Abstract 11211: CTCF and HMGA1 Opposite Directionally Regulate the Gene Expression of microRNAs at Scaffold/ Matrix-attachment Regions in the Arterial Smooth Muscle Cell
Purpose: Although specific microRNAs (miRNAs) critically regulate the phenotypic modulation of arterial smooth muscle cell (SMC), the mechanism of their gene expression is unknown. The nuclear matrix maintains the genome structure and becomes the hub of active transcriptional machinery. We hypothesized cis-acting Scaffold/Matrix-Attachment Regions (MARs) in the upstream regions of miRNA genes would regulate the gene expression through architectural transcription factor HMGA1 or CCCTC-binding factor (CTCF).
Methods and Results: In silico MARWiz predicted powerful conserved MAR elements in 10kb upstream regions of human miRNA genes with peak cut off=0.75 (two in miRNA-21 and three in miRNA-145). In cultured human arterial SMC knocking down of HMGA1 mRNA using specific siRNA reduced the expression of miRNA-145 to 43% and miRNA-21 to 70% by real-time PCR. On the other hand, knocking down of CTCF increased the expression of miRNA-145 to 310% and miRNA-21 to 198%. Microarray profiling analysis of miRNAs unexpectedly demonstrated significant reduction of the expression of no less than 185 miRNA genes including miRNA-145 after knocking down of HMGA1 and significant elevation of 119 miRNA genes including miRNA-145 after knocking down of CTCF. PCR amplification of the individual MAR element revealed its actual existence in the nuclear matrix fraction, not with the chromatin fraction. Moreover, the tight associations of HMGA1 and CCF proteins with the nuclear matrix were demonstrated both by immuno-fluorescence and western blot analysis. Either knocking down of HMGA1 and CTCF significantly suppressed the gene expression of down-stream SMC differentiation markers (α-SM actin, SM22α, SM-MHC), demonstrated by real-time PCR.
Conclusion: These results indicate that HMGA1 and CTCF opposite directionally regulate the gene expression of miRNAs governing SMC differentiation at MARs.
- © 2013 by American Heart Association, Inc.