MicroRNA-137通过Wnt信号通路对宫颈癌细胞增殖、凋亡及裸鼠移植瘤的作用研究

Inhibitory effect of microRNA-137 on proliferation and apoptosis of cervical cancer cells and transplanted tumor in nude mice through Wnt signaling pathway

摘要:

目的 探究microRNA-137（miR-137）通过Wnt信号通路对人宫颈癌细胞HeLa的增殖与凋亡，以及对裸鼠移植瘤的作用。方法 将人宫颈癌细胞HeLa分别转染miR-137-mimic质粒（miR-137组）和空载质粒（NC组），另设空白组只加入等量转染试剂不做其他处理。待细胞稳定转染后，采用CCK-8法检测细胞活性。采用流式细胞仪检测细胞凋亡。采用实时荧光定量聚合酶链反应（qRT-PCR）检测Wnt、基质金属蛋白酶-7（MMP-7）、分泌型蛋白Dickkopf-1（DKK1）、miR-137 mRNA相对表达量；采用Western blotting检测Wnt、MMP-7、DKK1蛋白的相对表达量。将稳定转染的细胞接种于裸鼠背部，观察裸鼠移植瘤的生长情况，绘制生长曲线并计算抑瘤率。结果 空白组、NC组、miR-137组12 h、24 h、48 h的OD值比较，采用重复测量设计的方差分析，结果 ①不同时间点的OD值有差异（P <0.05）；②3组的OD值有差异（P <0.05），miR-137组OD值较空白组和NC组低，相对细胞活性较低；③3组OD值的变化趋势有差异（P <0.05）。miR-137组细胞凋亡率高于空白组和NC组（P <0.05）。miR-137组细胞Wnt和MMP-7 mRNA相对表达量较空白组和NC组降低（P <0.05）；miR-137组细胞DKK1和miR-137 mRNA相对表达量较空白组和NC组升高（P <0.05）。miR-137组细胞Wnt、MMP-7蛋白相对表达量较空白组和NC组降低（P <0.05）；miR-137组细胞DKK1蛋白相对表达量较空白组和NC组升高（P <0.05）。NC组裸鼠移植瘤的抑瘤率与miR-137组比较，差异有统计学意义（P <0.05），miR-137组裸鼠移植瘤的抑瘤率增加。空白组、NC组、miR-137组裸鼠不同时间点的肿瘤体积比较，采用重复测量设计的方差分析，结果 ①不同时间点的肿瘤体积有差异（P <0.05）；②3组的肿瘤体积有差异（P <0.05），miR-137组与空白组和NC组相比，肿瘤生长缓慢，相对抑瘤效果较好；③3组肿瘤体积的变化趋势有差异（P <0.05）。结论 在人宫颈癌细胞中过表达miR-137可以促进癌细胞凋亡，抑制癌细胞增殖，该作用可能与Wnt及其上下游MMP-7/DKK1信号通路有关。

Abstract:

Objective To investigate the effect of microRNA-137 (miR-137) on the proliferation and apoptosis of human cervical cancer cell HeLa through Wnt signaling pathway and its effect on transplanted tumor in nude mice.Methods Human cervical cancer cell HeLa was transfected with miR-137-mic plasmid (miR-137 group) and empty plasmid respectively (NC group), another blank group was added with the same amount of transfection reagent without other treatment. After stable transfection, the cell activity was detected by CCK-8 method. Mining apoptosis was detected by flow cytometry. The relative expression of Wnt, matrix metalloproteinase-7 (MMP-7), secretory protein Dickkopf-1 (DKK1), and miR-137 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR); Western blotting was used to detect the relative expression of Wnt, MMP-7, and DKK1. Stably transfected cells were inoculated into the back of nude mice. The growth of transplanted tumor in nude mice was observed, the growth curve was drawn, and the tumor inhibition rate was calculated.Results The OD values of Blank group, NC group, miR-137 group in 12 h, 24 h, 48 h were compared and the ANOVA of repeated measurement design was used.Results (1) The OD values at different time points were different (P < 0.05). (2) The OD value of miR-137 group was lower than that of blank group and NC group (P < 0.05). (3) There are differences in OD values among the three groups (P < 0.05), the apoptosis rate of miR-137 group was higher than that of blank group and NC group (P < 0.05). The relative expressions of Wnt and MMP-7 mRNA in miR-137 group were lower than those in blank group and NC group (P < 0.05). The relative expression of DKK1 and miR-137 mRNA in miR-137 group was higher than that in blank group and NC group (P < 0.05). The relative expression of Wnt and MMP-7 protein in miR-137 group was lower than that in blank group and NC group (P < 0.05). The relative expression of DKK1 protein in miR-137 group was higher than that in blank group and NC group (P < 0.05). The tumor inhibition rate of NC group was significantly lower than that of miR-137 group (P < 0.05). The tumor volumes of nude mice in blank group, NC group, and miR-137 group were compared at different time points, and the repeated measurement design was used for ANOVA.Results (1) The tumor volumes at different time points were different (P <0.05). (2) The tumor volume of the three groups was different (P < 0.05). Compared with the blank group and the NC group, the tumor volume of the miR-137 group grew slowly, and the relative anti-tumor effect was better. (3) The trend of tumor volume change was different among the three groups (P < 0.05).Conclusion Overexpression of miR-137 in human cervical cancer cells can promote cancer cell apoptosis and inhibit cancer cell proliferation, which may be related to Wnt and MMP-7/DKK1 signaling pathway.