微小分子核糖核酸-449a调控CXC族趋化因子5抑制肝癌细胞迁移和侵袭

MicroRNA-449a suppresses liver cancer migration and invasion through targeting CXC chemo- kine ligand 5

目的探讨微小分子核糖核酸（miRNA，miR）-449a在肝癌中的表达和对肝癌细胞迁移、侵袭的影响及其调控机制。 方法收集18例肝癌组织及癌旁组织，反转录-聚合酶链反应（RT-PCR）检测组织和4种人肝癌细胞株、人正常肝细胞中miR-449a表达；将人肝癌细胞株转染miR-449a类似物（mimic）和阴性对照（NC）质粒，RT-PCR检测转染后miR-449a表达量；Transwell小室实验检测转染后两组细胞的侵袭和迁移细胞数，Western blot检测细胞中CXC族趋化因子5（CXCL5）蛋白表达。 结果与癌旁组织比较，肝癌组织中miR-449a表达量显著降低（0.957±0.165比0.462±0.221，t＝4.137，P＝0.021），与正常肝细胞比较，4种人肝癌细胞株中miR-449a表达量显著降低，差异均有统计学意义（P＝0.009、0.008、0.005、0.020）；选择miR-449a表达量中间的两种人肝癌细胞株BEL-7402和HepG2，采用miR-449a mimic转染后miR-449a表达量显著升高（BEL-7402：6.873±1.293、HepG2：7.246±1.320、NC：1.000，P＝0.012、0.017），且肝癌细胞的迁移（BEL-7402比NC：21.57±13.32比79.84±11.68，HepG2比NC：45.32±12.65比92.21±15.43，t＝24.331、21.109，P＝0.011、0.019）和侵袭细胞数显著降低（BEL-7402比NC：10.25±3.21比26.63±6.48，HepG2比NC：23.44±10.08比65.65±12.26，t＝11.039、12.224），与NC比较差异有统计学意义（P＝0.010、0.008）；过表达miR-449a后两种肝癌细胞中CXCL5蛋白表达显著降低（BEL-7402比NC：0.367±0.083比0.965±0.110，HepG2比NC：0.163±0.043比0.949±0.141，t＝20.176、25.158），与NC比较差异有统计学意义（P＝0.021、0.016）。 结论miR-449a在肝癌中低表达，可能作为抑癌基因通过靶向调控CXCL5影响肝癌细胞迁移和侵袭。

Objective To explore the expression of microRNA （miRNA, miR） -449a in liver cancer, the effect on migration and invasion, and the possible regulatory mechanism. Methods Eighteen cases of liver cancer tissues and adjacent tissues were collected. The expression of miR -449a in liver cancer tissues and adjacent tissues, 4 kinds of human liver cancer cell lines, and human normal hepato- cytes was detected by reverse transeriptase - polymerase chain reaction （ RT - PCR）. The human liver cancer cell lines were transfeeted with miR -449a mimic and the negative control （NC） plasmid. The ex- pression of miR - 449a after transfection was detected by RT - PCR. The cell number of invasion and mi- gration after transfection in the two groups was measured by Transwell assays. The expression of CXC che- mokine ligand 5 （ CXCL5 ） protein in the cells was detected by Western blotting. Results As compared with the adjacent tissues, the expression of miR -449a in liver cancer tissues was significantly reduced （0. 957 ±0. 165 vs. 0. 462 ±0. 221 ,t =4. 137,P =0. 021）. As compared with the human normal hepato- cytes, the expression of miR- 449a in four kinds of human liver cancer cell lines was significantly reduced too （ P = 0. 009, 0. 008, 0. 005, 0. 020 ）, the difference being statistically significant （ BEL - 7402 ： 6.873±1.293, HepG2：7.246 ± 1.320, NC： 1.000, P=0.012, 0.017）. Two kinds of human liver cancer cell lines with the middle expression of miR -449a were chosen, and the expression of miR -449a after transfection was statistically increased, and the cell number of invasion （ BEL - 7402 vs. NC： 21.57±13.32 vs. 79.84 ±11.68; HepG2 vs. NC： 45.32±12.65 vs. 92.21±15.43,t=24.331, 21.109, P=0.011, 0.019） and migration （BEL-7402 vs. NC： 10.25 ±3.21 vs. 26.63±6.48;HepG2 vs. NC： 23.44 ± 10.08 vs. 65. 65± 12. 26,t = 11. 039, 12. 224, P =0. 010, 0. 008） after trans- fection was significantly reduced. The expression of CXCL5 protein after transfection was statistically signif- icantly reduced （BEL -7402 vs. NC： 0. 367 ±0. 083 vs. 0. 965 ±0. 110; HepG2 vs. NC： 0. 163 ±0. 043 vs. 0.949±0. 141,t =20. 176, 25. 158） （P=0.021, 0.016）. Conclusion The miR -449a in liver cancer is expressed weakly, and can serve as a tumor suppressor to influence the migration and invasion of liver cancer cells through targeting CXCL5.