南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (05): 765-770.doi: 10.12122/j.issn.1673-4254.2020.05.25

• • 上一篇    

miR-144通过影响TLR/MyD88通路抑制肝癌SMMC-7721细胞侵袭

易青松，柳久久，艾热夏提·库尔班，李 竞，罗宏武，孙吉春   

  • 出版日期:2020-05-20 发布日期:2020-05-20
  • 基金资助:

Over-expression of miR-144 inhibits invasion of liver cancer SMMC-7721 cells in vitro by suppressing TLR/MyD88 pathway

  

  • Online:2020-05-20 Published:2020-05-20

HTML

PDF

705

摘要： 目的 探讨miR-144基因过表达对肝癌SMMC-7721细胞侵袭及Toll样受体（TLR）/髓样分化因子88（MyD88）免疫通路的影响。方法 培养人正常肝细胞HL-7702，肝癌细胞系SMMC-7721，采用荧光实时定量PCR法检测两组细胞miR-144基因表达差异，将SMMC-7721细胞分为空白组、miR-144 NC组、miR-144 mimics组，倒置荧光显微镜检测转染效率，qRT-PCR法检测转染后各组miR-144基因表达情况，通过细胞计数试剂盒（CCK8）法各组SMMC-7721细胞存活情况，通过Transwell法检测各组SMMC-7721细胞侵袭情况，应用免疫印迹法检测各组侵袭相关蛋白基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）以及TLR/MyD88通路相关蛋白表达情况，观察添加40 μmol/L MyD88抑制剂TJ-M2010-2组对SMMC-7721细胞TLR/MyD88通路相关蛋白表达情况。结果 与正常组相比，SMMC-7721组miR-144基因相对表达量显著降低（P<0.05）；与空白组、miR-144 NC组相比，miR-144 mimics组miR-144基因表达显著升高（P<0.05）；CCK-8实验显示，与对照组、miR-144 NC组相比，miR-144 mimics组转染48、72、96 h后细胞存活率显著降低（P<0.05）；与空白组、miR-144 NC组相比，miR-144 mimics组侵袭细胞数、MMP-2、MMP-9蛋白表达显著降低（P<0.05）；与空白组、miR-144 NC组相比，miR-144 mimics组、TJ-M2010-2组Toll样受体4（TLR4）、MyD88、磷酸化核因子-κB（p-NF-κB）/核因子-κB（NF-κB）蛋白表达显著降低（P<0.05），miR-144 mimics组与TJ-M2010-2组TLR4、MyD88、p-NF-κB/NF-κB蛋白表达差异无统计学意义（P>0.05）。结论 miR-144过表达可能通过抑制TLR/MyD88通路转导降低SMMC-7721细胞存活、抑制SMMC-7721细胞侵袭。

Abstract: Objective To investigate the effects of over-expression of miR-144 on invasion of SMMC-7721 cells and Toll-like receptor (TLR)/myeloid differentiation factor 88 (MyD88) pathway in hepatocellular carcinoma cells. Methods The expressions of miR-144 was examined in normal human hepatocyte line HL-7702 and hepatocarcinoma cell line SMMC-7721 using realtime quantitative PCR (qRT-PCR). SMMC-7721 cells were divided into blank group, miR-144 NC group and miR-144 mimics group, and the expressions of miR-144 in each group were detected with qRT-PCR. Cell count kit-8 (CCK8) was used to assess the survival of SMMC-7721 cells, and the cell invasion was evaluated using Transwell assay. The expressions of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and TLR/MyD88 pathway-related proteins in the cells were detected with Western blotting; the effect of 40 μ mol/L MyD88 inhibitor on TLR/MyD88 pathway-related proteins was examined in SMMC-7721 cells. Results Compared with normal human hepatocytes, SMMC-7721 cells expressed a significantly lower level of miR-144 (P<0.05). CCK-8 assay showed that test showed that miR-144 over-expression significantly decreased the cell survival rate (P<0.05), lowered the number of invasive cells, and decreased the expression of MMP-2 and MMP-9 in SMMC-7721 cells (P<0.05). The expressions of Toll-like receptor 4 (TLR4), MyD88, phosphorylated nuclear factor-kappa B (pNF-κB) and NF-κB protein decreased significantly in miR-144 mimics group and TJ-M2010-2 group (P<0.05) and were comparable between the two groups (P>0.05). Conclusion Overexpression of miR-144 decreases SMMC-7721 cell survival and invasion by inhibiting TLR/MyD88 pathway