miR-25靶向RECK促进宫颈癌HeLa细胞的增殖

发布时间：2018-03-18 16:03

本文选题：宫颈癌　切入点：He　出处：《细胞与分子免疫学杂志》2015年01期 　论文类型：期刊论文

【摘要】：目的探讨微小RNA-25(miR-25)对宫颈癌He La细胞增殖活性的影响及其与逆向诱导的带Kazal基序的富含半胱氨酸蛋白(RECK)的靶向关系。方法构建pc DNATM6.2-GW-pre-miR-25、pmir GLO-野生型RECK(RECK-WT)和突变型RECK(RECK-MT)真核表达载体及miR-25抑制剂(anti-miR-25),采用荧光实时定量PCR(qRT-PCR)检测pre-miR-25和anti-miR-25在He La细胞中的转染效率,并采用MTT法分析miR-25对He La细胞增殖活性的影响,双萤光素酶报告基因、qRT-PCR和Western blot法检测miR-25与RECK靶向关系。结果测序结果显示pc DNATM6.2-GW-pre-miR-25、pmir GLO-RECK-WT和pmir GLO-RECK-MT重组载体构建成功,qRT-PCR提示pre-miR-25与anti-miR-25在He La细胞中具有良好的转染效率。MTT结果显示miR-25能够明显促进He La细胞的增殖。双萤光素酶报告基因检测显示共转染pre-miR-25与RECK-WT的He La细胞其萤光活性显著下降,同时qRT-PCR及Western blot也显示anti-miR-25在转录和转录后水平均能够显著增加He La细胞中RECK的表达。结论miR-25能够靶向RECK,促进宫颈癌He La细胞的增殖。
[Abstract]:Objective to investigate the effect of minimal RNA-25 miR-25) on the proliferation activity of cervical cancer He-La cells and its targeting relationship with reverse-induced cysteine rich protein (RECK) with Kazal motif. Methods the eukaryotic expression of pc-DNATM6.2-GW-pre-miR-25miR-pmir GLO-wild-type RECK-WTand mutant RECK-MTK were constructed. The transfection efficiency of pre-miR-25 and anti-miR-25 in He-La cells was detected by real-time fluorescence quantitative PCR qRT-PCRR, and anti-miR-25, a miR-25 inhibitor, was used to detect the transfection efficiency of pre-miR-25 and anti-miR-25 in He-La cells. The effect of miR-25 on the proliferation of He-La cells was analyzed by MTT assay. Double luciferase reporter gene qRT-PCR and Western blot were used to detect the targeting relationship between miR-25 and RECK. Results the sequencing results showed that pc-DNATM6.2-GW-pre-miR-25 pmir GLO-RECK-WT and pmir GLO-RECK-MT recombinant vectors were successfully constructed, which indicated that pre-miR-25 and anti-miR-25 had good transfection efficiency in he La cells. The results showed that miR-25 could significantly promote the proliferation of He-La cells, and the fluorescence activity of He-La cells co-transfected with pre-miR-25 and RECK-WT was significantly decreased by double luciferase reporter gene assay. QRT-PCR and Western blot also showed that anti-miR-25 could significantly increase the expression of RECK in He-La cells at both transcriptional and post-transcriptional levels. Conclusion miR-25 can target Reck and promote the proliferation of He-La cells.
【作者单位】：河北省人民医院肿瘤二科;河北省人民医院肿瘤三科;河北省人民医院核医学科;河北省人民医院老年病二科;
【分类号】：R737.33

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