NOD1对甲状腺癌细胞BPCPA恶性生物学功能的影响及机制

Effect of NOD1 on the Biological Functions of BPCPA Thyroid Cancer Cells and Its Mechanism

目的:探讨核苷酸结合寡聚结构域1(nucleotide-binding oligomerization domain 1,NOD1)对甲状腺癌细胞BPCPA生物学行为的影响及其调控机制。方法:通过siRNA技术干扰甲状腺癌细胞株BPCPA中NOD1的表达水平,转染空载体质粒NC-siRNA的细胞命名为siNC组,转染NOD1干扰质粒NOD1-siRNA的细胞命名为siNOD1组。采用qRT-PCR和Western blot验证干扰效果;采用EdU增殖实验检测NOD1表达下调后细胞增殖的变化;流式细胞术检测NOD1表达下调后细胞周期和凋亡的变化;细胞划痕实验检测NOD1表达下调后细胞运动能力的变化;Transwell实验检测NOD1表达下调后细胞侵袭能力的变化;Western blot检测干扰NOD1对MAPK信号通路的影响。结果:转染NOD1-siRNA后,siNOD1组细胞中NOD1的mRNA和蛋白表达水平较siNC组明显下降(0.194±0.059 vs 1.00±0.000,t=19.220,P<0.001;0.073±0.020 vs 0.352±0.040,t=8.852,P<0.001);siNOD1组细胞的EdU阳性率高于siNC组(76.41%±3.75%vs 28.75%±3.79%,t=4.751,P<0.001);相较于siNC组细胞,siNOD1组G1期细胞比例下降(66.23%±4.28%vs 55.38%±3.11%,t=2.902,P=0.044),S期和G2/M期细胞比例增加(22.41%±2.87%vs 36.00%±3.49%,t=4.249,P=0.013;6.84%±0.50%vs 10.92%±1.08%,t=4.859,P=0.010);siNOD1组和siNC组细胞的凋亡率差异无统计学意义(8.47%±0.61%vs 8.74%±1.03%,t=0.322,P=0.763);siNOD1组细胞划痕愈合距离显著多于siNC组[(609.33±41.11)μm vs(316.63±24.05)μm,t=8.691,P=0.001];siNOD1组侵袭细胞数量为(545.3±24.5)个,显著多于siNC组(392.3±17.6)个(t=5.060,P=0.007);siNOD1组细胞的P38和ERK磷酸化水平较siNC组高(1.196±0.045 vs 0.357±0.030,t=21.961,P<0.001;0.764±0.039 vs 0.219±0.038,t=14.265,P<0.001)。结论:NOD1通过MAPK信号通路抑制甲状腺癌细胞BPCPA的增殖和转移,可能是甲状腺癌潜在的治疗靶点。

Objective: To investigate the effect of nucleotide-binding oligomerization domain 1(NOD1) on the biological behaviors of BPCPA thyroid cancer cells and its mechanism. Methods: The expression level of NOD1 in BPCPA cell line was interfered with by siRNA. Cells transfected with control plasmid NC-siRNA were named as siNC, and the cells transfected with NOD1-interfered plasmid, DON1-siRNA, were named as siNOD1. The interference effect was verified by qRT-PCR and Western blot. After NOD1 was down-regulation, EdU proliferation assay was performed to detect the changes in cell proliferation;flow cytometry was used to detect the changes in cell cycle and apoptosis;cell scratch assay was used to detect the changes in cell movement;transwell assay was used to detect the changes in cell invasion. Western blot was used to detect the effect of NOD1 on MAPK signaling pathway. Results: After being transfected with NOD1-siRNA, mRNA and protein expression levels of NOD1 in the siNOD1 group were significantly lower than those in the siNC group(0.194±0.059 vs 1.00±0.000, t=19.220, P<0.001;0.073±0.020 vs 0.352±0.040, t=8.852, P<0.001);the EdU-positive rate in the siNOD1 group was higher than that in the siNC group [76.41%±3.75% vs 28.75%±3.79%, P<0.001];the proportion of G1-phase cells in the siNOD1 group was lower that that in the siNC group(55.38%±3.11% vs 66.23%±4.28%, t=2.902, P=0.044);the proportion of S-phase and G2-phase cells were higher than those in the siNC group(22.41%±2.87% vs 36.00%±3.49%, t=4.249, P=0.013;6.84%±0.50% vs 10.92%±1.08%, t=4.859, P=0.010);there was no significant difference in apoptosis between siNOD1 and siNC groups(8.47%±0.61% vs 8.74%±1.03%, t=0.322, P=0.763);the gap in the scratch wound in the siNOD1 group was significantly wider than that in the siNC group [(609.33±41.11) μm vs(316.63±24.05) μm, t=8.691, P=0.001];the number of invasive cells in the siNOD1 group was 545.3±24.5, significantly more than that in the siNC group(392.3±17.6)(t=5.060, P=0.007);the phosphorylation levels of P38 and ERK in the siNOD1 group were higher than those in the siNC group(1.196±0.045 vs 0.357±0.030, t=21.961, P<0.001;0.764±0.039 vs 0.219±0.038, t=14.265, P<0.001). Conclusion: NOD1 inhibits the proliferation and metastasis of BPCPA thyroid cancer cells through MAPK signaling pathway, which may be a potential therapeutic target for thyroid cancer.