[Detection of RNA interference in nasopharyngeal carcinoma cell lines using reporter genes].
OBJECTIVE
RNA interference (RNAi) technique is now widely used in studies of gene function, signal transduction pathway, and gene therapy because it can effectively and specifically inhibit gene expression. This study was designed to synthesize small interfering RNA (siRNA) by in vitro transcription, and construct retrovirus vectors to express small hairpin RNA (shRNA), detect RNAi in nasopharyngeal carcinoma cell lines, and to develop a RNAi technique platform.


METHODS
siRNAs targeting green fluorescent protein (GFP) and luciferase (Luc) were synthesized by in vitro transcription, while shRNAs targeting GFP and Luc were constructed from pSUPER.retro. Cervical cancer cell line HeLa, nasopharyngeal carcinoma cell lines CNE1, CNE2, and 5-8F were co-transfected with siRNAs or shRNAs and reporter gene pEGFP-N1 or pGL3. The expression of GFP was detected by fluorescent microscopy and Western blot. The activity of luciferase was measured by Luciferase Enzyme Assay System.


RESULTS
siRNA duplexes with 3' UU overhangs and shRNA specifically silenced GFP expression, while antisense RNA and siRNA without 3' UU overhangs did not trigger RNA interference of GFP. Quantitative luciferase activity analysis showed that siRNA inhibited Luc expression in HeLa, CNE1, CNE2, and 5-8F cell lines with inhibition rates of 91.43%, 78.01%, 90.30%, and 62.85%, respectively. Similarly, the inhibition rate was 78.22% when shRNA targeting Luc was co-transfected into HeLa cell line.


CONCLUSIONS
Both siRNAs and shRNAs can induce RNAi. 3' UU overhangs of siRNA may play a role in RNAi. RNAi can be triggered in both nasopharyngeal carcinoma cell lines and HeLa cell line.
Yin
Zhi-Hua
ZH
Cancer Research Institute, Central South University, Changsha, Hunan, 410078, P.R.China.
Ren
Cai-Ping
CP
Li
Feng
F
Jiang
Wei-Hong
WH
Yang
Xu-Yu
XY
Feng
Xiang-Ling
XL
Yao
Kai-Tai
KT
D045744:Cell Line, Tumor; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009303:Nasopharyngeal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D008156:Luciferases