Construction of lentiviral vector of RNA interference and its effect on A549 and A549/DDP cells
- VernacularTitle:siMDR1重组慢病毒载体的构建及其对A549和A549/DDP细胞的影响
- Author:
Jianfeng HUANG
;
Zhenya SHEN
;
Lin XU
;
Yunsheng YU
;
Wenxue YE
;
Haoyue HUANG
- Publication Type:Journal Article
- Keywords:
siRNA;
RNA interference;
multidrug resistance gene;
A549/DDP cells
- From:China Oncology
2009;19(7):497-502
- CountryChina
- Language:Chinese
-
Abstract:
Background and purpose: Multidrug resistance (MDR) of tumor cell is the main reason for clinical chemotherapy failure as well as cancer recurrence and metastasis. This study was to construct a lentiveral vector of RNA interference of MDR1 gene and observe its inhibitive role on the expression of MDR1 in A549 and A549/DDP cells. Methods: Oligos of base pairs for hairpin RNA targeting MDR1 were chemically synthesized. Via annealing and inserting them into the down-stream of H1 promoter of linearized pSUPER, we obtained the siRNA constructs for MDR1, which were afterwards transfected into A549, a human lung cancer cell line expressing high level MDR1, the impact of constructs was observed on the expression and interference efficiency of siRNA against MDR1. The effective sequence of siRNA targeting MDR1 gene was confirmed. Both sense and antisence oligo DNA of the targeting sequence was designed, synthesized and cloned into the PTM vector, containing a promoter and a green fluorescent protein (GFP). The resulting lentiviral vector containing MDR1 siRNA was named PTM-siMDR1 and then transfected into A549 and A549/DDP cells after being confirmed by PCR and sequencing. Results: Restriction digestion and DNA sequencing showed that the siRNA constructs for MDR1 were successfully produced and the expressed siRNA could effectively down-regnlate the expression of MDRI. PCR demonstrated that the lentivirus RNAi vector of MDR1 producing PTM-siMDR1 was constructed successfully. The chemosensitivity of A549/DDP cells to cisplatin were enhanced obviously after trartsfection. Conclusion: The lentivirus RNAi vector of MDR1 can significantly revise the resistance ofA549/ DDP cells with eisplatin after infection.
