BRD4 inhibitor specifically inhibits the development of wild-type Kras differentiated thyroid carcinoma by regulating BRD4/miR-106b-5p/P21 axis

doi:10.3760/cma.j.cn371439-20200528-00089

Abstract

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

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.

Figures/Tables 18

References 24

[1]	陈爱民, 骆献阳. 分化型甲状腺癌侵犯喉气管食管临床分析[J]. 临床耳鼻咽喉头颈外科杂志, 2017, (10):802-803. DOI: 10.13201/j.issn.1001-1781.2017.10.016. doi: 10.13201/j.issn.1001-1781.2017.10.016
[2]	Qu R, Li J, Yang J, et al. Treatment of differentiated thyroid cancer: can endoscopic thyroidectomy via a chest-breast approach achieve similar therapeutic effects as open surgery?[J]. Surg Endosc, 2018,32(12):4749-4756. DOI: 10.1007/s00464-018-6221-1. doi: 10.1007/s00464-018-6221-1
[3]	De La Fouchardière C, Decaussin-Petrucci M, Berthiller J, et al. Predictive factors of outcome in poorly differentiated thyroid carcinomas[J]. Eur J Cancer, 2018,92:40-47. DOI: 10.1016/j.ejca.2017.12.027. doi: S0959-8049(18)30005-4 pmid: 29413688
[4]	Xu K, Chen D, Qian D, et al. AZD5153, a novel BRD4 inhibitor, suppresses human thyroid carcinoma cell growth in vitro and in vivo[J]. Biochem Biophys Res Commun, 2018,499(3):531-537. DOI: 10.1016/j.bbrc.2018.03.184. doi: 10.1016/j.bbrc.2018.03.184
[5]	Smeby J, Sveen A, Merok MA, et al. CMS-dependent prognostic impact of KRAS and BRAF^V600E mutations in primary colorectal cancer[J]. Ann Oncol, 2018,29(5):1227-1234. DOI: 10.1093/annonc/mdy085. doi: S0923-7534(19)34549-1 pmid: 29518181
[6]	Telechea-Fernández M, Rodríguez-Fernández L, García C, et al. New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis: effect of KRAS status[J]. Oncotarget, 2018,9(10):9100-9113. DOI: 10.18632/oncotarget.23888. doi: 10.18632/oncotarget.23888 pmid: 29507677
[7]	Duman BB, Kara OI, Uğuz A, et al. Evaluation of PTEN, PI3K, MTOR, and KRAS expression and their clinical and prognostic relevance to differentiated thyroid carcinoma[J]. Contemp Oncol (Pozn), 2014,18(4):234-240. DOI: 10.5114/wo.2014.43803. doi: 10.5114/wo.2014.43803
[8]	Dong X, Hu X, Chen J, et al. BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis[J]. Cell Death Dis, 2018,9(2):203. DOI: 10.1038/s41419-017-0181-6. doi: 10.1038/s41419-017-0181-6
[9]	Shi DM, Bian XY, Qin CD, et al. miR-106b-5p promotes stem cell-like properties of hepatocellular carcinoma cells by targeting PTEN via PI3K/Akt pathway[J]. Onco Targets Ther, 2018,11:571-585. DOI: 10.2147/ott.S152611. doi: 10.2147/ott.S152611
[10]	Wei K, Pan C, Yao G, et al. MiR-106b-5p promotes proliferation and inhibits apoptosis by regulating BTG3 in non-small cell lung cancer[J]. Cell Physiol Biochem, 2017,44(4):1545-1558. DOI: 10.1159/000485650. doi: 10.1159/000485650 pmid: 29197876
[11]	赵善民, 汤球, 刘志学, 等. K-Ras^G12D基因突变体慢病毒载体的构建及其鉴定[J]. 2014,14(2):223-225, 250. DOI: 10.13241/j.cnki.pmb.2014.02.006. doi: 10.13241/j.cnki.pmb.2014.02.006
[12]	Schlumberger M, Brose M, Elisei R, et al. Definition and management of radioactive iodine-refractory differentiated thyroid cancer[J]. Lancet Diabetes Endocrinol, 2014,2(5):356-358. DOI: 10.1016/s2213-8587(13)70215-8. doi: 10.1016/s2213-8587(13)70215-8
[13]	Riesco-Eizaguirre G, Galofré JC, Grande E, et al. Spanish consensus for the management of patients with advanced radioactive iodine refractory differentiated thyroid cancer[J]. Endocrinol Nutr, 2016,63(4):e17-e24. DOI: 10.1016/j.endonu.2015.08.007. doi: 10.1016/j.endonu.2015.08.007
[14]	Hedayati M, Zarif Yeganeh M, Sheikholeslami S, et al. Diversity of mutations in the RET proto-oncogene and its oncogenic mechanism in medullary thyroid cancer[J]. Crit Rev Clin Lab Sci, 2016,53(4):217-227. DOI: 10.3109/10408363.2015.1129529. doi: 10.3109/10408363.2015.1129529
[15]	Rossi M, Buratto M, Tagliati F, et al. Relevance of BRAF(V600E) mutation testing versus RAS point mutations and RET/PTC rearrangements evaluation in the diagnosis of thyroid cancer[J]. Thyroid, 2015,25(2):221-228. DOI: 10.1089/thy.2014.0338. doi: 10.1089/thy.2014.0338
[16]	B Byeon HK, Na HJ, Yang YJ, et al. c-Met-mediated reactivation of PI3K/AKT signaling contributes to insensitivity of BRAF(V600E) mutant thyroid cancer to BRAF inhibition[J]. Mol Carcinog, 2016,55(11):1678-1687. DOI: 10.1002/mc.22418. doi: 10.1002/mc.22418
[17]	Zhu X, Zhao L, Park JW, et al. Synergistic signaling of KRAS and thyroid hormone receptor β mutants promotes undifferentiated thyroid cancer through MYC up-regulation[J]. Neoplasia, 2014,16(9):757-769. DOI: 10.1016/j.neo.2014.08.003. doi: 10.1016/j.neo.2014.08.003
[18]	Wang ZD, Wei SQ, Wang QY. Targeting oncogenic KRAS in non-small cell lung cancer cells by phenformin inhibits growth and angiogenesis[J]. Am J Cancer Res, 2015,5(11):3339-3349.
[19]	Wong CC, Qian Y, Li X, et al. SLC25A22 Promotes proliferation and survival of colorectal cancer cells with KRAS mutations and xenograft tumor progression in mice via intracellular synjournal of aspartate[J]. Gastroenterology, 2016,151(5): 945-960.e6. DOI: 10.1053/j.gastro.2016.07.011. doi: 10.1053/j.gastro.2016.07.011
[20]	Yang L, Zhou Y, Li Y, et al. Mutations of p53 and KRAS activate NF-κB to promote chemoresistance and tumorigenesis via dysregulation of cell cycle and suppression of apoptosis in lung cancer cells[J]. Cancer Lett, 2015,357(2):520-526. DOI: 10.1016/j.canlet.2014.12.003. doi: 10.1016/j.canlet.2014.12.003
[21]	Deneka AY, Haber L, Kopp MC, et al. Tumor-targeted SN38 inhi-bits growth of early stage non-small cell lung cancer (NSCLC) in a KRas/p53 transgenic mouse model[J]. PLoS One, 2017,12(4):e0176747. DOI: 10.1371/journal.pone.0176747. doi: 10.1371/journal.pone.0176747
[22]	Kim H, Hwang H, Lee H, et al. L1 cell adhesion molecule promotes migration and invasion via JNK activation in extrahepatic cholangiocarcinoma cells with activating KRAS mutation[J]. Mol Cells, 2017,40(5):363-370. DOI: 10.14348/molcells.2017.2282. doi: 10.14348/molcells.2017.2282
[23]	Du L, Kim JJ, Shen J, et al. KRAS and TP53 mutations in inflammatory bowel disease-associated colorectal cancer: a meta-analysis[J]. Oncotarget, 2017,8(13):22175-22186. DOI: 10.18632/oncotarget.14549. doi: 10.18632/oncotarget.14549
[24]	Román M, Baraibar I, López I, et al. KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target[J]. Mol Cancer, 2018,17(1):33. DOI: 10.1186/s12943-018-0789-x. doi: 10.1186/s12943-018-0789-x

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'

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

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

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

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