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