超声辐照携带miR-520c-3p和miR-132微泡对体外培养人肝癌细胞Huh7的影响
| 作者: |
1潘青云,
2雷长江,
2李媛,
2胡福荣,
2李磊,
2黄剑彬,
2曾诚
1 江汉大学第二附属医院肿瘤科，武汉 430050 2 江汉大学第二附属医院普外科，武汉 430050 |
| 通讯: |
雷长江
Email: changjiang0118@163.com |
| DOI: | 10.3978/j.issn.2095-6959.2017.06.007 |
| 基金: | 第四批汉阳知音英 才计划 湖北省自然科学基金, 2016CFB591 武汉市科技局应用基础研究, 2015061701011630 |
摘要
目的：观察超声辐照携带miR-520c-3p和miR-132微泡对体外培养人肝癌细胞Huh7的影响。方法：以超声辐照携带miR-520c-3p和miR-132微泡转染体外培养人肝癌Huh7细胞株。实验分5组：对照组(A组)，miR-520c-3p mimics negative control+miR-132 mimics negative control+微泡造影剂+超声辐照(B组)，miR-520c-3p mimics+微泡造影剂+超声辐照(C组)，miR-132 mimic+微泡造影剂+超声辐照(D组)，miR-520c-3p mimics+miR-132 mimics+微泡造影剂+超声辐照(E组)。超声辐照条件：机械指数0.4，辐照参数2.0 W/cm2，连续辐照30 s，间隔60 s，共5次。采用实时荧光定量PCR检测miR-520c-3p，miR-132，磷脂酰肌醇蛋白多糖-3(GPC3)和转录辅助因子YAP mRNA表达，采用Western印迹检测GPC3和YAP蛋白表达，采用噻唑蓝比色法检测细胞增殖活性，采用流式细胞术检测细胞凋亡。结果：与A组、B组比较，C组、E组miR-520c-3p相对表达量升高(P<0.05)，而D组无明显改变(P>0.05)；D组、E组miR-132相对表达量升高(P<0.05)，而C组无明显改变(P>0.05)。与A组、B组比较，C组、E组GPC3蛋白相对表达量降低(P<0.05)，而其mRNA相对表达量无明显改变(P>0.05)。C组、D组、E组YAP蛋白以及其mRNA相对表达量均降低(P<0.05)。与A组、B组比较，C组、D组、E组细胞增殖率降低(P<0.05)，且E组降低最明显(P<0.05)；C组、D组、E组细胞凋亡率增加(P<0.05)，且E组凋亡最明显(P<0.05)。结论：超声辐照携带miR-520c-3p和miR-132微泡能显著抑制人肝癌Huh7细胞株增殖，并促进其凋亡，其机制可能与调节GPC3和YAP的表达有关。
关键词：
超声辐照
miR-520c-3p
miR-132
肝细胞癌
磷脂酰肌醇蛋白多糖-3
转录辅助因子YAP
Effect of ultrasound irradiation carrying miR-520c-3p and miR-132 microbubble on human hepatocellular carcinoma Huh7 cell line cultured in vitro
CorrespondingAuthor: LEI Changjiang Email: changjiang0118@163.com
DOI: 10.3978/j.issn.2095-6959.2017.06.007
Abstract
Objective: To investigate the effect of ultrasound irradiation carrying miR-520c-3p and miR-132 microbubble on human hepatocellular carcinoma Huh7 cell line cultured in vitro. Methods: Human hepatocellular carcinoma Huh7 cell line cultured in vitro was transfected with miR-520c-3p and miR-132 which were carried with ultrasound microbubble. The experiment was divided into 5 groups: control group (group A), miR-520c-3p mimics negative control+miR-132 mimics negative control+microbubble+ultrasound irradiation (group B), miR-520c-3p mimics+microbubble+ultrasound irradiation (group C), miR-132 mimics+microbubble+ultrasound irradiation (group D), miR-520c-3p mimics+miR-132 mimics+microbubble+ultrasound irradiation (group E). Ultrasonic irradiation conditions: mechanical index 0.4, irradiation parameters 2.0 w/cm2, continuous irradiation 30 s, interval 60 s, a total of 5 times. The expression of miR-520c-3p, miR-132, phosphatidylinositol-3 (GPC3) and transcription factor YAP mRNA were detected by real-time fluorescence quantitative PCR. The expression of GPC3 and YAP protein was detected by Western blotting. The cell proliferation was detected by flow cytometry. The cell apoptosis was detected by flow cytometry. Results: The relative expression of miR-520c-3p in group C and E was higher than that in group A and B (P<0.05), but there was no significant change in group D (P>0.05). The relative expression of miR-132 in group D and E was higher than that in group A and B (P<0.05), but there was no significant change in group C (P>0.05). Compared with group A and B, the relative expression of GPC3 protein in group C and E was decreased (P<0.05), but the expression of GPC3 mRNA was not changed (P>0.05). Compared with group A and B, the expression of YAP protein and its mRNA in group C, D and E were decreased (P<0.05). Compared with group A and B, the proliferation rate of group C, D and E decreased (P<0.05), and the group E decreased most obviously (P<0.05). The apoptosis rate of group C, D and E increased (P<0.05), and the group E apoptosis most obviously (P<0.05). Conclusion: Ultrasound irradiation carrying miR-520c-3p and miR-132 microbubble can significantly inhibit the proliferation of human hepatoma cell line Huh7 and promote its apoptosis, which may be related to the regulation of the expression of GPC3 and YAP.