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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。
Objective To investigate the effect of miR-25 on the proliferation of cervical cancer HeLa cells and its relationship with the reverse cascade of caspases (RECK) with Kazal motif. Methods The eukaryotic expression vector pcDNA-DNATM6.2-GW-pre-miR-25 and pmir GLO-RECK (RECK-WT) and RECK-MT were constructed and the anti-miR- 25). The transfection efficiency of pre-miR-25 and anti-miR-25 in HeLa cells was detected by real-time quantitative PCR (qRT-PCR). The proliferation of HeLa cells was analyzed by MTT assay Luciferase reporter gene, qRT-PCR and Western blot were used to detect the relationship between miR-25 and RECK. Results The sequencing results showed that the recombinant plasmids pcDNATM6.2-GW-pre-miR-25, pmir GLO-RECK-WT and pmir GLO-RECK-MT were successfully constructed and qRT-PCR suggested that pre-miR-25 and anti-miR- In He La cells with good transfection efficiency. MTT results showed that miR-25 can significantly promote He La cell proliferation. Luciferase reporter assay showed that the luciferase activity of HeLa cells co-transfected with pre-miR-25 and RECK-WT was significantly decreased, and qRT-PCR and Western blot also showed that anti-miR-25 in transcription and transcription After treatment, the expression of RECK in He La cells was significantly increased. Conclusion miR-25 can target RECK and promote the proliferation of Hela cells.