BRD4抑制剂通过BRD4/miR-106b-5p/P21分子轴特异性抑制野生型Kras分化型甲状腺癌发展

摘要/Abstract

摘要：

目的探讨含溴结合域蛋白4(BRD4)抑制剂对野生型Kras分化型甲状腺癌(DTC)的影响及其机制。方法选取DTC细胞株Kras^WTTPC-1,构建基因突变型Kras^G12DTPC-1细胞。采用CCK-8法检测BRD4抑制剂JQ-1对Kras^WTTPC-1细胞增殖活力的影响。用0.2 μmol/L JQ-1处理Kras^WTTPC-1细胞(JQ-1组),另设阴性对照(NC)组,分别采用Transwell侵袭实验、流式细胞术检测JQ-1对Kras^WTTPC-1细胞侵袭和凋亡的影响;检测JQ-1对BRD4、miR-106b-5p、P21表达的影响,以及P21抑制剂UC2288对P21、BRD4表达的影响。将Kras^WTTPC-1细胞分为JQ-1+NC-OE组、JQ-1+p21-OE组(过表达p21)及JQ-1+p21-OE+miR-106b-5p mimic组(同时过表达p21和miR-106b-5),检测各组细胞增殖、侵袭及凋亡情况。将TPC-1细胞分为Kras^WT组、Kras^WT+JQ-1组、Kras^G12D组及Kras^G12D+JQ-1组,检测各组细胞增殖、侵袭及凋亡情况。结果JQ-1以剂量和时间依赖方式抑制Kras^WTTPC-1细胞的增殖活力。在NC组和JQ-1组中,细胞侵袭数分别为124.67±9.61、82.67±8.02,细胞凋亡率分别为(5.91±0.34)%、(10.33±1.10)%,差异均具有统计学意义(t=5.812,P=0.004;t=6.653,P=0.003)。JQ-1显著抑制Kras^WTTPC-1细胞中BRD4及miR-106b-5p的表达并促进P21的表达。UC2288显著抑制P21表达,但对BRD4表达无显著影响。JQ-1+NC-OE组、JQ-1+p21-OE组及JQ-1+p21-OE+miR-106b-5p mimic组中,Kras^WTTPC-1细胞24 h增殖活力分别为0.46±0.03、0.35±0.04、0.44±0.03(F=8.720,P=0.017),JQ-1+p21-OE组较JQ-1+NC-OE组显著降低(P<0.05);3组细胞侵袭数分别为83.00±9.17、56.67±6.03、79.67±10.07(F=8.347,P=0.018),JQ-1+p21-OE组较JQ-1+NC-OE组显著减少(P=0.009);3组细胞凋亡率分别为(10.00±0.49)%、(15.39±1.14)%、(10.32±0.80)%(F=37.764,P<0.001),JQ-1+p21-OE组较JQ-1+NC-OE组显著增加(P<0.001);JQ-1+p21-OE+miR-106b-5p mimic组与JQ-1+NC-OE相比,细胞增殖活力、侵袭数及凋亡率差异均无统计学意义(均P>0.05)。在Kras^WT组、Kras^WT+JQ-1组、Kras^G12D组及Kras^G12D+JQ-1组中,24 h细胞增殖活力分别为0.50±0.05、0.39±0.04、0.68±0.08、0.64±0.05(F=17.776,P<0.001),与Kras^WT组相比,Kras^WT+JQ-1组增殖活力显著降低,Kras^G12D组增殖活力显著升高(均P<0.05);各组细胞侵袭数分别为129.33±11.50、86.00±9.54、161.67±13.01、146.33±13.20(F=22.598,P<0.001),与Kras^WT组相比,Kras^WT+JQ-1组侵袭数显著降低(P=0.002),Kras^G12D组侵袭数显著增加(P=0.010);各组细胞凋亡率分别为(6.17±0.50)%、(10.42±0.73)%、(3.43±0.47)%、(3.41±0.32)%(F=119.170,P<0.001),与Kras^WT组相比,Kras^WT+JQ-1组中细胞凋亡率显著增加(P<0.001),Kras^G12D组凋亡率显著降低(P<0.001);Kras^G12D+JQ-1组细胞增殖活力、侵袭数及凋亡率与Kras^G12D组相比差异均无统计学意义(均P>0.05)。结论BRD4抑制剂能够通过调节BRD4/miR-106b-5p/P21分子轴,特异性抑制Kras野生型DTC的发展,而对Kras突变型DTC肿瘤细胞的增殖、侵袭和凋亡无显著影响。

关键词:甲状腺肿瘤,BRD4抑制剂,miR-106b-5p,P21

Abstract:

ObjectiveTo explore the influence of bromodomain-containing protein 4 (BRD4) inhibitor on wild-type Kras differentiated thyroid carcinoma (DTC) and its mechanism.MethodsThe DTC cell line Kras^WTTPC-1 was selected and the mutant Kras^G12DTPC-1 cells were constructed. CCK-8 assay was used to detect the effect of BRD4 inhibitor JQ-1 on the proliferation activity of Kras^WTTPC-1 cells. Kras^WTTPC-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^WTTPC-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^WTTPC-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^WTgroup, Kras^WT+JQ-1 group, Kras^G12Dgroup and Kras^G12D+JQ-1 group, and the cell proliferation, invasion and apoptosis of each group were detected.ResultsJQ-1 inhibited the proliferation activity of Kras^WTTPC-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^WTTPC-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^WTTPC-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 (allP>0.05). In Kras^WTgroup, Kras^WT+JQ-1 group, Kras^G12Dgroup 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^WTgroup, cell proliferation activity in the Kras^WT+JQ-1 group was significantly decreased, while that in the Kras^G12Dgroup was significantly increased (bothP<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^WTgroup, the number of cell invasion in the Kras^WT+JQ-1 group was significantly decreased (P=0.002), and that in the Kras^G12Dgroup 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^WTgroup, the apoptosis rate in the Kras^WT+JQ-1 group was significantly increased (P<0.001), and that in the Kras^G12Dgroup 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^G12Dgroup (allP>0.05).ConclusionBRD4 inhibitor can specifically inhibit the development of wild-type Kras DTCviaregulating 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

冯志平, 杨传周, 陈婷, 朱家伦, 刘超, 吕娟, 陆建梅, 邓智勇. BRD4抑制剂通过BRD4/miR-106b-5p/P21分子轴特异性抑制野生型Kras分化型甲状腺癌发展[J]. 国际肿瘤学杂志, 2021, 48(8): 463-472.

Feng Zhiping, Yang Chuanzhou, Chen Ting, Zhu Jialun, Liu Chao, Lyu Juan, Lu Jianmei, Deng Zhiyong. BRD4 inhibitor specifically inhibits the development of wild-type Kras differentiated thyroid carcinoma by regulating BRD4/miR-106b-5p/P21 axis[J]. Journal of International Oncology, 2021, 48(8): 463-472.

图/表18

基因	引物
miR-106b-5p	正向:5'-TTCAGATCCCAGCGGTGCCTCT-3'
	反向:5'-AGGCACCGCTGGGATCTGAATT-3'
U6	正向:5'-CTCGCTTCGGCAGCACA-3'
	反向:5'-AACGCTTCACGAATTTGCGT-3'

表1

JQ-1 (mol/L)	0 h	24 h	48 h	72 h
0	0.28±0.03	0.52±0.04	0.84±0.07	1.00±0.09
0.2	0.25±0.04	0.42±0.04^a	0.61±0.05^a	0.72±0.07^a
1.0	0.26±0.03	0.38±0.05^b	0.53±0.06^b	0.66±0.05^b
5.0	0.25±0.03	0.30±0.05	0.38±0.05^c	0.56±0.05^c
F值	-	12.817	39.122	30.786
P值	-	0.002	<0.001	<0.001

表2

图1

图2

组别	caspase-3	Bax	Bcl-2	E-cadherin	N-cadherin	vimentin
NC组	0.99±0.10	0.97±0.06	1.00±0.09	1.00±0.09	0.99±0.10	1.00±0.11
JQ-1组	1.59±0.12	1.64±0.14	0.54±0.07	1.75±0.14	0.57±0.06	0.55±0.07
t值	6.813	7.691	7.328	8.140	6.321	6.144
P值	0.002	0.002	0.002	0.001	0.003	0.004

表3

图3

图4

图5

图6

组别	0 h	24 h	48 h	72 h
JQ-1+NC-OE组	0.23±0.03	0.46±0.03	0.62±0.04	0.70±0.05
JQ-1+p21-OE组	0.26±0.05	0.35±0.04^a	0.43±0.04^a	0.52±0.06^a
JQ-1+p21-OE+miR-106b-5p mimic组	0.26±0.03	0.44±0.03	0.58±0.04	0.67±0.04
F值	-	8.720	20.585	11.866
P值	-	0.017	0.002	0.008

表4

图7

图8

图9

组别	0 h	24 h	48 h	72 h
Kras^WT组	0.28±0.03	0.50±0.05	0.85±0.07	0.98±0.10
Kras^WT+JQ-1组	0.26±0.03	0.39±0.04^a	0.56±0.06^a	0.70±0.07^a
Kras^G12D组	0.27±0.03	0.68±0.08^a	1.00±0.08^a	1.33±0.09^a
Kras^G12D+JQ-1组	0.25±0.04	0.64±0.05	0.96±0.05	1.24±0.09
F值	-	17.776	25.101	32.655
P值	-	<0.001	<0.001	<0.001

表5

图10

图11

组别	caspase-3	Bax	Bcl-2	E-cadherin	N-cadherin	vimentin
Kras^WT组	1.00±0.11	1.00±0.09	1.01±0.10	0.99±0.10	1.01±0.09	1.01±0.10
Kras^WT+JQ-1组	1.80±0.15^a	1.75±0.14^a	0.62±0.05^a	1.51±0.14^a	0.58±0.07^a	0.52±0.07^a
Kras^G12D组	0.77±0.08^a	0.72±0.07^a	1.54±1.10^a	0.51±0.06^a	1.45±0.13^a	1.78±0.13^a
Kras^G12D+JQ-1组	0.91±0.08^b	0.84±0.09^b	1.41±0.13^b	0.58±0.06^b	1.33±0.12^b	1.65±0.13^b
F值	64.409	54.747	75.043	41.122	87.905	54.249
P值	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

表6

图12

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