国际肿瘤学杂志

国际肿瘤学杂志 ›› 2023, Vol. 50 ›› Issue (7): 398-406.doi: 10.3760/cma.j.cn371439-20230217-00078

• 论著 • 上一篇    下一篇

BHLHE40靶向HMGA2激活氧化磷酸化通路降低甲状腺癌细胞对顺铂敏感性的研究

张劲男1(), 刘邦卿2, 李军1, 刘晓辉2   

  1. 1唐山市人民医院头颈科，唐山 063000
    2唐山市人民医院胸外二科，唐山 063000
  • 收稿日期:2023-02-17 修回日期:2023-05-26 出版日期:2023-07-08 发布日期:2023-08-03
  • 通讯作者: 张劲男，Email： ga71x345@163.com

Research on BHLHE40 targets HMGA2 to reduce the sensitivity of thyroid cancer cells to cisplatin through activating the oxidative phosphorylation pathway

Zhang Jinnan1(), Liu Bangqing2, Li Jun1, Liu Xiaohui2   

  1. 1Department of Head and Neck，Tangshan People's Hospital，Tangshan 063000，China
    2The Second Department of Thoracic Surgery，Tangshan People's Hospital，Tangshan 063000，China
  • Received:2023-02-17 Revised:2023-05-26 Online:2023-07-08 Published:2023-08-03
  • Contact: Zhang Jinnan，Email： ga71x345@163.com

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摘要：

目的 探究BHLHE40能否通过靶向高迁移率族蛋白A2（HMGA2）激活氧化磷酸化（OXPHOS）通路进而影响甲状腺癌细胞对顺铂的敏感性。方法 通过在线数据库TCGA-甲状腺癌和hTFtarget分析HMGA2及上游转录因子BHLHE40在甲状腺癌组织中的mRNA表达情况。采用脂质体转染法将si-HMGA2、oe-HMGA2、oe-BHLHE40以及阴性对照si-NC、oe-NC转染至甲状腺癌细胞K1和SW579中，通过实时荧光定量PCR检测BHLHE40和HMGA2在甲状腺癌细胞SW579、FTC-133、K1和正常甲状腺细胞Nthy ori3-1中的mRNA表达水平，采用MTT法检测细胞活力，CCK-8法计算顺铂半抑制浓度（IC50）值，流式细胞术检测细胞凋亡水平，蛋白质印迹法检测OXPHOS复合体的表达，Seahorse XFe 96分析细胞的耗氧率。双荧光素酶报告实验和染色质免疫沉淀实验分析BHLHE40与HMGA2的结合关系。结果 TCGA 数据库结果表明，HMGA2和BHLHE40 mRNA在甲状腺癌组织中的表达（10.57±2.58、13.89±1.13）均较甲状腺正常组织高（4.82±1.69、12.28±1.01），差异均具有统计学意义（t=16.69，P<0.001；t=10.43，P<0.001）。实时荧光定量PCR结果发现，正常甲状腺细胞Nthy ori3-1、甲状腺癌细胞SW579、FTC-133和K1中HMGA2 mRNA相对表达量分别为1.00±0.13、2.94±0.23、4.71±0.41和6.29±0.49，BHLHE40 mRNA相对表达量分别为1.00±0.12、2.60±0.23、3.39±0.35和6.18±0.51，差异均具有统计学意义（F=130.50，P<0.001；F=125.20，P<0.001）。进一步两两比较发现，与正常甲状腺细胞相比，甲状腺癌细胞中HMGA2和BHLHE40 mRNA的表达水平均显著升高（均P<0.001）。MTT法检测显示，与si-NC组相比，si-HMGA2处理显著降低了K1细胞的细胞活力（均P<0.05），oe-HMGA2处理相对于oe-NC组显著增加了SW579细胞的细胞活力（均P<0.05）；与oe-NC+DMSO组相比，oe-HMGA2+DMSO组SW579细胞的细胞活力增强，而OXPHOS通路抑制剂Gboxin能够逆转过表达HMGA2对细胞活力的影响（均P<0.05）。流式细胞术和CCK-8实验结果显示，与si-NC组（凋亡水平：6.19%±0.28%；顺铂IC50值：17.47 μmol/L）相比，敲低HMGA2能增加K1细胞的凋亡水平（11.96%±0.32%；t=19.17，P<0.001）和顺铂敏感性（IC50值：1.49 μmol/L）；与oe-NC组（凋亡水平：9.98%±0.32%；顺铂IC50值：8.17 μmol/L）相比，过表达HMGA2显著降低了SW579细胞凋亡水平（4.32%±0.25%；t=19.65，P<0.001）和顺铂敏感性（IC50值：34.95 μmol/L）。双荧光素酶报告实验结果发现，与si-NC组相比，在人肾上皮细胞293T细胞中敲低BHLHE40的表达显著降低野生型HMGA2的荧光素酶活性（0.31±0.02比1.00±0.11；t=10.69，P=0.004），但对于突变型HMGA2的荧光素酶活性没有显著性影响（1.06±0.11比1.00±0.07；t=0.80，P=0.470）。染色质免疫沉淀实验结果显示，与IgG组（1.00±0.10）相比，anti-BHLHE40组K1细胞的HMGA2 mRNA表达水平显著增加（6.57±0.62；t=15.36，P<0.001）。与oe-NC+DMSO组相比，oe-HMGA2+DMSO组SW579细胞凋亡水平（P<0.05）和顺铂敏感性均降低，OXPHOS复合体Ⅰ~Ⅴ表达显著增强，细胞耗氧率升高（均P<0.05），oe-HMGA2+Gboxin处理可逆转过表达HMGA2的影响（均P<0.05）。回复实验表明，与oe-NC+si-NC组相比，过表达BHLHE40后SW579细胞的细胞活力和OXPHOS复合体Ⅰ~Ⅴ的表达增强，细胞耗氧率和顺铂IC50值显著增加，细胞凋亡水平下降（均P<0.05）；而同时敲低HMGA2可逆转过表达BHLHE40的影响（均P<0.05）。结论 BHLHE40可通过靶向调控HMGA2的表达激活氧化磷酸化通路，进而影响甲状腺癌细胞对顺铂的敏感性。

关键词: HMGA2蛋白, 甲状腺肿瘤, 氧化磷酸化, 顺铂

Abstract:

Objective To explore whether BHLHE40 can affect the sensitivity of thyroid cancer （TC） cells to cisplatin by activating oxidative phosphorylation （OXPHOS） pathway by targeting high mobility group A2 （HMGA2）. Methods The mRNA expression of HMGA2 and its upstream transcription factor BHLHE40 in TC tissues was analyzed by TCGA-THCA and hTFtarget online databases. The si-HMGA2，oe-HMGA2，oe-BHLHE40，negative control si-NC and oe-NC were transfected into TC cells （K1 and SW579） by liposome transfection method. The mRNA expression levels of BHLHE40 and HMGA2 in TC cells （SW579，FTC-133，and K1） and normal thyroid cells （Nthy ori3-1） were detected by real-time quantitative PCR （qRT-PCR）. The cell viability was detected by MTT assay，the half inhibitory concentration （IC50） value of cisplatin was calculated by CCK-8 assay，the apoptosis level was detected by flow cytometry，and the expression of OXPHOS complex was detected by Western blotting. Seahorse XFe 96 was used to analyze the oxygen consumption rate of the TC cells. Dual-luciferase assay and chromatin immunoprecipitation （ChIP） assay were used to analyze the binding relationship between BHLHE40 and HMGA2. Results TCGA database results showed that the mRNA expression levels of HMGA2 and BHLHE40 in TC tissues （10.57±2.58，13.89±1.13） were higher than those in normal thyroid tissues （4.82±1.69，12.28±1.01），with statistically significant differences （t=16.69，P<0.001； t=10.43，P<0.001）. The results of qRT-PCR showed that the relative mRNA expression levels of HMGA2 in normal thyroid cells （Nthy ori3-1） and TC cells （SW579，FTC-133，and K1） were 1.00±0.13，2.94±0.23，4.71±0.41 and 6.29±0.49，while those of BHLHE40 were 1.00±0.12，2.60±0.23，3.39±0.35 and 6.18±0.51 respectively，both with statistically significant differences （F=130.50，P<0.001； F=125.20，P<0.001）. Further pairwise comparison showed that mRNA expression levels of HMGA2 and BHLHE40 in TC cells were significantly higher than those in normal thyroid cells （all P<0.001）. According to MTT experimental results，si-HMGA2 treatment significantly reduced the cell viability of K1 cells compared to the si-NC group （all P<0.05）. In addition，compared to the oe-NC group，oe-HMGA2 treatment significantly increased the cell viability of SW579 cells （all P<0.05）. Compared to the oe-NC+DMSO group，the oe-HMGA2+DMSO group showed enhanced cell viability of SW579 cells，while the OXPHOS pathway inhibitor Gboxin was able to reverse the effect of overexpressing HMGA2 on cell viability （all P<0.05）. The results of flow cytometry and CCK-8 experiments showed that compared to the si-NC group （apoptosis level： 6.19%±0.28%； cisplatin IC50 value： 17.47 μmol/L），knocking down HMGA2 could increase the apoptosis level （11.96%±0.32%； t=19.17，P<0.001） and cisplatin sensitivity （IC50 value： 1.49 μmol/L） of K1 cells. In addition，compared to the oe-NC group （apoptosis level： 9.98%±0.32%； cisplatin IC50 value： 8.17 μmol/L），overexpressing HMGA2 significantly decreased the apoptosis level （4.32%±0.25%； t=19.65，P<0.001） and cisplatin sensitivity （IC50 value： 34.95 μmol/L） of SW579 cells. The results of dual-luciferase assay showed that compared with the si-NC group，knocking down the expression of BHLHE40 in human kidney epithelial 293T cells significantly reduced the luciferase activity of wild-type HMGA2 （0.31±0.02 vs. 1.00±0.11； t=10.69，P=0.004）. However，there was no significant effect on the luciferase activity of mutant-type HMGA2 （1.06±0.11 vs. 1.00±0.07； t=0.80，P=0.470）. ChIP results showed that the mRNA expression level of HMGA2 in K1 cells was significantly increased in the anti-BHLHE40 group （6.57±0.62） compared with the IgG group （1.00±0.10； t=15.36，P<0.001）. Compared to the oe-NC+DMSO group，the oe-HMGA2+DMSO group showed decreased apoptosis level （P<0.05） and cisplatin sensitivity of SW579 cells，with a significant increase in the expression of OXPHOS complexes Ⅰ-Ⅴ and cellular oxygen consumption rates （all P<0.05）. The effect of overexpressing HMGA2 was reversed by treatment with oe-HMGA2+Gboxin （all P<0.05）. The recovery experiment showed that compared to the oe-NC+si-NC group，overexpression of BHLHE40 in SW579 cells increased cell viability and the expression of OXPHOS complexes Ⅰ-Ⅴ，while decreasing apoptosis levels and increasing cellular oxygen consumption rates and cisplatin IC50 values （all P<0.05）. However，simultaneous knockdown of HMGA2 reversed the effect of overexpressing BHLHE40 （all P<0.05）. Conclusion BHLHE40 can activate the OXPHOS pathway by targeting and regulating the expression of HMGA2，thereby affecting the sensitivity of TC cells to cisplatin.

Key words: HMGA2 protein, Thyroid neoplasms, Oxidative phosphorylation, Cisplatin