Bhlhe40 protects cochlear hair cell-like HEI-OC1 cells against H_(2)O_(2) ‑triggered oxidative injury

Background:Cochlear hair cell injury is a common pathological feature of hearing loss.The basic helix-loop-helix family,member e40(Bhlhe40),a gene belonging to the basic helix-loop-helix(bHLH)family,exhibits strong transcriptional repression activity.Methods:Oxidative damage,in House Ear Institute-Organ of Corti 1(HEI-OC1)cells,was caused using hydrogen peroxide(H2O2).The Ad-Bhlhe40 particles were constructed to overexpress Bhlhe40 in HEI-OC1 cells.Various assays including cell counting kit-8(CCK-8),terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay(TUNEL),flow cytometry,immunofluorescence,and corresponding commercial kits were employed to investigate the impacts of Bhlhe40 on cell viability,apoptosis,oxidative stress levels,mitochondrial membrane potential and cellular senescence.Additionally,a dual-luciferase reporter assay was performed to confirm the targeting of the histone deacetylases 2(Hdac2)by Bhlhe40.Results:The results revealed that Bhlhe40 was downregulated in H_(2)O_(2)-treated HEI-OC1 cells,but its overexpression improved cell viability and mitigated H_(2)O_(2)-induced oxidative injury in HEI-OC1 cells with increase of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx)activities and decrease of reactive oxygen species(ROS)levels.Besides,overexpression of Bhlhe40 suppressed H_(2)O_(2)-triggered cell senescence,as evidenced by the fact that the upregulation of P53,P21,and P16 in HEI-OC1 cells treated with H2O2 were all alleviated by Bhlhe40 overexpression.And we further verified that overexpression of Bhlhe40 could inhibit the expression of Hdac2,which may be related to the repression of Hdac2 transcription.Conclusion:This study suggests that Bhlhe40 plays a protective role against senescence and oxidative damage in cochlear hair cells exposed to H2O2.

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

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:17.47μmol/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:1.49μmol/L);与oe-NC组(凋亡水平:9.98%±0.32%;顺铂IC50值:8.17μmol/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:34.95μmol/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。