miR-765通过Wnt/β-catenin信号通路调控甲状腺乳头状癌细胞生物学行为的研究

miR-765 regulates proliferation,migration,and invasion of papillary thyroid carcinoma cells via Wnt/β-catenin signalingpathwayyLi

目的探究miR-765在甲状腺乳头状癌(papillarythyroidcarcinoma,PTC)细胞中的作用及相关信号通路机制。方法利用qPCR检测miR-765在正常人甲状腺细胞系Nthy-ori3-1及人PTC细胞系B-CPAP和TPC-1中的表达。将PTC细胞分成空白对照组(blank control,BC)、阴性对照组(negative control,NC)和miR-mimic组,BC组不做特殊处理,NC组转染阴性对照序列,miRNA模拟物(miR-mimic)组转染miR-765模拟物上调其表达。分别利用CCK-8实验、平板克隆形成实验、划痕实验和Transwell侵实验检测转染miR-mimic后PTC细胞的增殖、迁移及侵袭能力。利用Westernblot实验检测转染miR-mimic后PTC细胞中Wnt/β-catenin信号通路关键蛋白β-catenin的核内表达。结果与Nthy-ori 3-1细胞相比,PTC细胞中的miR-765表达显著下降(B-CPAP,P=0.0003;TPC-1,P=0.0003)。转染miR-mimic可显著上调PTC细胞中miR-765的表达(B-CPAP,P<0.0001;TPC-1,P<0.0001)。CCK-8实验(B-CPAP,P<0.05;TPC-1,P<0.05)、平板克隆形成实验(B-CPAP,P=0.0001;TPC-1,P<0.0001)、划痕实验(B-CPAP,P=0.0006;TPC-1,P<0.0001)及Tran-Swell侵袭实验(B-CPAP,P=0.001;TPC-1,P=0.0014)结果显示,上调miR-765的表达可显著抑制PTC细胞的增殖、迁移和侵袭能力。Western实验结果显示上调miR-765的表达可显著抑制PTC细胞中Wnt/β-catenin信号通路的水平(B-CPAP,P=0.0039;TPC-1,P=0.0004)。结论上调miR-765可抑制Wnt/β-catenin信号通路并抑制PTC细胞的增殖、迁移和侵袭能力,这不仅说明miR-765是PTC治疗的潜在新靶点,还进一步揭示了其发挥调控作用的机制。

Objective To investigate the role of miR-765 in papillary thyroid carcinoma(PTC)cells and further uncover the associated signaling mechanism.Methods qPCR was used to assess miR-765 expression in normal human thyroid cell line(Nthy-ori 3-1)and human PTC cell lines(B-CPAP and TPC-1).PTC cells were di-vided into blank control group(BC)without special treatment,negative control group(NC)that was transfected with negative control sequences,and miR-mimic group that was transfected with miR-mimic.Transfection with miR-mimic was used to up-regulate the expression of miR-765 in PTC cells.CCK-8,plate colony formation,wound-healing,and Transwell invasion assays were used to assess the proliferation,migration,and invasion of PTC cells,respectively.Western blot assay was used to assess the level of nuclearβ-catenin,the key protein of the Wnt/β-catenin pathway,in PTC cells.Results The level of miR-765 expression of PTC cells was significantly lower than that of Nthy-ori 3-1 cells(B-CPAP,P=0.0003;TPC-1,P=0.0003).Transfection with miR-mimic significantly up-regulated miR-765 expression in PTC cells(B-CPAP,P<0.0001;TPC-1,P<0.0001).Results of CCK-8 assay(B-CPAP,P<0.05;TPC-1,P<0.05),plate colony formation assay(B-CPAP,P=0.0001;TPC-1,P<0.0001),wound-healing assay,and Transwell invasion assay(B-CPAP,P=0.001;TPC-1,P=0.0014)showed that up-regulating the expression of miR-765 significantly inhibited the proliferation,migration,and invasion of PTC cells.Western blot results showed that up-regulating the expression of miR-765 significantly reduced nuclearβ-catenin(B-CPAP,P=0.0039;TPC-1,P=0.0004).Conclusion up-regulating the expression of miR-765 inhibits the proliferation,migration,and invasion of PTC cells and the Wnt/β-catenin signaling pathway,which not only proposes miR-765 as a novel potential therapeutic target for PTC,but also further revealed the associated mechanism.