Construction and identification of siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene and its effect on the cell cycle of INS-1 cells.
- Author:
Hua-sheng LIANG
1
;
Yao-ming XUE
;
Yu-hua ZHONG
Author Information
- Publication Type:Journal Article
- MeSH: Cell Cycle; drug effects; Genetic Vectors; genetics; Glucose; pharmacology; Humans; Insulin-Secreting Cells; cytology; drug effects; metabolism; RNA, Small Interfering; genetics; Receptor, Parathyroid Hormone, Type 1; genetics; metabolism
- From: Journal of Southern Medical University 2010;30(9):2093-2096
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the expression vector of siRNA targeting parathyroid hormone 1 receptor (PTH1R) gene and evaluate its effect on the cell cycle of INS-1 cells.
METHODSThe sequences of PTH1R gene was retrieved from Genbank, and 4 pairs of oligonucleotides were synthesized and inserted into pSUPERretro RNAi, which was identified by RT-PCR and sequence analysis. The vectors were then transfected into INS-1 cells, in which the expression of PTH1R was observed by Western blotting to evaluate the transfection efficiency. The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry.
RESULTSRT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene. The vectors were successfully transfected into INS-1 cells, and the most effective vector was selected by Western blotting. Transfection with the siRNA for PTH1R gene silencing resulted in the inhibition of INS-1 form entering the S phase.
CONCLUSIONThe successful construction of the recombinant PTH1R-siRNA vectors establishes a basis for further study of protective role of the PTH1R gene in INS-1 cells in high glucose medium.