CAJ | 학술논문

目的：探讨hsa-microRNA-218（hsa-mir-218）对颗粒溶素（Granulysin，GLS）表达的影响。方法：抽提佛波酯（ Phorbol 12-myristate 13-acetatefor ， PMA）激活的THP-1细胞的总RNA，逆转录后PCR扩增GLS基因，将其克隆至pDsRed-Ex-press-C1，构建GLS表达质粒pDsRed-GLS。将pDsRed-GLS与pGenesil-mir-218（pGenesil-mir-control）共转染293T细胞，36 h后激光共聚焦显微镜观察两种质粒在细胞中的表达情况，48 h后提取细胞总RNA和蛋白，RT-PCR和Western blot检测hsa-mir-218对GLS表达的影响。结果：酶切和测序结果证实重组质粒pDsRed-GLS构建成功，且能与microRNA干扰质粒在293T细胞中共表达。与转染pGenesil-mir-control组细胞相比，Western blot 结果显示转染pGenesil-mir-218组细胞GLS蛋白表达下降约50％，而GLS mRNA表达无明显变化。结论：hsa-mir-218在转录后水平干扰了GLS的表达，为进一步探讨mir-218干扰GLS表达的机制打下基础。
목적：탐토hsa-microRNA-218（hsa-mir-218）대과립용소（Granulysin，GLS）표체적영향。방법：추제불파지（ Phorbol 12-myristate 13-acetatefor ， PMA）격활적THP-1세포적총RNA，역전록후PCR확증GLS기인，장기극륭지pDsRed-Ex-press-C1，구건GLS표체질립pDsRed-GLS。장pDsRed-GLS여pGenesil-mir-218（pGenesil-mir-control）공전염293T세포，36 h후격광공취초현미경관찰량충질립재세포중적표체정황，48 h후제취세포총RNA화단백，RT-PCR화Western blot검측hsa-mir-218대GLS표체적영향。결과：매절화측서결과증실중조질립pDsRed-GLS구건성공，차능여microRNA간우질립재293T세포중공표체。여전염pGenesil-mir-control조세포상비，Western blot 결과현시전염pGenesil-mir-218조세포GLS단백표체하강약50％，이GLS mRNA표체무명현변화。결론：hsa-mir-218재전록후수평간우료GLS적표체，위진일보탐토mir-218간우GLS표체적궤제타하기출。
Objective:To elucidate the effect of hsa-microRNA-218(hsa-mir-218)on exogenous granulysin (GLS) expression in 293T cells.Methods:Total RNA was extracted from THP-1 cells induced by phorbol 12-myristate 13-acetatefor (PMA), and GLS gene was amplified by RT-PCR, and then cloned into pDsRed-Express-C1 to construct the GLS expression vector pDsRed-GLS.Then 293T cells were co-transfected with pDsRed-GLS and pGenesil-mir-218 (pGenesil-mir-control) and laser confocal microscopy was per-formed 36 h later to detect their co-expression .Total RNA and protein were extracted 48 h post transfection , and RT-PCR and Western blot were performed to detect the effect of hsa-mir-218 on exogenous GLS expression .Results:The GLS expression vector pDsRed-GLS was constructed successfully and laser confocal microscopy indicated that it was co -expressed with the interference vector .Compared with that of cells transfected with pGenesil-mir-control, Western blot showed a markedly decrease of GLS protein expression (50%) in the cells transfected with pGenesil-mir-218.However, GLS mRNA expression remained unchanged .Conclusion: hsa-mir-218 nega-tively regulates GLS expression at a post-transcriptional level , and this provides an experimental basis for future study of mechanism of GLS expression regulated by mir-218 .