miR-217调控TIMP3的表达对喉癌细胞生物学行为的影响

Effects of miR-217 Regulation of TIMP3 expression on Biological Behavior of Laryngeal Carcinoma Cells

目的探讨miR-217调控金属肽酶抑制剂3(metallopeptidase inhibitor 3, TIMP3)的表达对喉癌细胞生物学行为的影响。方法选择喉癌并行喉部分切除术或喉全切除术的癌组织32例,同时取距离肿瘤2 cm的癌旁正常喉组织32例。选择人喉癌细胞株Hep2,并将miR-217 mimics、Control mimics转染至细胞,分别作为miR-217 mimics组与miR-217 NC组,再将TIMP3 3'-UTR野生型(Wt)质粒和TIMP3 3'-UTR突变型(Mut)质粒分别转染细胞。实时定量核酸扩增检测(qPCR)miR-217及TIMP3的表达水平,双荧光素酶实验检测验证miR-217与TIMP3在喉癌Hep2细胞株中的调控关系,MTT增殖实验检测细胞增殖数,Transwell检测细胞迁移、侵袭数,分析miR-217在喉癌组织中的表达与其临床特征的相关性。结果 qPCR检测结果显示,癌旁正常喉组织、喉癌组织的miR-217表达量分别为1.53±0.21、0.68±0.13,TIMP3表达量分别为1.18±0.15、0.31±0.08,比较差异有统计学意义(P<0.05)。双荧光素酶实验结果显示,miR-217 mimics+TIMP3 3'-UTR Wt组、miR-217 NC+TIMP3 3'-UTR Wt组、miR-217 mimics+TIMP3 3'-UTR Mut组、miR-217 NC+TIMP3 3'-UTR Mut组的荧光素酶活性分别为1.79±0.31、1.15±0.11、1.28±0.28、1.18±0.19,比较差异有统计学意义(t=12.09,P=0.019);miR-217 mimics+TIMP3 3'-UTR Wt组、miR-217 NC+TIMP3 3'-UTR Wt组双荧光素酶活性比较差异有统计学意义(P<0.05)。与miR-217 NC组比较,miR-217 mimics组喉癌细胞增殖数、迁移细胞数及侵袭细胞数降低,差异有统计学意义(P<0.05)。miR-217表达与喉癌的病理分期及淋巴结有无转移相关(P<0.05)。结论 miR-217调控TIMP3表达以抑制喉癌细胞的增殖、迁移和侵袭。

Objective To investigate the effect of miR-217 regulation of metallopeptidase inhibitor 3 (TIMP3) expression by on biological behavior of laryngeal carcinoma (LC) cells. Methods A total of 32 patients with LC who underwent partial or total laryngectomy were enrolled. Cancerous tissues and adjacent normal tissues 2 cm away from the tumor were collected from each patient. Human LC cell line Hep2 was selected, and miR-217 mimics and Control mimics were transfected into cells as miR-217 mimics group and miR-217 NC group, respectively, and TIMP3 3'-UTR wild type (Wt) plasmid and The TIMP3 3'-UTR mutant (Mut) plasmid was transfected into cells, respectively.Real-time Quantitative PCR (RT-qPCR) was used to detect the expression levels of miR-217 and TIMP3 in LC tissues. Dual luciferase reporter gene assay was used to verify the regulation of miR-217 and TIMP3 in LC cell line Hep2. The cell proliferation was detected by MTT proliferation assay, and the cell migration and invasion were detected by Transwell assay. In addition, the correlation between miR-217 expression in LC and its clinical features was analyzed. Results The results of qPCR assay showed that the expressions of miR-217 in adjacent normal tissues and LC tissues were 1.53±0.21 and 0.68±0.13, respectively, and the expression levels of TIMP3 were 1.18±0.15 and 0.31±0.08, respectively, suggesting significant differences ( P <0.05). The dual luciferase assay showed that luciferase activities in miR-217 mimics+TIMP3 3'-UTR Wt group, miR-217 NC+TIMP3 3'-UTR Wt group, miR-217 mimics+TIMP3 3'-UTR Mut group, and miR-217 NC+TIMP3 3'-UTR Mut group were 1.79±0.31, 1.15±0.11, 1.28±0.28 and 1.18±0.19, respectively. The difference was statistically significant ( t =12.09, P =0.019). The difference in the activity of the dual luciferase between the miR-217 mimics+TIMP3 3'-UTR Wt group and the miR-217 NC+TIMP3 3'-UTR Wt group was statistically significant ( P <0.05). Compared with the miR-217 NC group, the number of proliferative cells, migrant cells and invasive cells in the miR-217 mimics group was decreased, and the difference was statistically significant ( P <0.05). The expression of miR-217 was associated with pathological stage of LC and presence or absence of lymph node metastasis ( P <0.05). Conclusion miR-217 regulates the expression of TIMP3, thereby inhibiting the proliferation, migration and invasion of LC cells.