[miR-497通过靶向CDK6抑制喉鳞状细胞癌生长的机制研究]

[Study on the mechanism of miR-497-induced laryngeal squamous cell carcinoma growth inhibition by targeting CDK6].

作者信息

Niu J T, Liu S G, Yan P, Li C

机构信息

Department of Otorhinolaryngology Head and Neck Surgery, Second Hospital, Tianjin Medical University, Tianjin 300221, China.

Tianjin Medical University, Tianjin 300000, China.

出版信息

Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2019 Feb 7;54(2):126-132. doi: 10.3760/cma.j.issn.1673-0860.2019.02.006.

DOI:10.3760/cma.j.issn.1673-0860.2019.02.006

PMID:30776865

Abstract

To study the effects of miR-497 and CDK6 on the growth of laryngeal squamous cell carcinoma (LSCC). The expressions of CDK6 mRNA in fresh LSCC specimens, the adjacent normal mucosa of LSCC, and cell lines of LSCC were detected with quantitative real time polymerase chain reaction, pcDNA3.1(+) CDK6 plasmids were respectively transfected into the LSCC cells, and MTT assay and clone formation assay were performed to evaluate the growth of LSCC cells. Flow cytometry was employed for cell cycle analysis. SPSS17.0 software was used to analyze the data. CDK6 was highly expressed in LSCC=14.01, =0.009) and the overall survival rate of the patients with high CDK6 expression was less than that with low CDK6 expression, with a significant difference (=3.236, <0.001). Double luciferase reporter gene analysis showed that fluorescence activity in wild type CDK6 group was significantly different from that in control group (<0.01), while there was no significant difference in the fluorescence activity between mutant CDK6 group and control group (>0.05). (490) values were respectively 0.42±0.14 (Mean±SD) in siRNA Hep-2 group, 0.51±0.13 in siRNA TU-212 group; 0.98±0.16 in control Hep-2 group and 1.17±0.20 in control TU-212 group. Colonies were 55±4 in siRNA Hep-2 group, 51±3 in siRNA TU-212 group, 108±6 in control Hep-2 group and 105±7 in control TU-212 group, namely, cell growth and clone formation ability in CDK6 siRNA group were significantly lower than those in the control group. Cells cycle was blocked in G0/G1 phase (G0/G1: 65.20%±10.12% in siRNA Hep-2 group; 63.42%±8.97% in siRNA TU-212 group; 45.31%±7.55% in control Hep-2 group; and 42.37%±7.28% in control TU-212 group), and cells decreased obviously in S phase (S: 25.39%±5.51% in siRNA Hep-2 group; 27.21%±5.43% in siRNA TU-212 group; 42.87%±6.85% in control Hep-2 group; and 44.76%±7.02% in control TU-212 group). Compared with miR-497 group, cell growth and clone formation ability in miR-497/CDK6 group were partly restored (all <0.05). CDK6 expression in LSCC is upregulated, functioning as an oncogene. High expression of CDK6 is a predictor for poor prognosis. miR-497, functioning as a tumor suppressor gene, inhibits the growth of LSCC by targeting CDK6.

摘要

研究miR - 497和CDK6对喉鳞状细胞癌（LSCC）生长的影响。采用实时定量聚合酶链反应检测新鲜LSCC标本、LSCC癌旁正常黏膜及LSCC细胞系中CDK6 mRNA的表达，将pcDNA3.1(+) - CDK6质粒分别转染至LSCC细胞中，采用MTT法和克隆形成试验评估LSCC细胞的生长情况。采用流式细胞术进行细胞周期分析。使用SPSS17.0软件分析数据。CDK6在LSCC中高表达（t = 14.01，P = 0.009），CDK6高表达患者的总生存率低于低表达患者，差异有统计学意义（t = 3.236，P < 0.001）。双荧光素酶报告基因分析显示，野生型CDK6组荧光活性与对照组相比差异有统计学意义（P < 0.01），而突变型CDK6组与对照组荧光活性差异无统计学意义（P > 0.05）。siRNA - Hep - 2组（490）值分别为0.42±0.14（均数±标准差），siRNA - TU - 212组为0.51±0.13；对照Hep - 2组为0.98±0.16，对照TU - 212组为1.17±0.20。siRNA - Hep - 2组克隆数为55±4，siRNA - TU - 212组为51±3，对照Hep - 2组为108±6，对照TU - 212组为105±7，即CDK6 siRNA组细胞生长和克隆形成能力明显低于对照组。细胞周期阻滞于G0/G1期（G0/G1：siRNA - Hep - 2组为65.20%±10.12%；siRNA - TU - 212组为63.42%±8.97%；对照Hep - 2组为45.31%±7.55%；对照TU - 212组为42.37%±7.28%），S期细胞明显减少（S：siRNA - Hep - 2组为25.39%±5.51%；siRNA - TU - 212组为27.21%±5.43%；对照Hep - 2组为42.87%±6.85%；对照TU - 212组为44.76%±7.02%）。与miR - 497组相比，miR - 497/CDK6组细胞生长和克隆形成能力部分恢复（均P < 0.05）。CDK6在LSCC中表达上调，起癌基因作用。CDK6高表达是预后不良的预测指标。miR - 497作为抑癌基因，通过靶向CDK6抑制LSCC的生长。