BRAF激活的长链非编码RNA介导促甲状腺激素受体基因甲基化促进甲状腺乳头状癌细胞的增殖和迁移

BRAF-activated non-coding RNA mediated thyroid-stimulating hormone receptor gene methylation promotes the proliferation and migration of thyroid papillary carcinoma

目的探讨BRAF激活的长链非编码RNA(BANCR)调控甲状腺乳头状癌(PTC)促甲状腺激素受体(TSHR)基因甲基化及其表达的作用及其机制。方法应用实时定量荧光聚合酶链反应(qRT-PCR)法检测PTC细胞中BNACR表达;应用慢病毒载体构建ihBANCR、绿色荧光蛋白(GFP)和isBANCR的PTC细胞株并应用甲基化特异性聚合酶链式反应(MSP)法检测BANCR对TSHR甲基化水平的影响;通过细胞计数试剂盒(CCK-8)细胞增殖和细胞划痕实验探讨BANCR对PTC细胞增殖和迁移能力的影响;使用肿瘤基因组图谱(TCGA)数据库分析PTC数据集中TSHR甲基化相关酶,并通过蛋白质印迹法(Western blot)实验进行验证。组间比较采用t检验,χ^(2)检验及方差分析。结果BANCR在PTC细胞系(TPC-1和IHH-4)中高表达(F=7.410,P<0.05);BANCR可显著上调THSR基因甲基化水平(t=8.072,P<0.05);ihBANCR组的PTC细胞较GFP组其增殖能力(232.7±107.7比165.8±68.6,F=11.100,P<0.05)和迁移能力(0.058±0.041比0.307±0.033,t=4.669,P<0.05)显著增强,进一步应用甲基化酶抑制剂后可削弱上述结果;ihBANCR组的PTC细胞株中TSHR的表达水平较GFP组显著降低(2.095±0.045比3.375±0.191,F=51.540,P<0.05),进一步应用甲基化酶抑制剂后可减弱BANCR对TSHR表达的抑制作用(0.646±0.149比1.564±0.493,F=51.540,P<0.05)。生物信息学分析显示PTC中TSHR表达减低与TSHR基因甲基化水平(r=-0.351,P<0.05)及甲基化酶EZH2(r=-0.313,P<0.05)和DNMT1(r=-0.224,P<0.05)表达呈负相关,进一步上调BANCR后可显著增加甲基化酶EZH2(0.689±0.099比1.326±0.221,t=2.634,P<0.05)和DNMT1(0.617±0.068比1.427±0.066,t=8.500,P<0.05)。结论BANCR在PTC细胞中表达上调,并可通过TSHR基因甲基化促进PTC的增殖和迁移。

Objective To investigate the expression of BRAF-activated non-coding RNA(BANCR)in thyroid papillary carcinoma(PTC)cells,and to explore the mechanism of BANCR regulating the expression of thyroid stimulating hormone receptor(TSHR)and its promoter methylation.Methods The difference of BNACR expression levels between three human PTC cells and human normal thyroid cell line was detected by qRT-PCR.The cell lines of ihBANCR and green fluorescent protein(GFP)were constructed with lentiviral vectors in the cell line of TPC-1,and methylation-specific polymemse chain reaction(MSP)was used to detect the effect of BANCR on TSHR methylation levels.The effects of BANCR on the proliferation and migration capacity of TPC cell lines were investigated by cell counting kit-8(CCK-8)method and scratch experiments respectively.Finally,the correlation between TSHR expression and TSHR gene methylation and its associated enzymes in tumor tissue of PTC data set was compared and analyzed by The Cancer Genome Atlas(TCGA)database,and the Western blotting method was used in TPC-1 cell line.Results BANCR was significantly over expressed in PTC cell lines.BANCR can significantly increase the level of THSR gene methylation;The proliferation and migration of PTC cells with high expression of BANCR were significantly enhanced,and further application of methylase inhibitor could reduce the above effects of BANCR.Up-regulating BANCR significantly reduced the expression of TSHR and increased the expression of methylase.Conclusion BANCR is up-regulated in PTC cells and can increase the methylation level of TSHR gene by up-regulation of methylase,resulting in decreased expression of TSHR,thus promoting the proliferation and migration of PTC cells.