Smad3 Research Articles

A case report of sinonasal glomangiopericytoma: An important reminder to always collect specimen.

To present a case report of sinonasal glomangiopericytoma (GPC) in a female patient in her thirties and to highlight the importance of collecting pathology specimens even in routine sinus surgery cases. A case report detailing the diagnosis of GPC in a female in her thirties, including her initial presentation, treatment, and follow-up, along with a brief review of the literature. Pathology of the collected specimen revealed sinonasal GPC along with chronic rhinosinusitis. Immunohistochemistry was positive for SMA, beta-catenin, and cyclin D1; and negative for STAT6, ERG, pankeratin, SOX10, and S100. This diagnosis expands the knowledge around the demographic profile of GPC patients. GPC should be included in the differential diagnosis of sinonasal masses, even in younger patients. The case highlights the importance of collecting the entire pathology specimen in all cases, even of ones that seem routine and benign.

The Rare and Atypical Diabetes Network (RADIANT) Study: Design and Early Results.

The Rare and Atypical Diabetes Network (RADIANT) will perform a study of individuals and, if deemed informative, a study of their family members with uncharacterized forms of diabetes. The protocol includes genomic (whole-genome [WGS], RNA, and mitochondrial sequencing), phenotypic (vital signs, biometric measurements, questionnaires, and photography), metabolomics, and metabolic assessments. Among 122 with WGS results of 878 enrolled individuals, a likely pathogenic variant in a known diabetes monogenic gene was found in 3 (2.5%), and six new monogenic variants have been identified in the SMAD5, PTPMT1, INS, NFKB1, IGF1R, and PAX6 genes. Frequent phenotypic clusters are lean type 2 diabetes, autoantibody-negative and insulin-deficient diabetes, lipodystrophic diabetes, and new forms of possible monogenic or oligogenic diabetes. The analyses will lead to improved means of atypical diabetes identification. Genetic sequencing can identify new variants, and metabolomics and transcriptomics analysis can identify novel mechanisms and biomarkers for atypical disease.

Effectiveness of Apigenin, Resveratrol, and Curcumin as Adjuvant Nutraceuticals for Calvarial Bone Defect Healing: An In Vitro and Histological Study on Rats

Bone healing is a major clinical issue, especially in bone defects of critical dimensions. Some studies have reported in vivo positive effects on bone healing by some bioactive compounds, such as the phenolic derivatives found in vegetables and plants, such as resveratrol, curcumin, and apigenin. The aim of this work was (1) to analyze in vitro in human dental pulp stem cells the effects of these three natural compounds on the gene expression of related genes downstream to RUNX2 and SMAD5, key factor transcriptions associated with osteoblast differentiation, in order to better understand the positive effects that can occur in vivo in bone healing, and (2) to evaluate in vivo the effects on bone healing of critical-size defects in the calvaria in rats of these three nutraceuticals tested in parallel and for the first time administered by the gastric route. Upregulation of the RUNX2, SMAD5, COLL1, COLL4, and COLL5 genes in the presence of apigenin, curcumin, and resveratrol was detected. In vivo, apigenin induced more consistent significant bone healing in critical-size defects in rat calvaria compared to the other study groups. The study findings encourage a possible therapeutic supplementation with nutraceuticals during the bone regeneration process.

Downregulated FGFR3 Expression Inhibits In Vitro Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells of Mice with TBXT Gene Mutation.

We studied the effect of fibroblast growth factor receptor 3 (FGFR3) inhibitor BGJ-398 on the differentiation of bone marrow mesenchymal stem cells (BM MSC) into osteoblasts in wild type (wt) mice and in animals with mutation in TBXT gene (mt) and possible differences in the pluripotency of these cells. Cytology tests showed that the cultured BM MSC could differentiate into osteoblasts and adipocytes. The effect of different BGJ-398 concentrations on the expression of FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 were studied by quantitative reverse transcription PCR. The expression of RUNX2 protein was evaluated by Western blotting. BM MSC of mt and wt mice did not differ in pluripotency and expressed the same membrane marker antigens. BGJ-398 inhibitor reduced the expression of FGFR3 and RUNX2. In BM MSC from mt and wt mice have similar gene expression (and its changing) in FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 genes. Thus, our experiments confirmed the effect of decreased expression of FGFR3 on osteogenic differentiation of BM MSC from wt and mt mice. However, BM MSC from mt and wt mice did not differ in pluripotency and are an adequate model for laboratory research.

Molecular Characterization, Expression Profiles of SMAD4, SMAD5 and SMAD7 Genes and Lack of Association with Litter Size in Tibetan Sheep.

Simple SummarySelection of molecular markers used in the marker-assisted selection of the litter size of sheep is critically essential. In this study, we identified single nucleotide polymorphisms (SNPs), g.51537A>G in SMAD5 and g.319C>T in SMAD7, from Tibetan sheep, and they were genotyped. The expression patterns of SMAD4, SMAD5 and SMAD7 in the tissues of Tibetan sheep were determined. Furthermore, we also performed an association analysis of the SNPs with litter size. The results indicated that SMAD4, SMAD5 and SMAD7 were extensively expressed in the various tissues of Tibetan sheep. Sequence analysis showed that SMAD4 contained 1662 bp encoding 533 amino acids, SMAD5 contained 1667 bp encoding 465 amino acids and SMAD7 contained 1408 bp encoding 427 amino acids. Alignment analysis showed that the amino acid sequence of the three proteins shared high similarity with sheep, yak, cattle, dog, human, pig, chimpanzee, rhesus monkey and house mouse. Furthermore, association analysis showed that both g.51537A>G in SMAD5 and g.319C>T in SMAD7 were not significantly associated with litter size of Tibetan sheep.SMAD4, SMAD5 and SMAD7 belonging to the transforming growth factor β (TGF-β) superfamily are indispensable for oocyte formation and development, ovarian organogenesis and folliculogenesis. However, only a few studies have investigated the characteristics of SMAD4, SMAD5 and SMAD7 in Tibetan sheep and the effect of their polymorphism on litter size. In this study, we examined the expression of SMAD4, SMAD5 and SMAD7 in 13 tissues of Tibetan sheep by reverse transcription-quantitative polymerase chain reaction. Further, cDNA of these genes was cloned, sequenced and subjected to bioinformatics analysis. DNA sequencing was also used to detect single nucleotide polymorphisms (SNPs). However, iM-LDRTM technology was used for SNP genotyping. Associations between polymorphisms and litter size were analyzed using data from genotyping of 433 Tibetan sheep. The results showed that the expression of SMAD4, SMAD5 and SMAD7 genes was ubiquitous in the tissues of Tibetan sheep, such as the ovary, uterus and oviduct, hypothalamus, hypophysis, heart, liver, spleen, lung, kidney, rumen, duodenum and longissimus dorsi. However, the expression was unbalanced and upregulated in the spleen, lung, ovary and uterus and downregulated in the longissimus dorsi. The bioinformatics analysis showed that SMAD4, SMAD5 and SMAD7 in Tibetan sheep encoded proteins of 533, 465 and 427 amino acids, respectively. Sequence homology analysis of the three proteins among other animals showed that the sequences of SMAD4, SMAD5 and SMAD7 of Tibetan sheep were similar to those in sheep, yak, cattle, dog, human, pig, chimpanzee, rhesus monkey and house mouse. Two synonymous mutations, g.51537A>G and g.319C>T, were detected in SMAD5 and SMAD7, respectively. The associations of these SNPs and litter size were determined, and it was found that both g.51537A>G and g.319C>T have no significant effect on the litter size of Tibetan sheep. The results provided novel insights into the molecular characterization, expression profiles and polymorphisms of SMAD4, SMAD5 and SMAD7 in Tibetan sheep, but our results do not support associations between these genes and the litter size of Tibetan sheep.

Effect of genetic variants in the SMAD1 and SMAD5 genes promoter on growth and beef quality traits in cattle

The SMAD1 and SMAD5 genes belong to mothers against decapentaplegic proteins family, which participate in the BMP pathway to control skeletal myogenesis and growth. In the present study, we analyzed the associations between polymorphisms of SMAD1 and SMAD5 genes promoter and important economical traits in Qinchuan cattle. Four SNPs in the SMAD1 gene promoter and three SNPs in the SMAD5 promoter were identified by sequencing of 448 Qinchuan cattles. Allelic and frequency analyses of these SNPs resulted in eight haplotypes both in the promoters of the two genes promoter and identified potential cis-regulatory transcription factor (TF) components. In addition, correlation analysis showed that cattle SMAD1 promoter activity of individuals with Hap4 (P < 0.01) was stronger than that of individuals with Hap2. while the transcriptional activity of individuals with Hap3 within SMAD5 gene promoter was significantly (P < 0.01) higher followed by H2. Uniformly, diplotypes H4-H6 of SMAD1 gene and H1-H3 of SMAD5 gene performed significant (P < 0.01) associations with body measurement and improved carcass quality traits. All these results have indicated that polymorphisms in SMAD1 and SMAD5 genes promoter could impact the transcriptional regulation and then affect muscle content in beef cattle. Moreover, both the SMAD1 and SMAD5 genes were expressed ubiquitously in 10 tissues and had higher expression in the longissimus thoracis tissue from 6-month-old and 12-month-old cattle than in cattle of other ages. We can conclude that SMAD1 and SMAD5 genes may play an important role in muscle growth and development, and the variants mapped within SMAD1 and SMAD5 genes can be utilized in molecular marker-assisted selection for cattle carcass quality and body measurement traits in breed improvement programs of Qinchuan cattle.

Expression of transforming growth factor β pathway components in chronic graft-versus-host disease after allogeneic hematopoietic cell transplantation

Chronic graft-versus-host disease (cGvHD), an immunological complication of allogeneic cell transplantation, is the principal cause of non-relapse mortality and morbidity. Even though advances have been made in understanding the pathophysiology of this disorder, many questions remain. We sought to evaluate gene expression of transforming growth factor β (TGF-β) pathway components, through quantitative RT-PCR and PCR array, in patients with cGvHD with different disease activity. We observed an upregulation of SMAD3, BMP2, CDKN1A, IL6, and TGF-β2 genes in the clinical tolerance group, which had never developed cGvHD, or which had been withdrawn from all immunosuppressive treatments (IST) for at least 1 year. In addition, SMAD5 gene upregulation was observed in cGvHD patients undergoing IST, and ordinal regression showed a correlation between SMAD5 expression and disease severity. Our data support the evidence of the important role of TGF-β effects in the pathological process of cGvHD.

Predictive model based on gene and laboratory data for intravenous immunoglobulin resistance in Kawasaki disease in a Chinese population

BackgroundHere, we investigated the predictive efficiency of a newly developed model based on single nucleotide polymorphisms (SNPs) and laboratory data for intravenous immunoglobulin (IVIG) resistance in Kawasaki disease (KD) in a Chinese population.MethodsData relating to children with KD were acquired from a single center between December 2015 and August 2019 and used to screen target SNPs. We then developed a predictive model of IVIG resistance using previous laboratory parameters. We then validated our model using data acquired from children with KD attending a second center between January and December 2019.ResultsAnalysis showed that rs10056474 GG, rs746994GG, rs76863441GT, rs16944 (CT/TT), and rs1143627 (CT/CC), increased the risk of IVIG-resistance in KD patients (odds ratio, OR > 1). The new predictive model, which combined SNP data with a previous model derived from laboratory data, significantly increased the area under the receiver-operator-characteristic curves (AUC) (0.832, 95% CI: 0.776-0.878 vs 0.793, 95%CI:0.734-0.844, P < 0.05) in the development dataset, and (0.820, 95% CI: 0.730-0.889 vs 0.749, 95% CI: 0.652-0.830, P < 0.05) in the validation dataset. The sensitivity and specificity of the new assay were 65.33% (95% CI: 53.5-76.0%) and 86.67% (95% CI: 80.2-91.7%) in the development dataset and 77.14% (95% CI: 59.9-89.6%) and 86.15% (95% CI: 75.3-93.5%) in the validation dataset.ConclusionAnalysis showed that rs10056474 and rs746994 in the SMAD5 gene, rs76863441 in the PLA2G7 gene, and rs16944 or rs1143627 in the interleukin (IL)-1B gene, were associated with IVIG resistant KD in a Chinese population. The new model combined SNPs with laboratory data and improved the predictve efficiency of IVIG-resistant KD.

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Human Dental Pulp Stem Cell Osteogenic Differentiation Seeded on Equine Bone Block with Graphene and Melatonin

Equine bone blocks have osteogenic effects promoting bone regeneration with biocompatibility and osteoconductivity capacity. Human dental pulp stem cells (hDPSCs) can differentiate into osteoblasts enhancing biomineralization with such scaffolds. Melatonin is able to improve bone health and mediate bone formation. Collagenated equine bone blocks were coated with ammonia-functionalized graphene-oxide (G-N) at two different concentrations (2 μg/mL, G-N2; and 10 μg/mL, G-N10). The homogeneity of G-N coating was checked by Raman spectroscopy, whereas thermogravimetric analysis (TGA) allowed us to quantify the amount of G-N deposited on the blocks. The aim of this study was to investigate in vitro the effect of G-N-coated collagenated equine bone blocks on the proliferation and differentiation of hDPSCs with the addition of a melatonin. This evaluation was determined after 7, 14, and 21 days of culture by the expression of specific microRNAs, RUNX2 and SMAD5 gene expression, osteocalcin levels, and histological analysis. The results showed that equine blocks G-N2 and G-N10 and melatonin gave an optimal cell adhesion as shown by histological analysis, and an increase in the hDPSCs osteogenic potential as confirmed by microRNA and gene expression with an increase in osteocalcin levels. This study suggests that equine bone blocks coated with G-N2 and G-N10 and melatonin promote the osteogenic process.

MiR-21 regulates pulmonary hypertension in rats via TGF-β1/Smad2 signaling pathway.

The article "MiR-21 regulates pulmonary hypertension in rats via TGF-β1/Smad2 signaling pathway, by F. Ding, T. You, X.-D. Hou, K. Yi, X.-G. Liu, P. Zhang, X.-K. Wang, published in Eur Rev Med Pharmacol Sci 2019; 23 (9): 3984-3992-DOI: 10.26355/eurrev_201905_17828-PMID: 31115027" has been withdrawn from the authors. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/17828.

Reduced long noncoding RNA PGM5-AS1 facilitated proliferation and invasion of colorectal cancer through sponging miR-100-5p.

We aimed at investigating the expression of Long non-coding RNA (LncRNA) PGM5-AS1 and its facilitating effects on proliferation and invasion of colorectal cancer by sponging miR-100-5p. qRT-PCR was performed to detect the expressions of PGM5-AS1 and SMAD4 in human colorectal cancer tissues and cells. CCK-8 assay was performed to evaluate the SW403 cells proliferation and transwell assay was performed to evaluate the SW403 cells migration. The correlation between miR-100-5p and PGM5-AS1 was detected by statistical analysis. Bioinformatics prediction and Luciferase assay were performed to explore the interaction and binding site of PGM5-AS1 and miR-100-5p, miR-100-5p and SMAD4, respectively. We found that both PGM5-AS1 and SMAD4 were downregulated in human colorectal cancer tissues and cells. qRT-PCR and CCK-8 assay showed that PGM5-AS1 expression is associated with the proliferation of colorectal cancer cells. Transwell assay showed that PGM5-AS1 regulated the migration ability of colorectal cancer cells. The bioinformatics prediction and Luciferase assay demonstrated that by sponging miR-100-5p, PGM5-AS1 can serve as a molecular sponge to further regulate the expression of SMAD4. In this study, we found that lncRNA-PGM5-AS1 was low expressed in human colorectal cancer cells, which could promote tumor proliferation, migration and invasion by serving as a molecular sponge and by modulating the inhibitory effect of miR-100-5p on tumor suppressor gene SMAD4.

MiR-195 promotes myocardial fibrosis in MI rats via targeting TGF-β1/Smad.

The aim of this study was to investigate the effect of micro ribonucleic acid (miR)-195 on myocardial infarction (MI) in rats via regulating the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway. A total of 36 Sprague-Dawley rats were randomly divided into a normal group (n=12), a model group (n=12) and an miR-195 antagomir group (n=12). In the normal group, the heart was exposed only, and normal saline was intraperitoneally injected after operation. In the model group, the acute MI model was established. In the miR-195 antagomir group, the acute MI model was also established, and miR- 195 antagomir was intraperitoneally injected. The samples were collected at 2 weeks after surgery. Then cardiac function was detected via echocardiography, and the morphology of heart tissues was observed via hematoxylin and eosin (H&E) staining. Moreover, the expression of Collagen I was determined using immunohistochemistry, the protein expressions of TGF-β1, Smad3 and Smad7 were detected using Western blotting, and the expression of miR-195 was detected via quantitative polymerase chain reaction (qPCR). It was found by echocardiography that, compared with those in the normal group, left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) significantly declined, while left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs) significantly rose in the other two groups (P<0.05). In comparison with the model group, the miR-195 antagomir group had significantly increased LVEF and LVFS, and significantly decreased LVDd and LVDs (P<0.05). The immunohistochemistry results showed that the mean optical density of tissues with positively expressed Collagen I was obviously higher in the other two groups than that in the normal group (P<0.05), while it was obviously lower in the miR-195 antagomir group than that in the model group (P<0.05). According to the results of Western blotting, the protein expressions of TGF-β1 and Smad3 were evidently increased, while the protein expression of Smad7 was evidently decreased in the other two groups compared with those in the normal group (P<0.05). The opposite results were found in the miR-195 antagomir group compared with those in the model group (P<0.05). The results of qPCR manifested that the expression of miR-195 was markedly higher in the other two groups than that in the normal group (P<0.05), while it was markedly lower in the miR-195 antagomir group than in the model group (P<0.05). Moreover, it was observed using H&E staining that the myocardial fibers in the normal group had normal arrangement and intact structure, without obvious morphological abnormalities. In the model group, the myocardial fibers were arranged disorderly, and there were massive proliferating fibrous tissues, with a high degree of fibrosis. In themiR-195 antagomir group, the myocardial fibers were damaged and arranged less disorderly, and proliferation and fibrosis could be seen in some fibrous tissues, but to a lesser extent than the model group. In conclusion, miR-195 promotes myocardial fibrosis in MI rats via up-regulating the TGF-β1/Smad signaling pathway.

MiR-145-5p alleviates hypoxia/reoxygenation- induced cardiac microvascular endothelial cell injury in coronary heart disease by inhibiting Smad4 expression.

To investigate the effects and mechanism of miR-145-5p on hypoxia/reoxygenation (H/R)-induced cardiac microvascular endothelial cell (CMEC) injury in coronary heart disease (CHD). Patients with CHD (n=96) and healthy volunteers (n=96) were enrolled, and H/R injury model of CMECs was established. The expression of miR-145-5p and mothers against decapentaplegic homolog 4 (Smad4) mRNA in cells was quantified with reverse transcription polymerase chain reaction (RT-PCR). Then, miR-145-5p mimics and Smad4 inhibitor were transfected into CMECs. Cell counting kit-8 (CCK-8) was employed for proliferation detection, flow cytometry for apoptosis detection, and Western Blot for measuring the expression of apoptosis-related proteins and Smad4 protein. The expression of serum miR-145-5p in patients with CHD was significantly lower than that in healthy individuals. The area under the curve (AUC) of miR-145-5p in diagnosing CHD was 0.894, and the expression of miR-145-5p was negatively correlated with that of Smad4 (p<0.05). Over-expression of miR-145-5p promoted the proliferation, inhibited the apoptosis, and reduced inflammatory responses and oxidative stress in H/R-injured CMECs. Moreover, miR-145-5p might negatively regulate the expression of Smad4 in CMECs. Dual-Luciferase reporter assay determined the targeting relation between miR-145-5p and Smad4. MiR-145-5p is lowly expressed in patients with CHD, and its over-expression effectively alleviates H/R-induced CMEC injury by inhibiting Smad4.

MiR-34 promotes apoptosis of lens epithelial cells in cataract rats via the TGF-β/Smads signaling pathway.

To discuss the effect of micro ribonucleic acid (miR)-34 on the lens epithelial cell functions in the cataract rats. Differentially expressed miRNAs in the lens epithelial cells of the cataract rats were screened out by analyzing microarrays. The lens epithelial cells of the cataract rats transfected with miR-34 mimics were set as transfection group. Cells with silenced transforming growth factor-β (TGF-β) using RepSox were regarded as the transfection + inhibitor group, and the cells transfected with NC constituted control group. Relative expressions of miR-34, key genes in the TGF-β/Smads signaling pathway and apoptosis-related proteins [B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), Bcl-2 associated K protein (Bak), caspase-9 and surviving] in the control group, transfection group, and transfection + inhibitor group were detected. The proportions of apoptotic cells in the three groups were determined via flow cytometry. The differentially expressed miRNAs in the lens epithelial cells of the cataract rats included miR-5, miR-128, etc. Among the tested miRNAs, miR-34 presented remarkably downregulated expression [log2 fold change (FC) =-2.11, p=0.000]. After the lens epithelial cells of the cataract rats were transfected with miR-34 mimics, the expression of miR-34 was evidently elevated (p=0.000), while the expressions of TGF-β, Smad1, and Smad3 were significantly up-regulated. Following the treatment with the TGF-β inhibitor RepSox, the expressions of TGF-β, Smad1, and Smad3 were downregulated. After transfection of miR-34 mimics in lens epithelial cells of the cataract rats, upregulated Bax and Bak, downregulated Bcl-2 and surviving, and elevated apoptosis rate were observed. After the TGF-β inhibitor RepSox was added, the expressions of Bax and Bak declined prominently, while those of Bcl-2 and survivin were on the contrary, manifesting a declining cell apoptosis rate. The expression of caspase-9 had no significant change among the three groups. The proportion of apoptotic cells in control group, transfection group, and transfection + inhibitor group was 2.33%, 38.14%, and 16.88%, respectively, displaying differences among the three groups (p=0.002). MiR-34 can promote the apoptosis in lens epithelial cells of cataract rats via the TGF-β/Smads signaling pathway.

MiR-424 Inhibits neuronal apoptosis in rats with cerebral infarction through regulating TGF-β1/Smad3 signaling pathway.

It has been reported that micro ribonucleic acid (miR)-424 is an important molecule in cerebral ischemia. However, the precise mechanism of action and biological effects of miR-424 remain to be further explored. miR-424 mimic and miR-424 inhibitor were injected via the caudal vein in rats, and the effect of miR-424 expression on brain tissue damage induced by middle cerebral artery occlusion (MCAO) was detected. The miR-424 mimic-induced changes in genomic levels were detected via the gene chip assay, and the signaling pathways regulated by miR-424 and its potential targets were explored combined with target prediction. Then the effect of miR-424 mimic on apoptosis of PC12 cells induced by oxygen-glucose deprivation (OGD) was determined using Annexin V/PI assay. Finally, drosophila mothers against decapentaplegic protein 7 (Smad7) was overexpressed to further verify the mechanism of action of miR-424 mimic. Compared with that in the sham group, the expression of miR-424 in brain tissues significantly declined in the model group. The results of 2,3,5-triphenyltetrazolium chloride (TTC) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed that the miR-424 mimic obviously reduced the cerebral infarction area and apoptosis level of brain tissues, while the miR-424 inhibitor obviously increased the cerebral infarction area and apoptosis level of brain tissues. It was found, using bioinformatics and KEGG enrichment analysis, that differentially expressed genes induced by miR-424 were significantly enriched in the transforming growth factor-β (TGF-β) signaling pathway. According to the results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting, the miR-424 mimic could evidently lower the expression of Smad7, thus activating the TGF-β1/Smad3 signaling pathway. Overexpression of Smad7 could weaken the protective effect of miR-424 mimic on ischemic-hypoxic cells. Increasing the expression of miR-424 can inhibit Smad7 to activate the TGF-β1/Smad3 signaling pathway, thereby exerting a protective effect against the brain tissue damage induced by MCAO.

Upregulation of circular SMAD7 inhibits tumorigenesis of gastric cancer by reversing epithelial-to-mesenchymal transition.

Gastric cancer (GC) is one of the most ordinary malignant tumors. Recent studies have revealed that circular RNAs (circRNAs) play an important role in the progression of tumorigenesis. In this research, circ-SMAD7 was selected to identify how it functions in the progression of GC. Circ-SMAD7 expression in paired GC patients' tissue samples and cell lines was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The role of circ-SMAD7 in the metastasis of GC was detected through wound healing assay and transwell assay. Western blot assay and RT-qPCR were used to discover the function of circ-SMAD7 in epithelial-to-mesenchymal transition (EMT) process. Furthermore, tumor metastasis assay was also performed in vivo. In this study, RT-qPCR results showed that circ-SMAD7 expression was significantly lower in GC tissues compared to that in adjacent ones. Cell migration and invasion of GC were inhibited via upregulation of circ-SMAD7. Moreover, the results of further experiments revealed that the EMT-related proteins were regulated via upregulation of circ-SMAD7 in GC. Furthermore, tumor metastasis of GC was inhibited via upregulation of circ-SMAD7 in nude mice. These results indicate that upregulation of circ-SMAD7 inhibits GC cell migration and invasion via reversing the EMT process.

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Effect of astragalus polysaccharide combined with urine stem cell transplantation on type 2 diabetes in rats

Objective To study the effect of astragalus polysaccharides combined with hUSCs transplantation on type 2 diabetic rats. Methods Twenty-five SD rats were randomly selected into the normal control group, and the remaining 105 SD rats were used to establish type 2 diabetes model. The 100 rats successfully modeled were randomly divided into the diabetes group, astragalus polysaccharide treatment group, hUSCs treatment group, and astragalus polysaccharide + hUSCs treatment group, with 25 rats in each group. After 2 weeks of treatment, the FBG concentration, insulin and C-peptide concentrations, and body weight changes were measured in each group. The distribution and survival of PKH-26-labeled hUSCs in rat pancreatic tissue were observed by fluorescence microscopy. TUNEL method was used to detect the apoptosis of rat islet cells. Real-time quantitative PCR and Western Blot were used to detect the expression of TGF-β/Smad signaling pathway-related genes in rat pancreatic tissue. Results The FBG concentration of rats in the astragalus polysaccharide treatment group, hUSCs treatment group and astragalus polysaccharide+hUSCs treatment group were significantly decreased, and that in the combination treatment group was significantly lower those in the astragalus polysaccharide group and hUSCs group, and the differences were statistically significant(all P<0.05). Compared with the diabetic group, the insulin concentration, C-peptide concentration and body weight in the astragalus polysaccharide treatment group, hUSCs treatment group and combination treatment group rats were significantly increased, and those in the combination treatment group was significantly higher than those in the astragalus polysaccharide treatment group and in the hUSCs treatment group, the differences were statistically significant (all P<0.05). The results of fluorescence microscopy showed that the number of PKH-26 positive hUSCs in the combined treatment group was 74.64±9.75 in each high power field, which was significantly higher than that in the hUSCs treatment group (43.64±5.83), the difference was statistically significant (P<0.05). Compared with the diabetic group, the apoptotic rates of islet cells in the astragalus polysaccharide treatment group and the hUSCs treatment group were reduced, and the relative expressions levels of mRNA and protein of TGF-β1, Smad3, and Smad7 in the pancreatic tissue were also significantly reduced(all P<0.05). The reduction was more significant in the combination treatment group, and the differences were statistically significant (all P<0.05). Conclusions Astragalus polysaccharide combined with hUSCs transplantation can effectively reduce the FBG concentration, increase the concentration of insulin, C-peptide and body weight, reduce the apoptosis of pancreatic islet tissue, which may be related to the reduction of TGF-β/Smad in pancreatic tissue. Signaling pathways are involved in suppressing the inflammatory response. Key words: Astragalus polysaccharide; Human urine-derived stem cells; Transplantation; Diabetes mellitus, type 2; Rat

Next generation sequencing identifies novel potential actionable mutations for grade I meningioma treatment.

Meningiomas are common brain tumors that arise from the meningeal membranes that envelope the brain and spinal cord. The World Health Organization classifies these tumors into three histopathological grades. Because of tumor recurrence, treating meningiomas may be challenging even in well-differentiated grade I (GI) neoplasms. Indeed, around 5% of completely resected GI meningiomas relapse within 5 years. Therefore, identifying driver mutations in GI meningiomas through next generation sequencing (NGS) assays is paramount. The aim of this study was to validate the use of the 50-gene AmpliSeq Hotspot Cancer Panel v2 to identify the mutational status of 23 GI meningioma, namely, 12 non recurrent and 11 recurrent. In 18 out of the 23 GI meningiomas analyzed, we identified at least one gene mutation (78.2%). The most frequently mutated genes were c-kit (39.1%), ATM (26.1%), TP53 (26.1%), EGFR (26.1%), STK11 (21.7%), NRAS (17.4%), SMAD4 (13%), FGFR3 (13%), and PTPN11 (13%); less frequent mutations were SMARCB1 (8.7%), FLT3 (8.7%), KRAS (8.7%), FBWX7 (8.7%), ABL1 (8.7%), ERBB2 (8.7%), IDH1 (8.7%), BRAF (8.7%), MET (8.7%), HRAS (4.3%), RB1 (4.3%), CTNNB1 (4.3%), PIK3CA (4.3%), VHL (4.3%), KDR (4.3%), APC (4.3%), NOTCH1 (4.3%), JAK3 (4.3%), and SRC (4.3%). To our knowledge, mutations in all of these genes, except for TP53, STK11, SMARCB1, PIK3CA, VHL, and BRAF, have never been described before in meningiomas. Hence, these findings demonstrate the viability of NGS to detect new genetic alterations in GI meningiomas. Equally important, this technology enabled us to detect possible novel actionable mutations not previously associated with GI and for which selective inhibitors already exist.