RESUMEN
AIM: To construct an inducible artificial microRNA expression vector targeting eIF3g gene and use it to inhibit the expression of eIF3g in K562 cells. METHODS: The microRNA targeting human eIF3g was designed and obtained by PCR. After confirmed by sequencing, the microRNA was cloned into the pRevTRE2 plasmid. The Tet-off plasmids were transfected into K562 cells and selected for the stable tetracycline inducible K562/Tet-off transfectants. The pRevTRE2-eIF3g-miRNA plasmid was then transfected into stable K562/Tet-off transfectants and the expression of eIF3g was detected by Western blot. RESULTS: The microRNA targeting eIF3g was confirmed by sequencing. GFP fluorescence assay showed that the stable transfectants of Tet-off plasmids were under tetracycline control. Western blot results showed that the stable transfectants of pRevTRE2-eIF3g-microRNA inhibited eIF3g expression under the regulation of tetracycline. CONCLUSION: The tetracycline-inducible artificial microRNA expression vector targeting eIF3g gene has been successfully constructed and effectively inhibits eIF3g expression in K562 cells.
Asunto(s)
Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Vectores Genéticos/genética , MicroARNs/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/genética , Humanos , Células K562 , Inhibidores de la Síntesis de la Proteína/farmacología , Tetraciclina/farmacologíaRESUMEN
AIM: 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. CONCLUSION: 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.
