitro experiment in this study. The proportion of CD44＋/CD24-/low subpopulation was evaluated by flow cytometry. The p63 protein expression was detected by Western blotting. 24-well Transwell test was used to evaluate the ability of cell metastasis. Cell counting kit （CCK）-8 assays were performed to test the resistance to doxorubicin in the PTC cell lines. Meanwhile, quantitative reverse transciptase real time-polymerase chain reaction （qRT-PCR） was conducted to detect the TAp63 and ANp63 messenger RNA （mRNA） in PTC ceils. Results The proportion of 0D44＋/0D24-/low in PTC cells （TPC-1, BCPAP, GLAG-66 and NPA） and normal thyroid cells （Nthy-ori 3-1） were （34.59±1.88）%, （0.28±0.02）%, （44. 15±1.42）%, （56.96± 1.94）% and （0.25 ± 0.01 ）%, respectively. There were significant differences in the proportion of 0D44＋/CD24-/low between four PTC cell lines （all P〈0. 01）. Significant difference was also found beteeen three PTC cell lines （TPC-1, GLAG-66 and NPA） and Nthy-ori 3-1 （all P〈0. 05）. The p63 protein was found in all the PTC cells, but there was no expression in Nthy-ori 3-1. The number of NPA, GLAG-66, TPC-1 and BCPAP through the artificial basement membrane were （126.00 ±2.31）, （71.00 ±1.95）, （36.00± 3.05） and （12.00 ± 2.40）/high power field, respectively; and significant difference were found between them （all P〈0. 05）. No 3-1 Nthy-ori went through the artificial basement membrane. The doxorubicin solution had the lowest resistence against NPA growth, followed by GLAG-66, TPC-1, BCPAP and Nthy-ori 3-1. The expressions of ANp63 mRNA in TPC-1, BCPAP, GLAG-66 and NPA were 0. 000 570 ±0. 000 160, 0. 000 057 ± 0. 000 007,0. 000 800± 0. 000 085 and 0. 001 300±0. 000 091, respectively, and significant difference were found between them （all P〈0. 05）. There was no expression of TAp63 mRNA in the four PTC cell lines. Conclusion The migration and invasion of PTC cells are associated with the proportion of CD44＋/CD24-/low and p63 expression. The higher proportion of 0D44＋/CD24-/low Wand p63 expression in the cell lines, the stronger resistance to chemical treatment medicine. These two markers may be new targets for targeted therapy in the future.