RESUMO
OBJECTIVES: To improve the expression efficiency of recombinant hFIX, by enhancing its γ-carboxylation, which is inhibited by Calumenin (CALU), we used intronic artificial microRNAs (amiRNAs) for the CALU downregulation. METHODS: Two human CALU (hCALU)-specific amiRNAs were designed, validated and inserted within a truncated form of the hFIX intron 1, in either 3'- or 5'-untranslated regions of the hFIX cDNA, in an expression vector. After transfections of a human cell line with the recombinant constructs, processing of the miRNAs confirmed by RT-PCR, using stem-loop primers. The hFIX and hCALU expression assessments were done based on RT-PCR results. The Gamma(γ)-carboxylation of the expressed hFIX was examined by a barium citrate precipitation method, followed by Enzyme-Linked Immunosorbent Assay. RESULTS: Efficient CALU down regulations, with more than 30-fold decrease, occurred in the cells carrying either of the two examined the 3'-located amiRNAs. The CALU downregulation in the same cells doubled the FIX γ-carboxylation, although the transcription of the FIX decreased significantly. On the other hand, while the expression of the amiRNAs from the 5'-located intron had no decreasing effect on the expression level of CALU, the level of hFIX transcription in these cells increased almost twofold compared to the construct without amiRNA. CONCLUSION: The CALU downregulation, consistent with efficient hFIX γ-carboxylation, occurred in the cells carrying either of the two amiRNAs containing constructs, although it was affected by the locations of the amiRNA carrying introns, suggesting a possible need to optimize the conditions for the amiRNAs expression.
