The molecular mechanisms that regulate hADSC differentiation toward osteogenic precursors and subsequent bone-forming osteoblasts is unknown. Using osteoblast precursors obtained from subcutaneous human adipose tissue, we observed that microRNA-26a modulated late osteoblasts differentiation by targeting the SMAD1 transcription factor. Introduction: Elucidation of the molecular mechanisms guiding human adipose tissue-derived stem cells (hADSCs) differentiation is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify microRNA as a regulator of the osteogenic differentiation of hADSCs. Materials and Methods: Osteoblast differentiation of hADSCs was induced by treatment with dexamethasone, ascorbic acid, and -glycerol phosphate. The expression of osteoblastic phenotype was evaluated after the induction by simultaneous monitoring of alkaline phosphatase activity, the expression of genes involved in osteoblastic differentiation by real-time RT-PCR, and mineralization at the same time. MicroRNA expression was determined by Northern blot, and transfection of both antisense miR-RNA and sensor plasmids was done to validate the inhibitory role of microRNA during hADSC osteogenesis. Western blot was used to determine the expression levels of the SMAD1 protein. qRT-PCR analysis was used to compare the expression patterns of osteoblastic markers in transfected cells. Results and Conclusions:We analyzed the role of microRNA 26a (miR-26a) during differentiation of hADSCs. Northern blot analysis of miR-26a during hADSC differentiation showed increased expression, whereas expression of the SMAD1 protein was complementary to that of miR-26a. Because the highest expression of miR-26a and the lowest expression of SMAD1 protein were reached at hADSC terminal differentiation, we carried out our study during the late stages of hADSC differentiation. The inhibition of miR-26a, by 2Ј-O-methyl-antisense RNA, increased protein levels of its predicted target, SMAD1 transcription factor, in treated osteoblasts, upregulating bone marker genes and thus enhancing osteoblast differentiation. Our data suggest a role for miR-26a in the differentiation induced by treatment with dexamethasone, ascorbic acid, and -glycerol phosphate of hADSCs toward the osteogenic lineage by targeting its predicted target, the SMAD1 protein. This study contributes to a better knowledge of molecular mechanisms governing hADSC differentiation by proposing a microRNA-based control of late differentiation.
Several strands of evidence indicate that oestrogens exert a protective role against the development of colon cancer through indirect and direct effects on colonic epithelium. Oestrogen receptor b (ERb), the predominant ER subtype in human colon, is significantly decreased in colonic tumours compared with normal mucosa suggesting a potential role in the regulation of colon tumour growth.To investigate this hypothesis we engineered human colon cancer ERa-negative HCT8 cells in order to obtain ERb protein over-expression. Stably transfected cells were cloned and ERb expression and functionality were monitored by RT-PCR, Western blotting and transactivation in an assay using oestrogen-responsive reporter constructs.Over-expression of ERb inhibited cell proliferation and increased cell adhesion in a ligandindependent manner. Its constitutive activation is possibly due to cross-talk with intracellular signalling pathways, as epidermal growth factor and IGF-I were able to induce ERb transactivation.A possible mechanism by which ERb over-expression inhibits proliferation in HCT8 cells is by modulation of some key regulators of the cell cycle; there is a decrease in cyclin E and an increase in the cdk inhibitor p21CIP1. In fact, flow cytometry analysis provided evidence for blocking of the G1-S phase progression induced by ERb over-expression. The magnitude of this effect was affected by the level of ERb expression.These results provide the first direct evidence that ERb plays an important role in colon cancer as a regulator of cell proliferation through the control of key cell cycle modulators and arrest in G1-S phase transition. These findings are compatible with the hypothesis that the loss of ERb expression could be one of the events involved in the development or progression of colon cancer.
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, interplaying with transcription factors in complex regulatory networks. Menin is the product of the MEN1 oncosuppressor gene, responsible for multiple endocrine neoplasia type 1 syndrome. Recent data suggest that menin functions as a general regulator of transcription. Menin expression modulates mesenchymal cell commitment to the myogenic or osteogenic lineages. The microRNA 26a (miR-26a) modulates the expression of SMAD1 protein during the osteoblastic differentiation of human adipose tissue-derived stem cells (hADSCs). We used siRNA silencing against MEN1 mRNA and pre-miR-26 mimics to study the interplay between them and to investigate the interplay between menin and miR-26a as regulators of osteogenic differentiation in the hADSCs. We found that in hADSCs the siRNA-induced silencing of MEN1 mRNA resulted in a down regulation of miR-26a, with a consequent up-regulation of SMAD1 protein. Chromatin immunoprecipitation (ChIP) showed that menin occupies the miR-26-a gene promoter, thus inducing its expression and confirming that menin is a positive regulator of miR-26a. In conclusion, results from this study evidenced, for the first time, a direct interaction between menin transcription factor and miRNA, interaction that seems to play a pivotal role during the hADSCs osteogenesis, thus suggesting a novel target for bone disease RNA-based therapy.
The effects of phytoestrogens have been studied in the hypothalamic-pituitary-gonadal axis and in various non-gonadal targets. Epidemiologic and experimental evidence indicates a protective effect of phytoestrogens also in colorectal cancer. The mechanism through which estrogenic molecules control colorectal cancer tumorigenesis could possibly involve estrogen receptor beta, the predominantly expressed estrogen receptor subtype in colon mucosa.To validate this hypothesis, we therefore used an engineered human colon cancer cell line induced to overexpress estrogen receptor beta, beside its native cell line, expressing very low levels of ERbeta and not expressing ERalpha; as a phytoestrogenic molecule, we used kaempferide triglycoside, a glycosylated flavonol from a Dianthus caryophyllus cultivar. The inhibitory properties of this molecule toward vegetal cell growth have been previously demonstrated: however, no data on its activity on animal cell or information about the mechanism of this activity are available. Kaempferide triglycoside proved to inhibit the proliferation of native and estrogen receptor beta overexpressing colon cancer cells through a mechanism not mediated by ligand binding dependent estrogen receptor activation. It affected HCT8 cell cycle progression by increasing the G(0)/G(1) cell fraction and in estrogen receptor beta overexpressing cells increased two antioxidant enzymes. Interestingly, the biological effects of this kaempferide triglycoside were strengthened by the presence of high levels of estrogen receptor beta.Pleiotropic molecular effects of phytoestrogens may explain their protective activity against colorectal cancer and may represent an interesting area for future investigation with potential clinical applications.
Multiple Endocrine Neoplasia Type 2 (MEN2) is a rare hereditary complex disorder characterized by the presence of medullary thyroid carcinoma (MTC), unilateral or bilateral pheochromocytoma (PHEO) and other hyperplasia and/or neoplasia of different endocrine tissues within a single patient. MEN2 has been reported in approximately 500 to 1000 families worldwide and the prevalence has been estimated at approximately 1:30,000. Two different forms, sporadic and familial, have been described for MEN2. Sporadic form is represented by a case with two of the principal MEN2-related endocrine tumors. The familial form, which is more frequent and with an autosomal pattern of inheritance, consists of a MEN2 case with at least one first degree relative showing one of the characteristic endocrine tumors. Familial medullary thyroid carcinoma (FMTC) is a subtype of MEN2 in which the affected individuals develop only medullary thyroid carcinoma, without other clinical manifestations of MEN2. Predisposition to MEN2 is caused by germline activating mutations of the c-RET proto-oncogene on chromosome 10q11.2. The RET gene encodes a single-pass transmembrane tyrosine kinase that is the receptor for glial-derived neurotrophic growth factors. The combination of clinical and genetic investigations, together with the improved understanding of the molecular and clinical genetics of the syndrome, helps the diagnosis and treatment of patients. Currently, DNA testing makes possible the early detection of asymptomatic gene carriers, allowing to identify and treat the neoplastic lesions at an earlier stage. In particular, the identification of a strong genotype-phenotype correlation in MEN2 syndrome may enable a more individualized treatment for the patients, improving their quality of life. At present, surgical treatment offers the only chance of cure and therefore, early clinical and genetic detection and prophylactic surgery in subjects at risk are the main therapeutic goal. DefinitionMultiple Endocrine Neoplasia Type 2 (MEN2) (OMIM 171400) is a rare hereditary complex disorder characterized by the presence of medullary thyroid carcinoma (MTC), unilateral or bilateral pheochromocytoma (PHEO) and other hyperplasia and/or neoplasia of different endocrine tissues within a single patient. Two different forms, sporadic and familial, have been described for MEN2. Sporadic form is represented by a case with two of the principal MEN2-related endocrine tumors, while the
Multiple endocrine neoplasia type 1 (MEN1) syndrome is characterized by the occurrence of tumors of parathyroids, neuroendocrine cells of the gastro-enteropancreatic tract and anterior pituitary. MEN1 gene encodes menin-oncosuppressor protein. Loss of heterozygosity at 11q13 is typical of MEN1 tumors. We have analyzed the MEN1 mRNA and menin expression in fibroblasts from normal skin biopsies and from MEN1 patients (two with a frameshift 738del4 (exon 3) mutation, introducing a premature stop codon, and an individual with an R460X (exon 10) nonsense mutation). The expression of full-length menin protein did not differ between MEN1 and normal fibroblasts. Wild-type alleles mRNAs were expressed in MEN1 patients, whereas mutant alleles were partially degraded by nonsense-mediated mRNA decay pathway, suggesting a mechanism of compensation for allelic loss by the up-regulation of wild-type menin expression at a post-transcriptional level. Small-interfering RNA silencing of the wildtype mRNA allele abolished menin compensation, whereas the ribozyme silencing of the MEN1-mutated mRNA allele resulted in strongly enhanced wild-type menin expression. Gel-retardation analysis showed that in vitro-specific RNA-protein complexes bound to MEN1 mRNA. These findings contribute to the understanding of tumorigenesis in MEN1, offering the basis for the development of RNA-based therapies in MEN1 gene mutation carriers.
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