miRNA-381靶向HMGB1对IHH-4细胞生长侵袭及迁移的影响

目的探究微小RNA-381 (miR-381)调控高迁移率族蛋白B1 (HMGB1)对甲状腺乳头状瘤IHH-4细胞生长、侵袭及迁移的影响。方法购置甲状腺乳头状瘤IHH-4细胞及正常甲状腺细胞Nthy-ori-3各1株,取第3代细胞培养48 h后,采用实时定量PCR(qRT-PCR)检测miR-381和HMGB1在甲状腺乳头状瘤IHH-4细胞和正常甲状腺Nthy-ori-3细胞中的表达水平。利用Lipofectamine 2000转染miR-381 mimic后,检测IHH-4细胞中miR-381和HMGB1的表达水平。生物信息学预测miR-381和HMGB1的靶向关系,并用荧光素酶报告实验验证两者的靶向关系。每孔按2×105/m L细胞密度接种IHH-4细胞,分为IHH-4组(甲状腺乳头状瘤细胞IHH-4组)、miR-381 mimic组(miR-381 mimic转染组)、pc-HMGB1组(HMGB1过表达转染组)、mimic+pc-HMGB1组(miR-381 mimic和pc-HMGB1共同转染组),每组设3个复孔,采用CCK8法检测各组IHH-4细胞活性,Transwell及划痕实验分别检测IHH-4细胞侵袭及迁移能力,免疫印迹法检测IHH-4细胞中Ki67、基质金属蛋白酶-2(MMP-2)、MMP-9和晚期糖基化终产物受体(RAGE)的表达水平。结果与Nthy-ori-3细胞比较,IHH-4细胞中miR-381mRNA水平降低,HMGB1 mRNA水平升高,差异有统计学意义(P <0. 01)。与IHH-4组相比,miR-381 mimic组miR-381表达增加(P <0. 01)。miR-381和HMGB1存在靶向关系。与IHH-4组相比,miR-381 mimic组细胞增殖倍数、Ki67表达、侵袭细胞数、划痕闭合率、MMP-2、MMP-9和RAGE表达均降低(P均<0. 01),pc-HMGB1组上述指标均升高(P均<0. 01);与miR-381mimic组相比,mimic+pc-HMGB1组细胞增殖倍数、Ki67表达、侵袭细胞数、划痕闭合率、MMP-2、MMP-9和RAGE水平也明显上升(P均<0. 01)。结论 miR-381通过靶向下调HMGB1表达,抑制IHH-4细胞增殖、侵袭和迁移。

Effects of miR-381 on proliferation, invasion and migration of papillary thyroid carcinoma IHH-4 cells by targeting HMGB1

Objective To investigate the effect of miR-381 on proliferation, invasion and migration of papillary thyroid carcinoma cell IHH-4 via targeting high-mobility group box 1 protein (HMGB1). Methods Purchasing one of thyroid papilloma IHH-4 cells and normal thyroid Nthy-ori-3 cells, taking the third generation of cells and cultured 48 h, the mRNA level of miR-381 and HMGB1 in normal thyrocyte Nthy-ori-3, IHH-4 cell were determined by qRT-PCR; miR-381 and HMGB1 expression levels in IHH-4 cells were detected after transfection with Lipofectamine 2000. The targeted-relationship between miR-381 and HMGB1 was predicted by bioinformatics and conformed by luciferase reporter assay. After inoculating with 2×10⁵/mL cell density per well, IHH-4 cells were randomly divided into IHH-4 group (IHH-4 group of thyroid papilloma cells), miR-381 mimic group (miR-381 mimic transfection group), pc-HMGB1 group (HMGB1 overexpression transfection group), mimic+pc-HMGB1 group (miR-381 mimic and pc-HMGB1 co-transfection group), each group was established three wells. IHH-4 cell proliferation was determined by CCK8 assay, cell invasion and migration were determined by Transwell and wound healing assay respectively; expression level of Ki67, metalloproteinase-2 (MMP-2), MMP-9 and receptor for advanced glycation endproducts (RAGE) were determined by western blot. Results Compared with Nthy-ori-3 cells, the mRNA level of miR-381 in IHH-4 cells was decreased (P<0.01), but HMGB1 was increased (P<0.01). Compared with the IHH-4 group, the expression of miR-381 in the miR-381 mimic group was increased (P<0.01). There is a targeting relationship between miR-381 and HMGB1. Compared with the IHH-4 group, the proliferation folds, Ki67 expression, the number of invasive cells, scratch closure rate, the expressions of MMP-2, MMP-9 and RAGE in the miR-381 mimic group were all decreased (all P<0.01), while those in the pc-HMGB1 group were all increased significantly (all P<0.01). In addition, compared with miR-381 mimic group, the proliferation folds, Ki67 expression, the number of invasive cells, scratch closure rate, the expressions of MMP-2, MMP-9 and RAGE in mimic+pc-HMGB1 group were all increased significantly (all P<0.01). Conclusion MiR-381 inhibits cell proliferation, invasion and migration of IHH-4 cells by targeting down-regulation of HMGB1 experssion.