miR -150促进鼻咽癌细胞增殖和侵袭的机制研究

摘要

目的：探讨miR -150是否通过靶向调控程序性细胞死亡因子4（PDCD4）促进鼻咽癌细胞增殖和侵袭，进一步揭示miR -150在鼻咽癌中的癌基因作用。方法2014年3—6月，培养鼻咽癌细胞株 CNE -2，取生长良好的CNE -2用于实验。设计合成 PDCD4野生型引物序列以及突变型引物序列，连接到含有荧光素酶载体质粒上，检测荧光素酶活性。分别瞬时转染 Vector、pcDNA3.1（＋）- PDCD4、miR - ctr、miR -150 mimics 联合 pcDNA3.1（＋）-PDCD4于 CNE -2，48 h 后采用 Western blotting 法检测 PDCD4表达水平。分别瞬时转染 Vector、pcDNA3.1（＋）-PDCD4、miR -150 inhibitors、miR -150 mimics 联合 pcDNA3.1（＋）- PDCD4于 CNE -2，分别于培养24、48、72、96 h 后测定其吸光度（ OD）值，反映其增殖能力。分别瞬时转染 Vector、pcDNA3.1（＋）- PDCD4、miR -150 inhibitors、miR -150 mimics 联合 pcDNA3.1（＋）- PDCD4于 CNE -2，采用 Transwell 侵袭实验检测 CNE -2侵袭能力。结果无miR -150转染的 PDCD4野生型细胞株荧光素酶活性为（0.975±0.112），miR -150转染的 PDCD4野生型细胞株荧光素酶活性为（0.588±0.042），差异有统计学意义（t ＝7.853，P ＝0.018）。无miR -150转染的 PDCD4突变型细胞株荧光素酶活性为（0.992±0.135），miR -150转染的 PDCD4突变型细胞株荧光素酶活性为（0.875±0.095），差异无统计学意义（t ＝1.461，P ＝0.281）。转染 Vector、pcDNA3.1（＋）- PDCD4、miR - ctr、miR -150 mimics 联合 pcDNA3.1（＋）- PDCD4的 CNE -2 PDCD4表达水平分别为（0.655±0.058）、（1.147±0.152）、（0.704±0.068）、（0.313±0.036），差异有统计学意义（F ＝43.410，P ﹤0.001）；其中，转染 Vector、miR - ctr、miR -150 mimics 联合 pcDNA3.1（＋）- PDCD4的 CNE -2 PDCD4表达水平均低于转染 pcDNA3.1（＋）- PDCD4的 CNE -2，差异有统计学意义（P ﹤0.05）。转染物和时间对 CNE -2增殖能力存在交互作用，转染物和时间对 CNE -2增殖能力均存在主效应（ P ﹤0.001）。转染 Vector、pcDNA3.1（＋）- PDCD4、miR -150 inhibitors、miR -150 mimics 联合pcDNA3.1（＋）- PDCD4的 CNE -2穿膜细胞数分别为（56.6±7.5）、（26.5±3.7）、（30.5±4.7）、（55.2±6.9）个，差异有统计学意义（ F ＝18.550，P ＝0.014）；其中，转染 pcDNA3.1（＋）- PDCD4、miR -150 inhibitors 的CNE -2穿膜细胞数少于转染 Vector、miR -150 mimics 联合 pcDNA3.1（＋）- PDCD4的 CNE -2，差异有统计学意义（P ﹤0.05）。结论 miR -150通过抑制 PDCD4的表达，继而增强鼻咽癌细胞的增殖能力和侵袭能力。%Objective To investigate whether miR - 150 promotes cell proliferation and invasion by target regulating of programmed cell death 4 ( PDCD4 ) or not, and further reveal the oncogeme function of miR - 150 in nasopharyngeal carcinoma. Methods From March to June 2014,we cultured the nasopharyngeal carcinoma CNE - 2,and selected the well -grown ones in the experiment. We designed and synthesized PDCD4 of wild type primer sequence and mutant primer sequence, which would be linked to the carrier vector with luciferase reporter gene,and luciferase activity was detected. Vector,pcDNA3. 1 ( + ) - PDCD4,miR - ctr,and miR - 150 mimics combined with pcDNA3. 1( + ) - PDCD4 were transient transfected into CNE - 2 respectively,and the expression level of PDCD4 was detected after 48 h by Western blotting method. Vector, pcDNA3. 1( + ) - PDCD4,miR - 150 inhibitors and miR - 150 mimics combined with pcDNA3. 1( + ) - PDCD4 were transient tranfected into CNE - 2 respectively,and respectively cultured for 24,48,72,and 96 h to detect the absorbance (OD)value to reflect its ascending ability. Vector,pcDNA3. 1( + ) - PDCD4,miR - 150 inhibitors and miR - 150 mimics combined with pcDNA3. 1( + ) - PDCD4 were transient transfected into CNE - 2 respectively,and the invasion ability of CNE- 2 was reflected by Transwell invasion experiment. Results The luciferase activity of PDCD4 of wild type cell lines that without miR - 150 transfection was(0. 975 ± 0. 112),and luciferase activity of PDCD4 of wild type cell lines that with miR - 150 transfection was(0. 588 ± 0. 042),which showed significant differences( t = 7. 853,P = 0. 018). The luciferase activity of PDCD4 of mutant type cell lines that without miR - 150 transfection was(0. 992 ± 0. 135),mutant type cell lines that with miR - 150 transfection was( 0. 875 ± 0. 095 ),which showed no significant differences( t = 1. 461,P = 0. 281 ) . The expression levels of PDCD4 that were transient transfected with Vector,pcDNA3. 1( + ) - PDCD4,miR - ctr,miR - 150 mimics combined with pcDNA3. 1( + ) - PDCD4 were(0. 665 ± 0. 058), (1. 147 ± 0. 152), (0. 074 ± 0. 068),and (0. 313 ± 0. 036)respectively,which showed significant differences(F = 43. 410,P ﹤ 0. 011);the expression level of CNE -2 PDCD4 that had been transfected with Vector,miR - ctr,miR - 150 mimics combined with pcDNA3. 1( + ) - PDCD4 was lower than that of the CNE - 2 with transfection of pcDNA3. 1( + ) - PCDC4,which showed significant differences( P ﹤0. 05). The transfected objects and time played an interactive role in the proliferation capacity of CNE - 2,and both of them had a main effect in the proliferation capacity of CNE - 2(P ﹤ 0. 001). The numbers of transmembrane cells of CNE - 2 that were transient transfected with Vector,pcDNA3. 1( + ) - PDCD4,miR - 150 inhibitors and miR - 150 mimics combined with pcDNA3. 1( + ) - PDCD4 were(56. 6 ± 7. 5), (26. 5 ± 3. 7), (30. 5 ± 4. 7),and(55. 2 ± 6. 9),which showed significant differences(F = 18. 550,P = 0. 014);the number of transmembrane cells of CNE - 2 that were transfected with pcDNA3. 1( + ) - PDCD4,and miR - 150 inhibitors was less than that of CNE - 2 that were transfected with Vector, miR - 150mimics combined with pcDNA3. 1( + ) - PDCD4,which showed significant differences(P ﹤ 0. 05). Conclusion miR - 150 enhances the proliferation and invasion capacity of nasopharyngeal carcinoma cells by inhibiting PDCD4 expression.