关键词: 甲状腺肿瘤, BRD4抑制剂, miR-106b-5p, P21

Abstract:

Objective To explore the influence of bromodomain-containing protein 4 (BRD4) inhibitor on wild-type Kras differentiated thyroid carcinoma (DTC) and its mechanism.Methods The DTC cell line Kras^WT TPC-1 was selected and the mutant Kras^G12D TPC-1 cells were constructed. CCK-8 assay was used to detect the effect of BRD4 inhibitor JQ-1 on the proliferation activity of Kras^WT TPC-1 cells. Kras^WT TPC-1 cells were treated with 0.2 μmol/L JQ-1 (JQ-1 group), and a negative control group (NC group) was set. Transwell invasion assay and flow cytometry were used to detect the effect of JQ-1 on the invasion and apoptosis of Kras ^WT TPC-1 cells. The effect of JQ-1 on the expressions of BRD4, miR-106b-5p and P21, and the effect of P21 inhibitor UC2288 on the expressions of P21 and BRD4 were detected. Kras^WT TPC-1 cells were divided into JQ-1+NC-OE group, JQ-1+p21-OE group (overexpression of p21) and JQ-1+p21-OE+miR-106b-5p mimic group (overexpression of p21 and miR-106b-5 at the same time), and the proliferation, invasion and apoptosis of cells in each group were detected. TPC-1 cells were divided into Kras^WT group, Kras^WT+JQ-1 group, Kras^G12D group and Kras^G12D+JQ-1 group, and the cell proliferation, invasion and apoptosis of each group were detected. Results JQ-1 inhibited the proliferation activity of Kras^WT TPC-1 cells in a dose-dependent and time-dependent manner. In the NC group and JQ-1 group, the numbers of cell invasion were 124.67±9.61 and 82.67±8.02, and the apoptosis rates were (5.91±0.34)% and (10.33±1.10)%, respectively, with statistically significant differences (t=5.812, P=0.004; t=6.653, P=0.003). JQ-1 significantly inhibited the expressions of BRD4 and miR-106b-5p, and promoted the expression of P21 in Kras^WT TPC-1 cells. UC2288 significantly inhibited P21 expression, but had no significant effect on BRD4 expression. In the JQ-1+NC-OE group, JQ-1+p21-OE group and JQ-1+p21-OE+miR-106b-5p mimic group, the proliferation activities at 24 h of Kras^WT TPC-1 cells was 0.46±0.03, 0.35±0.04 and 0.44±0.03 (F=8.720, P=0.017), and the proliferation activity of JQ-1+p21-OE group was significantly lower than that of the JQ-1+NC-OE group (P<0.05). The numbers of cell invasion in the three groups were 83.00±9.17, 56.67±6.03 and 79.67±10.07 (F=8.347, P=0.018), and the number of cell invasion in the JQ-1+p21-OE group was significantly lower than that in the JQ-1+NC-OE group (P=0.009). The apoptosis rates of the three groups were (10.00±0.49)%, (15.39±1.14)% and (10.32±0.80)% (F=37.764, P<0.001), and the apoptosis rate of the JQ-1+p21-OE group was significantly higher than that in the JQ-1+NC-OE group (P<0.001). There were no significant differences in cell proliferation activity, invasion number and apoptosis rate between JQ-1+p21-OE+miR-106b-5p mimic group and JQ-1+NC-OE group (all P>0.05). In Kras^WT group, Kras^WT+JQ-1 group, Kras^G12D group and Kras^G12D+JQ-1 group, the cell proliferation activities at 24 h were 0.50±0.05, 0.39±0.04, 0.68±0.08 and 0.64±0.05 (F=17.776, P<0.001). Compared with the Kras^WT group, cell proliferation activity in the Kras^WT+JQ-1 group was significantly decreased, while that in the Kras^G12D group was significantly increased (both P<0.05). The numbers of cell invasion in the four groups were 129.33±11.50, 86.00±9.54, 161.67±13.01 and 146.33±13.20 (F=22.598, P<0.001). Compared with the Kras ^WT group, the number of cell invasion in the Kras^WT+JQ-1 group was significantly decreased (P=0.002), and that in the Kras^G12D group was significantly increased (P=0.010). The apoptosis rates in the four groups were (6.17±0.50)%, (10.42±0.73)%, (3.43±0.47)% and (3.41±0.32)% (F=119.170, P<0.001). Compared with the Kras^WT group, the apoptosis rate in the Kras^WT+JQ-1 group was significantly increased (P<0.001), and that in the Kras^G12D group was significantly decreased (P<0.001). There were no significant differences in cell proliferation activity, invasion number and apoptosis rate between Kras^G12D+JQ-1 group and Kras^G12D group (all P>0.05). Conclusion BRD4 inhibitor can specifically inhibit the development of wild-type Kras DTC via regulating the molecular axis of BRD4/miR-106b-5p/P21, but has no significant effect on the proliferation, invasion and apoptosis of mutant Kras DTC tumor cells.

Key words: Thyroid neoplasms, BRD4 inhibitor, miR-106b-5p, P21