[Application of copy number variation sequencing in patients with intellectual disability/developmental delay and autistic spectrum disorder].

OBJECTIVE: To assess the value of copy number variation sequencing (CNV-seq) for the diagnosis of children with intellectual disability (ID), developmental delay (DD), and autistic spectrum disorder (ASD). METHODS: Forty patients with ID/DD/ASD referred to Nanshan Maternity and Child Health Care Hospital from September 2018 to January 2022 were enrolled. G-banded karyotyping analysis was carried out for the patients. Genomic DNA was extracted from peripheral blood samples and subjected to CNV-Seq analysis to detect chromosome copy number variations (CNVs) in such patients. ClinVar, DECIPHER, OMIM and other database were searched for data annotation. RESULTS: Among the 40 patients (including 30 males and 10 females), 16, 15 and 6 were diagnosed with ID, DD and ASD, respectively. One patient had combined symptoms of ID and DD, whilst the remaining two had combined ID and ASD. Four patients were found with abnormal karyotypes, including 47,XY,+mar, 46,XY,inv(8)(p11.2q21.2), 46,XX,del(5)(p14) and 46,XX[76]/46,X,dup(X)(p21.1q12). Chromosome polymorphism was also found in two other patients. CNV-seq analysis has detected 32 CNVs in 20 patients (50.0%, 20/40). Pathogenic CNVs were found in 10 patients (25.0%), 15 CNVs of uncertain clinical significance were found in 12 patients (30.0%), and 7 likely benign CNVs were found in 4 patients (10.0%). CONCLUSION: Chromosome CNVs play an important role in the pathogenesis of ID/DD/ASD. CNV-seq can detect chromosomal abnormalities including microdeletions and microduplications, which could provide a powerful tool for revealing the genetic etiology of ID/DD/ASD patients.

Erratum: PI3K/Akt/HIF-1α signaling pathway mediates HPV-16 oncoprotein-induced expression of EMT-related transcription factors in non-small cell lung cancer cells: Erratum.

[This corrects the article DOI: 10.7150/jca.26112.].

Erratum: Cytochalasin H Inhibits Angiogenesis via the Suppression of HIF-1α Protein Accumulation and VEGF Expression through PI3K/AKT/P70S6K and ERK1/2 Signaling Pathways in Non-Small Cell Lung Cancer Cells: Erratum.

[This corrects the article DOI: 10.7150/jca.29933.].

Erratum: Cytochalasin H isolated from mangrove-derived endophytic fungus inhibits epithelial-mesenchymal transition and cancer stemness via YAP/TAZ signaling pathway in non-small cell lung cancer cells: Erratum.

[This corrects the article DOI: 10.7150/jca.50512.].

Cytochalasin H isolated from mangrove-derived endophytic fungus inhibits epithelial-mesenchymal transition and cancer stemness via YAP/TAZ signaling pathway in non-small cell lung cancer cells.

Our previous studies have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove and found that CyH induced apoptosis and inhibited migration and angiogenesis in non-small cell lung cancer (NSCLC) cells. In this study, we further investigated the effect of CyH on epithelial-mesenchymal transition (EMT) and cancer stemness of A549 and NCI-H460 NSCLC cells and the underlying mechanisms, especially the role of YAP/ TAZ signaling pathway in the process. Our results showed that CyH significantly inhibited invasive ability and the sphere formation of NSCLC cells. The expression of E-cadherin, an EMT epithelial marker, was obviously up-regulated, while the expression of Vimentin and N-cadherin, the EMT mesenchymal markers, was dramatically down-regulated by CyH treatment in NSCLC cells. Moreover, the expression of EMT-associated transcription factors including Slug, Twist1, and Snail1 and stemness markers including Nanog, Sox-2, and Oct-4 was significantly down-regulated by CyH treatment in NSCLC cells. Additionally, CyH significantly down-regulated YAP and TAZ expression and up-regulated LAST1/2 and MST1/2 expression, and CyH inhibited the interaction between YAP and TEAD. Furthermore, YAP knockdown abolished the effect of CyH on the expression of EMT- and stemness-related markers in NSCLC cells. Taken together, these results suggest that CyH inhibits EMT and cancer stemness of NSCLC cells via the regulation of YAP/TAZ signaling pathway.

Cytochalasin H Inhibits Angiogenesis via the Suppression of HIF-1α Protein Accumulation and VEGF Expression through PI3K/AKT/P70S6K and ERK1/2 Signaling Pathways in Non-Small Cell Lung Cancer Cells.

Our previous study has demonstrated that cytochalasin H (CyH) isolated from mangrove-derived endophytic fungus induces apoptosis and inhibits migration in A549 non-small cell lung cancer (NSCLC) cells. In this study, we further explored the effect of CyH on angiogenesis in NSCLC cells and the underlying molecular mechanisms. A549 and H460 NSCLC cells were treated with different concentrations of CyH for 24 h. The effects of CyH on NSCLC angiogenesis in vitro and in vivo were investigated. Hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in xenografted NSCLC of nude mice was analyzed by immunohistochemistry. ELISA was used to analyze the concentration of VEGF in the conditioned media derived from treated and untreated NSCLC cells. Western blot was performed to detect the levels of HIF-1α, p-AKT, p-P70S6K, and p-ERK1/2 proteins, and RT-qPCR was used to determine the levels of HIF-1α and VEGF mRNA in A549 and H460 cells. Our results showed that CyH significantly inhibited angiogenesis in vitro and in vivo, and suppressed the hemoglobin content and HIF-1α and VEGF protein expression in xenografted NSCLC tissues of nude mice. Meanwhile, CyH inhibited the secretion of VEGF protein and the expression of HIF-1α protein in A549 and H460 cells. Moreover, CyH had a significant inhibitory effect on VEGF mRNA expression but had no effect on HIF-1α mRNA expression, and CyH inhibited HIF-1α protein expression by promoting the degradation of HIF-1α protein in A549 and H460 cells. Additionally, CyH dramatically inhibited AKT, P70S6K, and ERK1/2 activation in A549 and H460 cells. Taken together, our results suggest that CyH can inhibit NSCLC angiogenesis by the suppression of HIF-1α protein accumulation and VEGF expression through PI3K/AKT/P70S6K and ERK1/2 signaling pathways.

Increased expression of monoamine oxidase A is associated with epithelial to mesenchymal transition and clinicopathological features in non-small cell lung cancer.

Monoamine oxidase A (MAOA), a mitochondrial enzyme, is closely associated with neurological disorders. Recently, MAOA has been linked to the progression of prostate cancer, hepatocellular carcinoma, and cholangiocarcinoma. However, MAOA was reported to have different effects on the progression of these types of cancer, and the role of MAOA in non-small cell lung cancer (NSCLC) progression remains unclear. The present study determined the expression of MAOA and epithelial to mesenchymal transition (EMT) markers in 45 pairs of NSCLC and matched non-tumor adjacent lung tissues, and further analyzed the correlation between MAOA expression and the EMT or the development of clinicopathological features. The results demonstrated that protein and mRNA expression levels of MAOA in NSCLC tissues were higher than those observed in the matched non-tumor adjacent lung tissues. Furthermore, the increased MAOA expression in NSCLC tissues was positively correlated with N-cadherin (r=0.525, P=0.002), Slug (r=0.515, P=0.001), and Twist (r=0.448, P=0.008) expressions, but negatively correlated with E-cadherin expression (r=-0.387, P=0.01). Additionally, the elevated MAOA expression in NSCLC tissues was associated with late stage NSCLC (Z=-2.596, P=0.029) and lymph node metastases (Z=-2.378, P=0.020). These findings suggest that MAOA may have a role in promoting NSCLC progression by mediating EMT.

Cytochalasin H isolated from mangrove­derived endophytic fungus induces apoptosis and inhibits migration in lung cancer cells.

Cytochalasin H (CyH) has been shown to exhibit promising anticancer activities against various types of cancers; however, the underlying mechanisms remain unknown. In a previous study, we isolated CyH from the mangrove­derived endophytic fungus Phomopsis sp. in Zhanjiang, China. In the present study, we further explored the effect of CyH on apoptosis and migration in the human lung adenocarcinoma cell line A549. Cell Counting kit­8 (CCK­8) assay was used to observe the effects of CyH on the growth of A549 cells. The cell cycle and apoptosis were determined using flow cytometry. The effect of CyH on cell migration was observed by scratch wound healing and chamber migration assays. Western blotting was used to detect the expression of apoptosis­ and metastasis­associated proteins. Our results showed that CyH exhibited cytotoxicity to A549 cells. The treatment of CyH arrested A549 cells at the G2/M phase. Furthermore, sub­G1 peaks and fragmented DNA ladders were observed, and the mitochondrial transmembrane potential was also decreased in CyH­treated A549 cells. CyH significantly increased Bax, P53, and cleaved caspase­3 (17 kDa) protein expression and decreased Bcl­xL, Bcl­2, and full­length caspase­3 (35 kDa) protein expression, resulting in an increased ratio of the pro­apoptosis/anti­apoptosis proteins Bax/Bcl­2. Additionally, CyH treatment inhibited the migration ability of A549 cells in a dose­dependent manner. Taken together, our results suggest that CyH may be a potential chemopreventive drug for the treatment of lung cancer.

PI3K/Akt/HIF-1α signaling pathway mediates HPV-16 oncoprotein-induced expression of EMT-related transcription factors in non-small cell lung cancer cells.

Background: Our previous studies have demonstrated that human papillomaviruse (HPV)-16 oncoproteins promoted epithelial-mesenchymal transition (EMT), leading to non-small cell lung cancer (NSCLC) progression, but the underlying molecular mechanisms still remain unclear. PI3K/Akt/HIF-1α signaling pathway has been reported to mediate hypoxia-induced EMT. In this study, we further explored the role of PI3K/Akt/HIF-1α signaling pathway in HPV-16 oncoprotein-induced EMT in NSCLC cells. Methods: A549 and NCI-H460 NSCLC cells were transiently transfected with pEGFP-HPV-16 E6 or E7 constructs. Western blotting and RT-qPCR were respectively performed to determine the protein and mRNA expression of EMT-related transcription factors. HPV-16 E6 or E7-transfected NSCLC cells were co-transfected with specific HIF-1α-siRNA or pretreated with different concentrations of LY294002, a specific PI3K inhibitor, followed by the analysis of expression of EMT-related transcription factors. The correlation between HIF-1α and EMT-related transcription factors in NSCLC tissues was analyzed by immunohistochemical staining and Spearman rank correlation coefficient. Results: HPV-16 E6 and E7 oncoproteins upregulated the expression of Slug and Twist1, the EMT-related transcription factors, at both protein and mRNA levels in A549 and NCI-H460 cells. The co-transfection with specific HIF-1α-siRNA, but not the non-specific (NS)-siRNA, significantly abrogated HPV-16 oncoprotein-induced upregulation of ZEB1, Snail1, Slug, and Twist1 at both protein and mRNA levels. Additionally, pretreatment with LY294002 obviously blocked HPV-16 E6- and E7-induced Snail1, Slug, and Twist1 protein expression in A549 and NCI-H460 cells. Further analysis of clinical specimens showed that HIF-1α protein was strongly expressed in NSCLC tissues, which was positively correlated with ZEB1, Snail1, Slug, and Twist1 protein expression. Conclusions: PI3K/Akt/HIF-1α may contribute to the progression of HPV-associated NSCLC via mediating the expression of EMT-related transcription factors in NSCLC cells.

Overexpression of Human Papillomavirus Type 16 Oncoproteins Enhances Epithelial-Mesenchymal Transition via STAT3 Signaling Pathway in Non-Small Cell Lung Cancer Cells.

The human papillomavirus (HPV) infection may be associated with the development and progression of non-small cell lung cancer (NSCLC). However, the role of HPV-16 oncoproteins in the development and progression of NSCLC is not completely clear. Epithelial-mesenchymal transition (EMT), a crucial step for invasion and metastasis, plays a key role in the development and progression of NSCLC. Here we explored the effect of HPV-16 oncoproteins on EMT and the underlying mechanisms. NSCLC cell lines, A549 and NCI-H460, were transiently transfected with the EGFP-N1-HPV-16 E6 or E7 plasmid. Real-time PCR and Western blot analysis were performed to analyze the expression of EMT markers. A protein microarray was used to screen the involved signaling pathway. Our results showed that overexpression of HPV-16 E6 and E7 oncoproteins in NSCLC cells significantly promoted EMT-like morphologic changes, downregulated the mRNA and protein levels of EMT epithelial markers (E-cadherin and ZO-1), and upregulated the mRNA and protein levels of EMT mesenchymal markers (N-cadherin and vimentin) and transcription factors (ZEB-1 and Snail-1). Furthermore, the HPV-16 E6 oncoprotein promoted STAT3 activation. Moreover, WP1066, a specific signal transducer and activator of transcription 3 (STAT3) inhibitor, reversed the effect of HPV-16 E6 on the expression of ZO-1, vimentin, and ZEB-1 in transfected NSCLC cells. Taken together, our results suggest that overexpression of HPV-16 E6 and E7 oncoproteins enhances EMT, and the STAT3 signaling pathway may be involved in HPV-16 E6-induced EMT in NSCLC cells.