| 伊丽娜,魏 欢,赵方新,张 烜,李文希,孙 鹏.过表达miR-455促进肝癌细胞诱导的管型形成[J].,2018,(7):1248-1252 |
| 过表达miR-455促进肝癌细胞诱导的管型形成 |
| Overexpression of miR-455 Promotes the Tube Formation Induced by Liver Cancer Cells |
| 投稿时间：2017-12-06 修订日期：2017-12-25 |
| DOI：10.13241/j.cnki.pmb.2018.07.010 |
| 中文关键词: miR-455 真核表达载体 肝癌细胞 微管形成 |
| 英文关键词: miR-455 Construction of eukaryotic expression vector Liver cancer cell Tube formation |
| 基金项目:国家自然科学基金项目(81660482)；内蒙古医科大学科技百万工程项目(YKD2015KJBW005) |
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| 中文摘要: |
| 摘要 目的：构建miR-455前体的真核表达载体，探讨其对肝癌细胞诱导管型形成的影响。方法：采用PCR扩增miR-455前体序列，通过双酶切将其连接到真核表达载体pcDNA3中。连接产物转化入大肠杆菌内进行扩增，采用菌落PCR、双酶切和测序鉴定重组子。将构建的质粒转染SMMC-7721细胞，通过real-time PCR检测成熟miR-455的表达，采用ELISA方法检测培养液上清中VEGF的表达。用该上清处理人脐静脉内皮细胞HUVEC后，检测管型形成的情况。结果：本实验成功构建了miR-455前体的真核表达载体，将其瞬时转染SMMC-7721细胞后，real-time PCR检测结果显示miR-455表达水平显著升高(P<0.01)。过表达miR-455后，SMMC-7721细胞上清VEGF表达水平呈时间依赖性升高(24 h P<0.05, 48 h和72 h P<0.01)。人脐静脉内皮细胞分别用转染pcDNA3和pcDNA3-pre-455的肝癌细胞培养液上清(72 h)重悬，接种于基质胶Matrigel上，转染pcDNA3-pre-455组形成典型的微管，管样结构明显完整。结论：过表达miR-455可促进肝癌细胞诱导的微管形成。 |
| 英文摘要: |
| ABSTRACT Objective: To establish the eukaryotic expression vector of miR-455 precursors and investigate the role of miR-455 on tube formation induced by liver cancer cells. Methods: The miR-455 precursor was amplified by PCR and inserted into the eukaryotic ex- pression vector pcDNA3. The recombinant plasmid was identified by colony PCR, enzyme digestion and sequencing. After transient transfection with the recombinant plasmid, the matured miR-455 expression of SMMC-7721 cell was detected using real-time PCR; the VEGF expression in cell culture supernant was measured by ELISA; the tube formation of HUVEC was observed after treating with transfected cell culture supernant. Results: The pre-miR-455 eukaryotic expression vectors were successfully constructed. The real-time PCR results showed that miR-455 expression was significantly increased(P<0.01) after transfecting into SMMC-7721 cells. The expres- sion of VEGF in SMMC-7721 cells increased in a time-dependent manner (P<0.05 for 24 h, P <0.01 for 48 h and 72 h) after overexpres- sion of miR-455. The human umbilical vein endothelial cells were resuspended with hepatica carcinoma cell supernatant (72 h) which was transfected by pcDNA3 and pcDNA3-pre-455 and seeded on Matrigel. The group which transfected with pcDNA3-pre-455 forms typical microtubules and the tubular structure is obviously complete. Conclusion: Overexpression of miR-455 promoted the tube forma- tion induced by liver cancer cells. |
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