miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

Exploring the Effect of Jiawei Buguzhi Pills on TGF-β-Smad Pathway in Postmenopausal Osteoporosis Based on Integrated Pharmacological Strategy

Objective. To explore the effect of Jiawei Buguzhi Pills (JWBGZP) on the TGF-β-Smad pathway in postmenopausal osteoporosis (PMO) based on integrated pharmacological strategy. Method. The ETCM database was used to collect JWBGZP. GeneCards and OMIM databases were utilized to obtain PMO-related genes. Cytoscape was used for network construction and analysis, and DAVID was used for GO and KEGG enrichment analysis of key targets. Animal experiments and cell experiments were conducted to further explore the mechanism. The bone mass density was detected by dual-energy X-ray bone densitometer. The TGF-β1 and Smad4 mRNA in bone tissue were detected by RT-qPCR. The TGF-β1 and Smad4 protein in bone tissue were detected by the western blot. The TGF-β1 and Smad4 protein in osteoblasts were determined by immunohistochemistry. Result. A total of 721 JWBGZP potential targets and 385 PMO-related genes were obtained. The enrichment analysis showed that JWBGZP may regulate the TGF-beta signaling pathway, oxidation-reduction process, aging, response to hypoxia, response to ethanol, negative regulation of cell proliferation, PI3K-Akt, HIF-1, and other signaling pathways. The animal experiments showed that compared with the model group, the femoral bone mineral density and lumbar bone mineral density of the JWBGZP group increased (P < 0.05); the expression levels of TGF-β1 and Smad mRNA and proteins in the JWBGZP group were significantly higher (P < 0.05). The cell experiment results showed a large number of osteoblast stained blue-purple and orange-red calcified nodules. The expression levels of TGF-β1 and Smad proteins in the JWBGZP group were significantly higher than those in the blank control group and the sham operation group, and the protein expression levels in the model group were the lowest (P < 0.05). Conclusion. JWBGZP may be involved in PI3K-Akt, HIF-1, estrogen, prolactin, and other signaling pathways and regulate MAPK1, AKT1, PIK3CA, JAK2, and other gene targets, regulate bone metabolism, and thereby treat PMO.

SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors

Background Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear. Methods Utilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/− mouse model was used to detect entero-endocrine cell hyperplasia with IHC. Results Analyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/− mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease. Conclusions Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors

Background Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.MethodsUtilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/- mouse model was used to detect entero-endocrine cell hyperplasia with IHC.ResultsAnalyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/- mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease.Conclusions Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

SMAD4 Overexpression in Patients with Sleep Apnoea May Be Associated with Cardiometabolic Comorbidities

Obstructive sleep apnoea (OSA) is associated with several diseases related to metabolic and cardiovascular risk. Although the mechanisms involved in the development of these disorders may vary, OSA patients frequently present an increase in transforming growth factor beta (TGFβ), the activity of which is higher still in patients with hypertension, diabetes or cardiovascular morbidity. Smad4 is a member of the small mother against decapentaplegic homologue (Smad) family of signal transducers and acts as a central mediator of TGFβ signalling pathways. In this study, we evaluate Smad4 protein and mRNA expression from 52 newly diagnosed OSA patients, with an apnoea–hypopnoea index (AHI) ≥30 and 26 healthy volunteers. These analyses reveal that OSA patients exhibit high levels of SMAD4 which correlates with variation in HIF1α, mTOR and circadian genes. Moreover, we associated high concentrations of Smad4 plasma protein with the presence of diabetes, dyslipidaemia and hypertension in these patients. Results suggest that increased levels of SMAD4, mediated by intermittent hypoxaemia and circadian rhythm deregulation, may be associated with cardiometabolic comorbidities in patients with sleep apnoea.

SMAD4 Haploinsufficiency in Small Intestinal Neuroendocrine Tumors

Background Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations in a single gene, CDKN1B, with heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.MethodsUtilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/- mouse model was used to detect entero-endocrine cell hyperplasia with IHC.ResultsAnalyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/- mouse model. Conclusions Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.