Most of the biological effects of 1a, 25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) are elicited by the binding to vitamin D receptor (VDR), which regulates gene expression. Earlier studies reported no correlation between the VDR protein and mRNA levels, suggesting the involvement of posttranscriptional regulation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through translational repression or mRNA degradation. A potential miR-125b recognition element (MRE125b) was identified in the 3 0 -untranslated region of human VDR mRNA. We investigated whether VDR is regulated by miR-125b. In luciferase assays using a plasmid containing the MRE125b, the antisense oligonucleotide for miR-125b significantly increased (130% of control) the reporter activity in KGN cells, whereas the precursor for miR-125b significantly decreased (40% of control) the reporter activity in MCF-7 cells, suggesting that miR-125b functionally recognized the MRE125b. By electrophoretic mobility shift assays, it was demonstrated that the overexpression of miR-125b significantly decreased the endogenous VDR protein level in MCF-7 cells to 40% of control. 1,25(OH) 2 D 3 drastically induced the CYP24 mRNA level in MCF-7 cells, but the induction was markedly attenuated by the overexpression of miR-125b. In addition, the antiproliferative effects of 1,25(OH) 2 D 3 in MCF-7 cells were significantly abolished by the overexpression of miR-125b. These results suggest that the endogenous VDR level was repressed by miR125b. In conclusion, we found that miR-125b posttranscriptionally regulated human VDR. Since the miR-125b level is known to be downregulated in cancer, such a decrease may result in the upregulation of VDR in cancer and augmentation of the antitumor effects of 1,25(OH) 2 D 3 .

We clarified that the single nucleotide polymorphisms in the promoter region or introns in the CYP2B6 affect the potency of cyclophosphamide activation to 4-hydroxycyclophosphamide. This information would be valuable for predicting adverse reactions and the clinical efficacy of cyclophosphamide.

Human vitamin D 3 hydroxylase (CYP24) catalyzes the inactivation of 1␣,25-dihydroxyvitamin D 3 (calcitriol), which exerts antiproliferative effects. CYP24 has been reported to be overexpressed in various cancers in which microRNA levels are dysregulated. In silico analysis identified a potential miR-125b recognition element (MRE125b) in the 3Ј-untranslated region of human CYP24 mRNA. We investigated whether CYP24 is regulated by miR-125b. In luciferase assays using a reporter plasmid containing MRE125b, transfection of the antisense oligonucleotide (AsO) for miR-125b increased the reporter activity in KGN cells, and transfection of precursor miR-125b decreased the reporter activity in MCF-7 cells. The endogenous CYP24 protein level was also increased by AsO for miR-125b in KGN cells and was decreased by precursor miR-125b in MCF-7 cells. These results suggested that human CYP24 is regulated by miR-125b. Immunohistochemical analysis revealed that the CYP24 protein levels in human breast cancer were higher than in adjacent normal tissues, without an accompanying CYP24 mRNA increase. On the other hand, the expression levels of miR-125b in cancer tissues were significantly (P Ͻ 0.0005) lower than those in normal tissues. It is noteworthy that the CYP24 protein levels in cancer tissues were inversely associated with the cancer/normal ratios of the miR-125b levels, indicating that the decreased miR-125b levels in breast cancer tissues would be one of the causes of the high CYP24 protein expression. In conclusion, this study clearly demonstrates that miR-125b post-transcriptionally regulates the CYP24, which serves as a possible mechanism for the high CYP24 expression in cancer tissues.Human CYP24 is a key enzyme involved in the inactivation of 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ; calcitriol].

Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ERalpha gene have been reported to be associated with breast cancer, endometrial cancer, and psychiatric diseases. However, functional analyses for most mutants with amino acid changes are still lacking. In the present study, to investigate the effects of point mutations on the function, gel-shift assays and luciferase assays were performed for eight kinds of mutated ERalpha proteins, including a single nucleotide change of C207G (N69K), G478T (G160C), T887C (L296P), A908G (K303R), C926T (S309F), A1058T (E353V), A1186G (M396V), and G1231deletion (411fsX7). The mutated ERalpha expression plasmids were constructed by site-directed mutagenesis. With gel-shift assays using in vitro translated ERalpha proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ERalpha proteins except ERalpha (G160C) and ERalpha (411fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ERalpha (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ERalpha (411fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ERalpha expression plasmids were co-transfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10pM-100nM E2) of the mutant ERalpha proteins except ERalpha (E353V) and ERalpha (411fsX7) were similar to that of wild-type ERalpha. However, at a low level of E2 (100pM), the mutants ERalpha (N69K), ERalpha (L296P), ERalpha (S309F), and ERalpha (M396V) showed a significant decrease of transactivation compared with that of the wild-type ERalpha. The mutants ERalpha (E353V) and ERalpha (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (4OHT) and ICI 182,780. The mutant ERalpha (S309F) showed decreased responsiveness for the antiestrogenicity of 4OHT. In conclusion, we found that some of the naturally occurring human ERalpha mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens.