[Clinical phenotype and genetic analysis of six Chinese patients affected with Acromicric dysplasia due to variants of FBN1 gene].

OBJECTIVE: To retrospectively analyze the clinical and genetic characteristics of six patients with Acromicric dysplasia due to variants of the FBN1 gene. METHODS: Six patients who had visited the Affiliated Hospital of Qingdao University between February 2018 and October 2020 were selected as the study subjects. Clinical data of the patients were collected. High-throughput sequencing was carried out. And candidate variants were verified by Sanger sequencing. RESULTS: All of the six patients had presented with severe short stature (< 3s), brachydactyly, short and broad hands and feet. Other manifestations included joint stiffness, facial dysmorphism, delayed bone age, liver enlargement, coracoid femoral head, and lumbar lordosis. Genetic testing revealed that all had harbored heterozygous variants of the FBN1 gene. Patient 1 had harbored a c.5183C>T (p.A1728V) missense variant in exon 42, which had derived from his father (patient 2). Patient 3 had harbored a c.5284G>A (p.G1762S) missense variant in exon 43, which had derived from her mother (patient 4). Patient 5 had harbored a c.5156G>T (p.C1719F) missense variant in exon 42, which was de novo in origin. Patient 6 had harbored a c.5272G>T (p.D1758Y) missense variant in exon 43, which was also de novo in origin. The variants carried by patients 1, 3 and 6 were known to be pathogenic. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the FBN1: c.5156G>T was rated as a pathogenic variant (PS2+PM1+PM2_Supporting +PM5+PP3). CONCLUSION: All of the six patients had severe short stature and a variety of other clinical manifestations, which may be attributed to the variants of the FBN1 gene.

Six1 Promotes Epithelial-Mesenchymal Transition in Bronchial Epithelial Cells via the TGFß1/Smad Signalling Pathway.

INTRODUCTION: The homeodomain transcription factor sine oculis homeobox homolog 1 (Six1) plays a crucial role in embryogenesis and is not expressed in normal adult tissue but is expressed in many pathological processes, including airway remodelling in asthma. The current study aimed to reveal the effects of Six1 in regulating the airway remodelling and its possible mechanism. METHODS: A mouse model of ovalbumin-induced asthma-associated airway wall remodelling and a bronchial epithelial cell (16HBE) model of transforming growth factor ß1 (TGFß1)-induced epithelial-mesenchymal transition (EMT) were used to investigate the role of Six1. Then, 16HBE cells were transformed with Six1 expression vectors and treated with a TGFß1 pathway inhibitor to determine the role of Six1 in EMT. The effect of Six1 and its possible mechanism were assessed by immunohistochemistry, RT-PCR, and Western blot. RESULTS: Six1 expression was elevated in the lungs in an OVA mouse model of allergic asthma and in 16HBE cells treated with TGFß1. Six1 overexpression promoted an EMT-like phenotype with a decreased protein expression of E-cadherin and increased protein expression of α-smooth muscle actin (α-SMA) as well as fibronectin in 16HBE cells; these effects appeared to promote TGFß1 and phospho-Smad2 (pSmad2) production, which are the main products of the TGFß1/Smad signalling pathway, which could be reduced by a TGFß1 inhibitor. CONCLUSION: These data reveal that Six1 and TGFß1 are potentially a part of an autocrine feedback loop that induces EMT, and these factors can be reduced by blocking the TGFß1/Smad signalling pathway. As such, these factors may represent a promising novel therapeutic target for airway remodelling in asthma.

Mutations in ASH1L confer susceptibility to Tourette syndrome.

Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by repetitive motor movements and vocal tics. The clinical manifestations of TS are complex and often overlap with other neuropsychiatric disorders. TS is highly heritable; however, the underlying genetic basis and molecular and neuronal mechanisms of TS remain largely unknown. We performed whole-exome sequencing of a hundred trios (probands and their parents) with detailed records of their clinical presentations and identified a risk gene, ASH1L, that was both de novo mutated and associated with TS based on a transmission disequilibrium test. As a replication, we performed follow-up targeted sequencing of ASH1L in additional 524 unrelated TS samples and replicated the association (P value = 0.001). The point mutations in ASH1L cause defects in its enzymatic activity. Therefore, we established a transgenic mouse line and performed an array of anatomical, behavioral, and functional assays to investigate ASH1L function. The Ash1l+/- mice manifested tic-like behaviors and compulsive behaviors that could be rescued by the tic-relieving drug haloperidol. We also found that Ash1l disruption leads to hyper-activation and elevated dopamine-releasing events in the dorsal striatum, all of which could explain the neural mechanisms for the behavioral abnormalities in mice. Taken together, our results provide compelling evidence that ASH1L is a TS risk gene.

MiR-204-5p Inhibits Transforming Growth Factor-ß1-Induced Proliferation and Extracellular Matrix Production of Airway Smooth Muscle Cells by Regulating Six1 in Asthma.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1)-in-duced proliferation of airway smooth muscle cells plays critical roles in the development of airway remodeling. Six1 (sine oculis homeobox homolog 1) has been demonstrated to be involved in airway inflammation and remodeling in asthmatic mice. OBJECTIVES: The aim of this work was to investigate the potential role of miR-204-5p in the proliferation and extracellular matrix (ECM) production of airway smooth muscle cells in asthma. METHODS: Real-time PCR was used to measure the expression of miR-204-5p in asthmatic airway smooth muscle cells. Cell viability and apoptosis were detected to evaluate the effect of miR-204-5p on airway smooth muscle cells. Dual-luciferase reporter experiments were applied to identify the target genes of miR-204-5p. RESULTS: MiR-204-5p was downregulated notably in asthmatic airway smooth muscle cells as well as cells stimulated with TGF-ß1. Overexpression of miR-204-5p markedly suppressed the TGF-ß1-induced proliferation of airway smooth muscle cells and the deposition of ECM, whereas the inhibition of miR-204-5p significantly enhanced the proliferation of airway smooth muscle cells and upregulated the level of fibronectin and collagen III. Furthermore, subsequent analyses demonstrated that Six1 was a direct target of miR-204-5p, and Western blot further indicated that miR-204-5p negatively regulated the expression of Six1. Most importantly, the restoration of Six1 expression reversed the inhibitory effect of miR-204-5p on TGF-ß1-induced proliferation and ECM production. CONCLUSIONS: MiR-204-5p inhibits TGF-ß1-in-duced proliferation and ECM production of airway smooth muscle cells by regulating Six1, identifying a potential therapeutic target for preventing airway remodeling in asthma.

Correlation between oxidative stress and NF-κB signaling pathway in the obesity-asthma mice.

In this study, a mice model of obesity-asthma was established. We investigated the correlation between oxidative stress and NF-κB signaling pathway in the lung tissues, together with the effects of acetylcysteine. The animals were fed on a high-fat diet, and then ovalbumin (OVA) sensitization was utilized to establish the obesity-asthma model. N-acetylcysteine was used to treat asthma, animals treated with budesonide served as control. The malondialdehyde (MDA) in the lung tissues was determined, together with the activity of glutathione (GSH). EMAS assay was utilized to measure the nuclear factor-κB-P65 (NF-κB-P65) in lung tissues. Western blot analysis was performed to determine the expression of inhibitor kappa B-α (IκB-α) and inhibitor kappa B kinase-ß (IKK-ß). The MDA in the asthma groups showed significantly elevation (P < 0.01), and the GSH showed significant decrease (P < 0.01), especially in the obesity-asthma group. The efficiency of N-acetylcysteine was superior to that of the budesonide in the decline of MDA and elevation of GSH (P < 0.01). In both asthma groups, the expression of IKK-ß and transcription of NF-κB-P65 in the lung tissues showed significant elevation (P < 0.01), and IκB-α showed significant decline (P < 0.01), especially in the obesity-asthma group. There was decline of IKK-ß and NF-κB-P65 and elevation of IκB-α in the N-acetylcysteine group, which was even significantly in the Budesonide group (P < 0.01). There was a positive correlation between MDA and NF-κB activation in the lung tissues in all the asthma groups and treatment groups (P < 0.05). Obesity-asthma mice showed higher oxidative stress and activation of NF-κB compared with that of the asthma mice. There was a positive correlation between MDA and NF-κB activation in the lung tissues in the asthma groups. N-acetylcysteine was more effective in reducing the oxidative stress compared to the budesonide.

[Analysis of clinical features and genetic variants in an infant with Bloom syndrome].

OBJECTIVE: To analyze the clinical features and genetic variants in a 13-month-old child with Bloom syndrome. METHODS: Clinical data of the child was collected. Genetic variants were detected by high-throughput sequencing and Sanger sequencing. RESULTS: The child was born at full term but was small for gestational age. His clinical features included loss of appetite, severe growth retardation, microcephaly, and small mandible. Genetic testing found that he had carried compound heterozygous c.1068+3A>C and c.1069-1G>C variants of the BLM gene, both of which were unreported previously. CONCLUSION: Bloom syndrome is mainly characterized by severe growth retardation in infancy. The novel variants have expanded the variant spectrum of the BLM gene.

MiR-448-5p inhibits TGF-ß1-induced epithelial-mesenchymal transition and pulmonary fibrosis by targeting Six1 in asthma.

MicroRNAs (miRNAs) are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. The aim of this study was to explore how miR-448-5p affects airway remodeling and transforming growth factor-ß1 (TGF-ß1)-stimulated epithelial-mesenchymal transition (EMT) by targeting Sine oculis homeobox homolog 1 (Six1) in asthma. Asthmatic mice models with airway remodeling were induced with ovalbumin solution. MiRNA expression was evaluated using quantitative real-time polymerase chain reaction. Transfection studies of bronchial epithelial cells were performed to determine the target genes. A luciferase reporter assay system was applied to identify whether Six1 is a target gene of miR-448-5p. In the current study, we found that miR-448-5p was dramatically decreased in lung tissues of asthmatic mice and TGF-ß1-stimulated bronchial epithelial cells. In addition, the decreased level of miR-448-5p was closely associated with the increased expression of Six1. Overexpression of miR-448-5p decreased Six1 expression and, in turn, suppressed TGF-ß1-mediated EMT and fibrosis. Next, we predicted that Six1 was a potential target gene of miR-448-5p and demonstrated that miR-448-5p could directly target Six1. An SiRNA targeting Six1 was sufficient to suppress TGF-ß1-induced EMT and fibrosis in 16HBE cells. Furthermore, the overexpression of Six1 partially reversed the protective effect of miR-448-5p on TGF-ß1-mediated EMT and fibrosis in bronchial epithelial cells. Taken together, the miR-448-5p/TGF-ß1/Six1 link may play roles in the progression of EMT and pulmonary fibrosis in asthma.

miR-135b promotes proliferation and metastasis by targeting APC in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. The aim of our study was to investigate the functional role of microRNA-135b (miR-135b) in TNBC. A real-time polymerase chain reaction assay was used to quantify miR-135b expression levels in 90 paired TNBC tissue and adjacent normal tissue samples. Wound-healing and transwell assays were performed to evaluate the effects of miR-135b expression on the migration and invasion of TNBC cells. Luciferase reporter and western blot analyses were used to verify whether the mRNA encoding APC is a major target of miR-135b. In the current study, we found that miR-135b was highly expressed in TNBC tissue and cells, and the expression levels were correlated with lymph node status and TNM stage. In TNBC cells, the ectopic expression of miR-135b promoted cell proliferation and invasion in vitro. In addition, our study proved that the overexpression of miR-135b significantly suppressed APC expression by targeting the 3'-untranslated region of APC, whereas enhanced APC expression could partially abrogate the miR-135b-mediated promotion of carcinogenic traits in TNBC cells. Taken together, our study demonstrated that miR-135b expression promoted the proliferation and invasion of TNBC by downregulating APC expression, indicating that miR-135b may serve as a promising target for the treatment of TNBC patients.

RETRACTED: Quercetin ameliorates lipopolysaccharide-caused inflammatory damage via down-regulation of miR-221 in WI-38 cells

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article "… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

MiR-212-5p Suppresses the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Targeting Prrx2.

BACKGROUND/AIMS: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of miR-212-5p in TNBC. METHODS: Realtime PCR was used to quantify miR-212-5p expression levels in 30 paired TNBC samples and adjacent normal tissues. Wound healing and Transwell assays were used to evaluate the effects of miR-212-5p expression on the invasiveness of TNBC cells. Luciferase reporter and Western blot assays were used to verify whether the mRNA encoding Prrx2 is a major target of miR-212-5p. RESULTS: MiR-212-5p was downregulated in TNBC, and its expression levels were related to tumor size, lymph node status and vascular invasion in breast cancer. We also observed that the miR-212-5p expression level was significantly correlated with a better prognosis in TNBC. Ectopic expression of miR-212-5p induced upregulation of E-cadherin expression and downregulation of vimentin expression. The expression of miR212-5p also suppressed the migration and invasion capacity of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. Moreover, our study observed that miR-212-5p overexpression significantly suppressed Prrx2 by targeting its 3'-untranslated region (3'-UTR) region, and Prrx2 overexpression partially abrogated miR-212-5p-mediated suppression. CONCLUSIONS: Our study demonstrated that miR-212-5p inhibits TNBC from acquiring the EMT phenotype by downregulating Prrx2, thereby inhibiting cell migration and invasion during cancer progression.

Silencing of Prrx1b suppresses cellular proliferation, migration, invasion and epithelial-mesenchymal transition in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired-related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial-mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up-regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal-like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/ß-catenin signaling pathway and induced the mesenchymal-epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.

Alterations in the gut microbiota of toddlers with cow milk protein allergy treated with a partially hydrolyzed formula containing synbiotics: A nonrandomized controlled interventional study.

Formulas containing intact cow milk protein are appropriate alternatives when human milk (HM) is not feasible. However, for babies with a physician-diagnosed cow milk protein allergy (CMPA), hydrolyzed formulas are needed. We conducted a 3-month, open-label, nonrandomized concurrent controlled trial (ChiCTR2100046909) between June 2021 and October 2022 in Qingdao City, China. In this study, CMPA toddlers were fed with a partially hydrolyzed formula containing synbiotics (pHF, n = 43) and compared with healthy toddlers fed a regular intact protein formula (IF, n = 45) or HM (n = 21). The primary endpoint was weight gain; the secondary endpoints were changes in body length and head circumference of both CMPA and healthy toddlers after 3-month feeding; and the exploratory outcomes were changes in gut microbiota composition. After 3 months, there were no significant group differences for length-for-age, weight-for-age, or head circumference-for-age Z scores. In the gut microbiota, pHF feeding increased its richness and diversity, similar to those of IF-fed and HM-fed healthy toddlers. Compared with healthy toddlers, the toddlers with CMPA showed an increased abundance of phylum Bacteroidota, Firmicutes, class Clostridia, and Bacteroidia, and a decreased abundance of class Negativicutes, while pHF feeding partly eliminated these original differences. Moreover, pHF feeding increased the abundance of short-chain fatty acid producers. Our data suggested that this pHF partly simulated the beneficial effects of HM and shifted the gut microbiota of toddlers with CMPA toward that of healthy individuals. In conclusion, this synbiotic-containing pHF might be an appropriate alternative for toddlers with CMPA.

Mesothelial cells differentiate into fibroblast-like cells under the scirrhous gastric cancer microenvironment and promote peritoneal carcinomatosis in vitro and in vivo.

Peritoneal metastases are one reason for the poor prognosis of scirrhous gastric cancer (SGC), and myofibroblast provides a favorable environment for the peritoneal dissemination of gastric cancer. The aim of this study was to determine whether myofibroblast originates from peritoneal mesothelial cells under the influence of the tumor microenvironment. Immunohistochemical studies of peritoneal biopsy specimens from patients with peritoneal lavage cytological (+) status demonstrate the expression of the epithelial markers cytokeratin in fibroblast-like cells entrapped in the stroma, suggesting that these cells stemmed from local conversion of mesothelial cells. To confirm this hypothesis in vitro, we co-incubated mesothelial cells with SGC or non-SGC to investigate morphology and function changes. As we expected, mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype with loss of epithelial morphology and decrease in the expression of cytokeratin and E-cadherin when exposed to conditioned medium from HSC-39, and the induction of mesothelial cells can be abolished using a neutralizing antibody to transforming growth factor-beta1 (TGF-ß1) as well as by pre-treatment with SB431542. Moreover, we found that these mesothelial cells-derived cells exhibit functional properties of myofibroblasts, including the ability to increase adhesion and invasion of SGC. In summary, our current data demonstrated that mesothelial cells are a source of myofibroblasts under the SGC microenvironment which provide a favorable environment for the dissemination of gastric cancer; TGF-ß1 produced by autocrine/paracrine in peritoneal cavity may play a central role in this pathogenesis.

[Retracted] Inhibition airway remodeling and transforming growth factor­ß1/Smad signaling pathway by astragalus extract in asthmatic mice.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the P­smad2 western blotting data shown in Fig. 7 were strikingly similar to data appearing in different form (namely, the bands appeared in the reverse orientation) in Fig. 4A in another article [Lv Z­D, Na D, Liu F­N, Du Z­M, Sun Z, Li Z, Ma X­Y, Wang Z­N and Xu H­M: Induction of gastric cancer cell adhesion through transforming growth factor­beta1­mediated peritoneal fibrosis. J Exp Clin Cancer Res 29: 139, 2010], which was written by mostly different authors at different research institutes (the author Zheng­Hai Qu did appear as an author on both papers). Owing to the fact that the contentious data in the above article had already been published prior to its submission to International Journal of Molecular Medicine, and due to a lack of overall confidence in the presented data, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 29: 564­568, 2012; DOI: 10.3892/ijmm.2011.868].

A Review of Three Chinese Cases of Acromicric/Geleophysic Dysplasia with FBN1 Mutations.

OBJECTIVE: This study aims to explore the clinical features and molecular diagnosis of FBN1-related acromelic dysplasia in Chinese patients. METHODS: The clinical and genetic features of three FBN1-related acromicric dysplasia (AD)/geleophysic dysplasia (GD) Chinese patients from two families were reviewed, and comprehensive medical evaluations were performed. Targeted next-generation sequencing was used to detect genetic mutations associated with short statures, including FBN1. Sanger sequencing was used to determine the de novo mutation origin. RESULTS: Patient 1 presented with short stature, short and stubby hands and feet, mild facial dysmorphism, hepatomegaly, delayed bone age and beak-like femoral heads. Patient 2 and this patient's father merely presented with short stature, wide and short hands, and beak-like femoral heads. One novel mutation, c.5272G>T(p.D1758Y), and one known mutation, c.5183C>T(p.A1728V), were identified in these patients. CONCLUSION: The clinical features varied among these patients. The variant c.5272G>T(p.D1758Y) is a novel mutation.

DLC-3 suppresses cellular proliferation, migration, and invasion in triple-negative breast cancer by the Wnt/ß-catenin pathway.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of DLC-3 in TNBC. The expression of DLC-3 was assessed by immunohistochemistry in TNBC to evaluate the clinicopathologic significance of DLC-3. Recombinant lentiviral vectors encoding the DLC-3 gene were constructed for transfection into MDA-MB-231. Real-time qPCR and western blot analysis were employed to evaluate the expression of DLC-3, ß-catenin, GSK-3ß and c-myc in DLC-3-transfected cells. Moreover, cell proliferation assays, cell colony formation assays, and cell migration and invasion assays were performed to elucidate the role of DLC-3 in TNBC development and progression. Our data revealed that DLC-3 was downregulated in TNBC, and its expression level was associated with lymph node status and differentiation grade in breast cancer. Both real-time qPCR and western blot analyses showed that the DLC-3 gene and protein were overexpressed in the DLC-3-transfected MDA-MB-231 cells. In addition, the expression of GSK-3ß was upregulated and the expression of ß-catenin and c-myc gene was downregulated in the DLC-3-transfected cells. Furthermore, DLC-3 overexpression inhibited cell proliferation, colony formation, migration, and invasion in vitro. DLC-3, functioning as a tumor-suppressor gene, inhibits cell growth and invasion in TNBC, possibly through regulation of the Wnt/ß-catenin signaling pathway.

p53-independent role of MYC mutant T58A in the proliferation and apoptosis of breast cancer cells.

Myc proto-oncogene (MYC) is an oncoprotein that promotes proliferation and apoptosis. MYC mutations frequently disrupt the apoptotic processes during tumorigenesis. In the present study, the effects of the MYC point mutation T58A on the progression of a cellular tumor antigen p53 (p53)-/- human breast cancer cell line was analyzed, and the mechanism of p53-independent MYC-induced apoptosis was investigated. HCC1937 cells were transfected with mutant (T58A) or wild-type (WT) MYC using lentiviral vectors. The proliferation of transfected cells was evaluated by colony formation and MTT assays, and apoptosis was analyzed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays. WT MYC was transfected into HCC1937 cells exhibiting p14/p21 silencing through lentivirus-mediated RNA interference. The expression levels of Bim were detected by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Mutant MYC proteins retained the ability to stimulate the proliferation of HCC1937 cells, although they were defective at promoting apoptosis due to a failure to induce the Bcl-2 homology 3 domain-only protein Bim. When p14 was silenced, the effects of mutant MYC on proliferation and apoptosis were weakened. When p21 was silenced, the effects of mutant MYC were strengthened. Breast cancer-derived T58A MYC mutations are unable to activate Bim due to their failure to regulate p14/p21. It was concluded that mutant MYC was more effective compared with WT MYC at promoting the progression of breast cancer.