[Construction and application of an artificial microRNA expression vector for inhibiting PAR4].
OBJECTIVE
To construct artificial microRNA expression vector targeting PAR4 and suppress the expression of PAR4 in human colorectal cancer SW620 cells with the artificial microRNA.


METHODS
Artificial microRNA was designed and amplified by two rounds of PCR and cloned into pMD-19T vector. The sequence of the cloned artificial microRNA was verified by DNA sequencing. Eight tandemly-repeated artificial microRNAs were subcloned into mammalian expression vector pcDNA3.1(+) to make the artificial microRNA- expressing vector pcDNA3.1(+)-8xPAR4-microRNA. The vector was transfected into human colorectal cancer SW620 cells, and stable transfectants were selected by G418. The expression of PAR4 was examined by Western blot.


RESULTS
DNA sequencing showed that the sequence of the cloned artificial microRNA targeting PAR4 was correct. Western blot result showed that the expression of PAR4 in SW620 cells stably transfected with pcDNA3.1(+)-8xPAR4-microRNA was markedly downregulated when compared to SW620 parental cells.


CONCLUSIONS
Artificial microRNA expression vector targeting PAR4 is successfully constructed with significant suppression effect on PAR4 expression in SW620 cells. This provides the basis for future studies on the function of PAR4 and potential cancer gene therapy targeting PAR4.
Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou 310016, China.
Na
N
Han
Li-sha
LS
Chu
Jiang
J
Cao
D001483:Base Sequence; D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D034622:RNA Interference; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors* / Q000235:genetics
D035683:MicroRNAs; D018179:Receptors, Thrombin; C112908:protease-activated receptor 4