Effect of hsa-microRNA-218 on granulysin expression
10.3969/j.issn.1000-484X.2014.05.005
- VernacularTitle:hsa-microRNA-218对颗粒溶素表达影响的探讨
- Author:
Yu FAN
;
Chun YANG
;
Luyu ZHANG
;
Jing YANG
;
Yonglin HE
;
Lei XU
;
Xin FENG
;
Chunyan ZHANG
;
Liuqing MU
- Publication Type:Journal Article
- Keywords:
Granulysin;
293T cells;
RNA interference;
Laser confocal microscopy
- From:
Chinese Journal of Immunology
2014;(5):596-599
- CountryChina
- Language:Chinese
-
Abstract:
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 .
