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目的人宫颈癌细胞(Hela)中表达Ⅱ型单纯疱疹病毒(HSV-2)潜伏相关转录体(LAT)编码的microRNA-H3(miR-H3),检测该mRNA对Hela细胞活性的影响。方法 PCR扩增microRNA-H3的前体序列,将其插入到pmRmcherry(cherry)载体中,并酶切测序同时验证;重组质粒转染Hela细胞,q PCR检测microRNA的相对表达水平,转染受体细胞,并用放线菌素D(Act D)诱导凋亡,MTT法、流式细胞术和Edu染色共同检测Hela细胞活性。结果双酶切、测序表明cherry/miR-H3重组质粒构建成功,q PCR检测miR-H3大量表达,MTT法、Edu染色和流式细胞术检测表明转染重组质粒cherry/miR-H3并经诱导凋亡组细胞的活性明显比空质粒转染组和未转染组细胞活性高,具有统计学意义,而与正常细胞对照组无显著差异。结论 cherry/miR-H3真核表达载体可在Hela细胞中高效表达,并具有抗Act D诱导的细胞凋亡作用。
Objective To detect the expression of microRNA-H3 (miR-H3) encoded by latent virus-associated herpesvirus type 2 (HSV-2) in Hela, and to detect the effect of this mRNA on the activity of Hela cells. Methods The precursor sequence of microRNA-H3 was amplified by PCR and inserted into pmRmcherry (cherry) vector. The recombinant plasmid was transfected into Hela cells. The relative expression level of microRNA was detected by q PCR. The cells were stained with Act D and the apoptosis of Hela cells was detected by MTT assay, flow cytometry and Edu staining. The results of double digestion, sequencing showed that cherry / miR-H3 recombinant plasmid was successfully constructed, q-PCR detection of miR-H3 large number of expression, MTT assay, Edu staining and flow cytometry showed that transfected recombinant plasmid cherry / miR- The activity of apoptotic cells was significantly higher than that of empty plasmid transfected and untransfected cells, which was statistically significant, but not significantly different from that of normal cells. Conclusion The cherry / miR-H3 eukaryotic expression vector is highly expressed in Hela cells and has anti-Act D-induced apoptosis.