[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].
OBJECTIVE
To 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.


METHODS
The 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.


RESULTS
RT-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.


CONCLUSIONS
The 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.
Liang
Hua-sheng
HS
Department of Endocrinology, Ninth Affiliated Hospital of Guangxi Medical University, Beihai 536000, China. flowchaos@yahoo.com.cn
Xue
Yao-ming
YM
Zhong
Yu-hua
YH
D002453:Cell Cycle / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics* / Q000378:metabolism
D034741:RNA, Small Interfering; D044168:Receptor, Parathyroid Hormone, Type 1; D005947:Glucose