A GC polymorphism was reported to be located on the precursor of miR-146a, another well known tumor suppressor, which could alter mature miR-146a www.selleckchem.com/products/Bortezomib.html expression, and was associated with risk for cervical cancer and adult glioma (42, 43). To illuminate the mechanisms contributing to down-regulation of miR-99a in HCC, we computationally mapped CpG islands upstream of the miR-99a gene, but no CpG-enriched region had been found. It offered little evidence for DNA methylation but still a chance for histone modification of the miR-99a gene. However, the gene encoding miR-99a was found residing within an intron of C21orf34. C21orf34 is located in chromosome 21q21, the region reported harboring a putative tumor suppressor gene in lung cancer (25).

Intriguingly, we found positive correlations within RNA levels of mature miR-99a, primary miR-99a, and their host gene C21orf34 in HCC tissues (supplemental Fig. 6). This result suggests that miR-99a may be co-transcripted with C21orf34, and reduced miR-99a expression seems less likely due to dysfunction of the maturation process from primary to mature miRNA. We also analyzed possible transcription factors binding 3 kb upstream of the miR-99a gene and C21orf34, which were Evi-1, STATx, NKx2.5, c-Rel, and Elk-1. Those may help to address further studies in the future. A total of 142 HCC patients were studied for relationships between reduced expression of miR-99a and prognosis of HCC patients, and a lower miR-99a expression level was identified to significantly correlate with shorter DFS.

Cox proportional hazards regression analysis further confirmed miR-99a to be an independent predictor. Although combined down-regulation of 19 miRNAs, including miR-99a, had been described to be correlated with shorter HCC survival time (44), here, we demonstrate for the first time the individual lower miR-99a expression in HCC tissues significantly correlated with shorter survival of HCC patients, and down-regulated miR-99a was confirmed as an independent predictor for poorer prognosis of HCC patients. As reported previously, abnormal expressions of miR-122, miR-26, miR-29, and miR-139 AV-951 also correlate with the prognosis of HCC patients (12, 14, 45, 46). Hence, we propose that the combined detection on levels of these miRNA in HCC, including miR-99a, may help to identify the prognosis of HCC patients more precisely. With informatics prediction and sequential experimental demonstration, IGF-1R and mTOR were identified as direct targets of miR-99a in our study. mTOR signaling pathway can be activated following upstream activation of receptors to multiple extracellular signals, including IGF-1R, and plays a key role in cell growth, protein translation, metabolism, cell invasion, and apoptosis (33, 34).