[Prenatal diagnosis of a case with Congenital myasthenic syndrome due to compound heterozygous variants of SCN4A gene].

OBJECTIVE: To explore the clinical and genetic characteristics of a fetus diagnosed with Congenital myasthenic syndrome type 16 (CMS16). METHODS: A couple who had visited Tianjin Medical University General Hospital in February 2018 due to "adverse outcome of two pregnancies" was selected as the study subject. Clinical data was gathered. Peripheral blood and amniotic fluid samples were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. Low-depth whole-genome sequencing was carried out to detect copy number variation (CNV) in the fetus. RESULTS: The couple's first pregnancy had resulted in a miscarriage at 27+5 weeks, when ultrasound had revealed pleural effusion and polyhydramnios in the fetus. Their second pregnancy was terminated at 30+5 weeks due to fetal hand malformations, polyhydramnios and pleural fluid. Both couple had denied family history of genetic conditions. For their third pregnancy, no CNV abnormality was detected, whilst a compound heterozygous variants, including a maternally derived c.3172C>T (p.R1058W) and paternal c.1431delG (p.K477fs*89) in the SCN4A gene were detected. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.3172C>T (p.R1058W) was predicted as a likely pathogenic variant (PM1+PM2_supporting+PP3+PP4), whilst the c.1431delG (p.K477fs*89) was predicted as a pathogenic variant (PVS1+PM2_supporting+PP4). CONCLUSION: The c.3172C>T (p.R1058W) and c.1431delG (p.K477fs*89) compound heterozygous variants of the SCN4A gene probably underlay the CMS16 in the third fetus.

Identification of SRSF10 as a promising prognostic biomarker with functional significance among SRSFs for glioma.

AIMS: The serine/arginine-rich splicing factor (SRSF) protein family members are essential mediators of the alternative splicing (AS) regulatory network, which is tightly implicated in cancer progression. However, the expression, clinical correlation, immune infiltration, and prognostic value of SRSFs in gliomas remain unclear. MATERIALS AND METHODS: Glioma samples were extracted from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) datasets. Several databases, such as HPA, DAVID, UALCAN were used to comprehensively explore the roles of SRSFs. In addition, experimental validation of SRSF10 was also conducted. KEY FINDINGS: Here, we found the expression alterations of the SRSF family in glioma samples using data from the TCGA and CGGA_325 datasets. Among the 12 genes, most were found to be closely associated with glioma clinical features, which linked to poor prognosis in glioma patients. Interestingly, survival analysis identified only SRSF10 as a potential independent risk prognostic biomarker for glioma patients. Immune analysis indicated that glioma patients with high SRSF10 expression may respond well to immunotherapies targeting immune checkpoint (ICP) genes. Finally, knocking down SRSF10 reduced glioma cell viability, induced G1 cell cycle arrest, and induced the exclusion of bcl-2-associated transcription factor 1 (BCLAF1) exon 5a. SIGNIFICANCE: Overall, this study uncovers the oncogenic roles of most SRSF family members in glioma, with the exception of SRSF5, while highlighting SRSF10 as a potential novel independent prognostic biomarker for glioma.

Wilms' tumour gene 1 (WT1) enhances non-small cell lung cancer malignancy and is inhibited by microRNA-498-5p.

BACKGROUND: Wilms' tumour gene 1 (WT1) is clearly recognized as a tumour promoter in diversiform of human malignancies. Nevertheless, knowledge of its expression, functions and potential molecular mechanisms in non-small cell lung cancer (NSCLC) remains elusive. METHODS: Differential expression of WT1 mRNA and protein between NSCLC and normal tissues were assessed by analyzing RNA-seq data from Oncomine and protein data from Human Protein Atlas, respectively. Subsequently, prognosis significance and immune cell infiltration were analyzed by Kaplan-Meier plotter and CIBERSORT. 60 pairs of local NSCLC tissues were involved to validate WT1 expression by quantitative PCR (qPCR) and Western blot. Moreover, Cell Counting Kit-8 (CCK-8), colony formation, transwell, dual luciferase reporter assays and in vivo xenograft tumour growth experiments were conducted to explore the function and mechanism of WT1 in NSCLC. RESULTS: Our solid data indicated that WT1 was increased in NSCLC tissues and cell lines in comparison with their matched controls. In particular, its upregulation correlated with worse prognosis and immune infiltration of the patients. Functional assays demonstrated that knockdown of WT1 inhibited NSCLC malignancy, including inhibiting cell proliferation, survival and invasion. Further exploration discovered that microRNA-498-5p (miR-498-5p) was the upstream suppressor of WT1 by directly targeting the 3' untranslated region (UTR) of WT1 mRNA. Moreover, expression of miR-498-5p was notably decreased and inversely correlated with WT1 in NSCLC tissues. Finally, we proved that miR-498-5p was a potent tumour suppressor in NSCLC by suppressing cell proliferation, survival and invasion, while WT1 restoration could in turn disrupt this suppression both in vitro and in vivo. CONCLUSION: The abnormal increase in WT1 contributes to the malignant properties of NSCLC cells, and miR-498-5p is a natural inhibitor of WT1. Our findings might facilitate the development of novel therapeutic strategies against NSCLC in the future.

The miR-29 family members induce glioblastoma cell apoptosis by targeting cell division cycle 42 in a p53-dependent manner.

BACKGROUND: Emerging evidence has shown that miR-29 is a promising biomarker and therapeutic target for malignancies. The roles of miR-29a/b/c in glioma pathogenesis remain need further investigation. METHODS: The expression levels of miR-29a/b/c and CDC42 were systematically analysed, and prognostic significance was evaluated by Kaplan-Meier survival and Cox regression analyses. The roles of miR-29a/b/c in apoptosis and the underlying mechanisms were explored via an alkaline single-cell gel electrophoresis assay, caspase 3/7 activity assays and Western blotting. RESULTS: miR-29a/b/c expression decreased progressively with the elevation of the WHO grade in our 147 human glioma specimens, compared with 20 non-tumour control brain tissues, and decreased miR-29a/b/c expression was associated with more aggressive phenotypes. Kaplan-Meier and Cox regression analyses demonstrated that lower miR-29a/b/c expression was correlated with worse prognosis, which was confirmed by analysis of 198 glioma patients from the CGGA cohort. These all indicate that miR-29a/b/c were independent predictors of prognosis in glioma patients. miR-29a/b/c induced apoptosis in GBM cells by silencing CDC42. Further detailed mechanistic investigation revealed that miR-29a/b/c promoted apoptosis in a p53-dependent manner by suppressing the CDC42/PAK/AKT/MDM2 pathway. CONCLUSIONS: miR-29a/b/c are independent predictors of prognosis in glioma patients. They induce glioblastoma cell apoptosis via silencing of CDC42 and suppression of downstream PAK/AKT/MDM2 signalling in a p53-dependent manner.

Recruitment of LEF1 by Pontin chromatin modifier amplifies TGFBR2 transcription and activates TGFß/SMAD signalling during gliomagenesis.

Synergies of transcription factors, chromatin modifiers and their target genes are vital for cell fate determination in human cancer. Although the importance of numerous epigenetic machinery for regulating gliomagenesis has been previously recognized, how chromatin modifiers collaborate with specific transcription factors remains largely elusive. Herein we report that Pontin chromatin remodelling factor acts as a coactivator for LEF1 to activate TGFß/SMAD signalling, thereby contributing to gliomagenesis. Pontin is highly expressed in gliomas, and its overexpression paralleled the grade elevation and poor prognosis of patients. Functional studies verified its oncogenic roles in GBM cells by facilitating cell proliferation, survival and invasion both in vitro and in vivo. RNA sequencing results revealed that Pontin regulated multiple target genes involved in TGFß/SMAD signalling. Intriguingly, we found that Pontin amplified TGFßR2 gene transcription by recruiting LEF1, thereby activating TGFß/SMAD signalling and facilitating gliomagenesis. Furthermore, higher TGFßR2 expression conferred worse patient outcomes in glioma. To conclude, our study revealed that the Pontin-LEF1 module plays a crucial role in driving TGFßR2 gene transcription, which could be exploited to target TGFß/SMAD signalling for anti-glioma therapy.

Pulmonary mucoepidermoid carcinoma: A clinicopathological study of 45 patients.

Pulmonary mucoepidermoid carcinoma (PMEC) is uncommon. The purpose of this study was to evaluate the clinicopathological features, diagnostic criteria, treatment options, and prognostic factors relating to primary PMEC. Clinical data on 45 patients with primary PMEC were collected and analyzed retrospectively at Tianjin Medical University General Hospital and the First People' Hospital of Longquanyi District Chengdu from January 2008 to December 2020. The 45 patients (25 males and 20 females) ranged in age from 22 to 72 years, with a median age of 49 and an average age of 47.7. All the patients underwent surgery, with 32 receiving only surgery and 13 receiving both surgery and postoperative chemotherapy. A total of 34 instances of low-grade tumors and 11 cases of high-grade tumors were discovered during postoperative pathological diagnosis. Forty-five patients were followed for 13 to 78 months, and four died during this period. In all four instances, a lung infection unrelated to the tumor was determined to be the cause of death. The MAML2 gene translocation was detected in 40 of 45 patients, with 34 of them testing positive. Radical surgery with lymph node dissection is an efficient treatment for PMEC. The prognosis is poor for patients with advanced disease, a negative MAML2 gene translocation, lymph node metastases, and high-grade tumors.

The ATPase Pontin is a key cell cycle regulator by amplifying E2F1 transcription response in glioma.

Pontin (RUVBL1) is a highly conserved ATPase of the AAA + (ATPases Associated with various cellular Activities) superfamily and is implicated in various biological processes crucial for oncogenesis. Its overexpression is observed in multiple human cancers, whereas the relevance of Pontin to gliomagenesis remains obscure. To gain insights into Pontin involvement in glioma, we performed bioinformatics analyses of Pontin co-expressed genes, Pontin-affected genes, and carried out experimental studies. The results verified that Pontin was upregulated in gliomas. Its higher levels might predict the worse prognosis of glioma patients. The Pontin co-expressed genes were functionally enriched in cell cycle progression and RNA processing. In the nucleus, Pontin promoted cell growth via facilitating cell cycle progression. Using RNA-seq, we found that Pontin knockdown resulted in altered expression of multiple genes, among which the E2F1 targets accounted for a large proportion. Mechanistic studies found that Pontin interacted with E2F1 and markedly amplified the E2F1 transcription response in an ATPase domain-dependent manner. By analyzing the RNA-seq data, we also found that Pontin could impact on the alternative splicing (AS). Both differential expressed genes and AS events affected by Pontin were associated with cell cycle regulation. Taken together, our findings provide novel insights of the importance of Pontin in gliomagenesis by regulating cell cycle and AS, and shed light on the possible application of Pontin as an antineoplastic target in glioma.

Improved Antiglioblastoma Activity and BBB Permeability by Conjugation of Paclitaxel to a Cell-Penetrative MMP-2-Cleavable Peptide.

In order to solve the problems of receptor promiscuity and poor blood-brain barrier (BBB) penetration in the treatment of glioblastomas (GBM), a novel dual-functional nanocomplex drug delivery system is developed based on the strategy of peptide-drug conjugates. In this study, SynB3-PVGLIG-PTX is designed and screened out by matrix metalloproteinase-2 (MMP-2), to which it exhibits the best affinity. The MMP-2-sensitive peptide (PVGLIG) and a cell-penetration peptide (SynB3) are combined to form a dual-functional peptide. Moreover, as a drug-peptide nanocomplex, SynB3-PVGLIG-PTX exhibited a high potential to form an aggregation with good solubility that can release paclitaxel (PTX) through the cleavage of MMP-2. From a functional perspective, it is found that SynB3-PVGLIG-PTX can specifically inhibit the proliferation, migration, and invasion of GBM cells in vitro in the presence of MMP-2, in contrast to that observed in MMP-2 siRNA transfected cells. Further investigation in vivo shows that SynB3-PVGLIG-PTX easily enters the brain of U87MG xenograft nude mice and can generate a better suppressive effect on GBM through a controlled release of PTX from SynB3-PVGLIG-PTX compared with PTX and temozolomide. Thus, it is proposed that SynB3-PVGLIG-PTX can be used as a novel drug-loading delivery system to treat GBM due to its specificity and BBB permeability.

Eucalyptal A inhibits glioma by rectifying oncogenic splicing of MYO1B mRNA via suppressing SRSF1 expression.

Glioma is the most common primary intracranial tumor, in which glioblastoma (GBM) is the most malignant and lethal. However, the current chemotherapy drugs are still unsatisfactory for GBM therapy. As the natural products mainly extracted from Eucalyptus species, phloroglucinol-terpene adducts have the potential to be anti-cancer lead compounds that attracted increasing attention. In order to discover the new lead compounds with the anti-GBM ability, we isolated Eucalyptal A with a phloroglucinol-terpene skeleton from the fruit of E. globulus and investigated its anti-GBM activity in vitro and in vivo. Functionally, we verified that Eucalyptal A could inhibit the proliferation, growth and invasiveness of GBM cells in vitro. Moreover, Eucalyptal A had the same anti-GBM activity in tumor-bearing mice as in vitro and prolonged the overall survival time by maintaining mice body weight. Further mechanism research revealed that Eucalyptal A downregulated SRSF1 expression and rectified SRSF1-guided abnormal alternative splicing of MYO1B mRNA, which led to anti-GBM activity through the PDK1/AKT/c-Myc and PAK/Cofilin axes. Taken together, we identified Eucalyptal A as an important anti-GBM lead compound, which represents a novel direction for glioma therapy.

The RNA-binding protein SRSF1 is a key cell cycle regulator via stabilizing NEAT1 in glioma.

The relevance of RNA processing has been increasingly recognized in a variety of diseases. We previously identified serine/arginine-rich splicing factor 1 (SRSF1) as an oncodriver in glioma via splicing control. However, its splicing-independent roles and mechanisms are poorly defined in glioma. In this study, by integrating the data mining of SRSF1-co-expressed genes, SRSF1-affected genes and experimental studies, we demonstrated that SRSF1 was the most highly expressed SRSF in the 9 tumor types tested, and it was a crucial cell cycle regulator in glioma. Importantly, we identified nuclear paraspeckle assembly transcript1 (NEAT1), an upregulated long non-coding RNA (lncRNA) in glioma, as a target of SRSF1. Endogenous NEAT1 inhibition resembled the effect of SRSF1 knockdown on glioma cell proliferation by retarding cell cycle. Mechanistically, we proved that SRSF1 bound to NEAT1 and facilitated its RNA stability. The positive correlation between SRSF1 and NEAT1 levels in cancers further supported the positive regulation of NEAT1 by SRSF1. Collectively, our results provide novel insights on the splicing-independent mechanisms of SRSF1 in glioma, and confirm that NEAT1, whose stability maintained by SRSF1, implicates gliomagenesis by regulating cell cycle. Both SRSF1 and NEAT1 may serve as promising targets for antineoplastic therapies.

The novel chromatin architectural regulator SND1 promotes glioma proliferation and invasion and predicts the prognosis of patients.

BACKGROUND: Upregulation of staphylococcal nuclease domain-containing protein 1 (SND1) is a common phenomenon in different human malignant tissues. However, little information is available on the underlying mechanisms through which SND1 affects glioma cell proliferation and invasion. METHODS: SND1, Ras homolog family member A (RhoA), and marker of proliferation Ki-67 (MKI67) were analyzed in 187 gliomas by immunostaining. The correlation between those markers and patients' prognoses was assessed using the Kaplan-Meier estimator. Gene Ontology, chromatin immunoprecipitation, electrophoretic mobility shift assay, and chromosome conformation capture were applied to identify SND1-activated target genes. We also used MTT, colony formation, transwell and orthotopic implantation assays to investigate SND1 function in glioma cell proliferative and invasive activity. RESULTS: We identified SND1 and RhoA as independent predictors of poor prognosis in glioma patients. SND1 knockdown significantly suppressed the proliferation and invasion of glioma cells. Mechanistically, we discovered that SND1 facilitated malignant glioma phenotypes by epigenetically inducing chromatin topological interaction, which activated downstream RhoA transcription. RhoA sequentially regulated expression of CCND1, CCNE1, CDK4, and CDKN1B and accelerated G1/S phase transition in glioma cell proliferation. CONCLUSIONS: Our findings identify SND1 as a novel chromatin architectural modifier and promising prognostic indicator for glioma classification and treatment.

miR-135a-5p Functions as a Glioma Proliferation Suppressor by Targeting Tumor Necrosis Factor Receptor-Associated Factor 5 and Predicts Patients' Prognosis.

miR-135a-5p has been reported as a tumor suppressor in several extracranial tumors. However, its exact roles in gliomagenesis and relevance to the patients' prognoses are largely unknown. Herein, we detected the miR-135a-5p and tumor necrosis factor receptor-associated factor 5 (TRAF5) levels in 120 human glioma specimens and 20 nontumoral brain tissues; we found the miR-135a-5p level decreased, whereas the TRAF5 level increased, with the elevation of glioma grade. Their labeling indexes were inversely correlated with each other and showed strong negative (miR-135a-5p) and positive (TRAF5) correlation with the Ki-67 index. Cox regression demonstrated that both of their expression levels were independent survival predictors, whereas Kaplan-Meier analysis showed that subgrouping the glioma patients according to their levels could perfectly reflect the patients' prognoses regardless of the similarities in pathologic, molecular, and clinical features. In the following in vitro and in vivo studies, it was demonstrated that miR-135a-5p induced G1 arrest and inhibited the proliferation of glioma cells by targeting TRAF5 and subsequently blocking AKT phosphorylation as well as c-Myc and cyclin D1 expression. These effects could be reversed by TRAF5 overexpression and simulated by specific TRAF5 silencing. This study highlights the importance of miR-135a-5p and TRAF5 in gliomagenesis and progression and implies their potential prognostic and therapeutic values in malignant glioma.

Splicing factor SRSF1 promotes gliomagenesis via oncogenic splice-switching of MYO1B.

Abnormal alternative splicing (AS) caused by alterations to splicing factors contributes to tumor progression. Serine/arginine splicing factor 1 (SRSF1) has emerged as a key oncodriver in numerous solid tumors, leaving its roles and mechanisms largely obscure in glioma. Here, we demonstrate that SRSF1 is increased in glioma tissues and cell lines. Moreover, its expression was correlated positively with tumor grade and Ki-67 index, but inversely with patient survival. Using RNA-Seq, we comprehensively screened and identified multiple SRSF1-affected AS events. Motif analysis revealed a position-dependent modulation of AS by SRSF1 in glioma. Functionally, we verified that SRSF1 promoted cell proliferation, survival, and invasion by specifically switching the AS of the myosin IB (MYO1B) gene and facilitating the expression of the oncogenic and membrane-localized isoform, MYO1B-fl. Strikingly, MYO1B splicing was dysregulated in parallel with SRSF1 expression in gliomas and predicted the poor prognosis of the patients. Further investigation revealed that SRSF1-guided AS of the MYO1B gene increased the tumorigenic potential of glioma cells through the PDK1/AKT and PAK/LIMK pathways. Taken together, we identify SRSF1 as an important oncodriver that integrates AS control of MYO1B into promotion of gliomagenesis and represents a potential prognostic biomarker and target for glioma therapy.

miR-29s function as tumor suppressors in gliomas by targeting TRAF4 and predict patient prognosis.

Robust proliferation and apoptosis inhibition of tumor cells are responsible for the high mortality and poor outcome of patients with high-grade gliomas. miR-29a/b/c have been reported to be important suppressors in several human tumor types. However, their exact roles in gliomagenesis and their relevance to patient prognosis remain unclear. In this study, using 187 human glioma specimens and 20 nontumoral brain tissues, we demonstrated that the expression of miR-29a/b/c decreased progressively as the grade of glioma and the Ki-67 index increased. However, the expression of TRAF4, the functional target of miR-29a/b/c, exhibited the inverse trend, and its level was inversely correlated with the levels of miR-29a/b/c. A Kaplan-Meier analysis demonstrated that the miR-29a/b/c and TRAF4 levels were closely associated with patient survival even in patients with the same tumor grade and identical IDH gene status. A functional study verified that miR-29a/b/c induced apoptosis and suppressed the proliferation of glioma cells by directly targeting TRAF4. An investigation of the mechanism revealed that miR-29a/b/c promoted apoptosis through the TRAF4/AKT/MDM2 pathway in a p53-dependent manner, while miR-29a/b/c induced G1 arrest and inhibited tumor cell proliferation by blocking the phosphorylation of AKT and GSK-3ß, and the expression of cyclin D1 and c-Myc. Furthermore, TRAF4-knockdown perfectly simulated the anti-glioma effects of miR-29a/b/c. These findings enrich our understanding of gliomagenesis, highlight the prognostic value of miR-29a/b/c and TRAF4, and imply their potential therapeutic roles in malignant gliomas.

miR-361-5p inhibits glioma migration and invasion by targeting SND1.

BACKGROUND: Downregulation of miR-361-5p contributes to epithelial-mesenchymal transition of glioma cells. However, the relevance of miR-361-5p to migration and invasion of gliomas remains unknown. MATERIALS AND METHODS: The relationship between miR-361-5p and SND1 expression was analyzed in 120 human gliomas and 8 glioma cell lines by in situ hybridization, immunohistochemistry, and Western blot. Dual-luciferase reporter assay was used to identify SND1 as a target of miR-361-5p. The mechanisms through which miR-361-5p inhibits glioma cell migration and invasion were studied by in vitro assays. RESULTS: miR-361-5p expression was significantly downregulated in glioma tissues and glioma cell lines, and was inversely correlated with glioma grades. However, SND1 expression was positively correlated with glioma grades and inversely correlated with miR-361-5p expression. miR-361-5p overexpression suppressed glioma cell migration and invasion through targeting SND1 and subsequently decreasing MMP-2 expression. In glioma cell lines, SND1 overexpression could partly reverse the antitumor effects of miR-361-5p. CONCLUSION: The findings provide evidence that miR-361-5p directly targets SND1 to degradation and then reduces MMP-2 gene transcription, thus inhibiting glioma migration and invasion. miR-361-5p is an important tumor suppressor and a novel diagnostic biomarker of glioma, and miR-361-5p and SND1 are potential therapeutic candidates for malignant gliomas.

CASH: a constructing comprehensive splice site method for detecting alternative splicing events.

RNA-sequencing (RNA-seq) can generate millions of reads to provide clues for analyzing novel or abnormal alternative splicing (AS) events in cells. However, current methods for exploring AS events are still far from being satisfactory. Here, we present Comprehensive AS Hunting (CASH), which constructs comprehensive splice sites including known and novel AS sites in cells, and identifies differentially AS events between cells. We illuminated the versatility of CASH on RNA-seq data from a wide range of species and also on simulated in silico data, validated the advantages of CASH over other AS predictors and exhibited novel differentially AS events. Moreover, we used CASH to identify SRSF10-regulated AS events and investigated the evolution of SRSF10-regulated splicing. The results showed that SRSF10-regulated splicing events are highly evolvable from chickens, mice to humans. However, SRSF10-regulated splicing model was observed to be immutable, in which SRSF10 binding to cassette exon promotes exon inclusion while binding to downstream exon induces exon skipping. Altogether, CASH can significantly improve the detection of AS events and facilitate the study of AS regulation and function in cells; the SRSF10 data first demonstrate a flexibility of SRSF10 with their regulated splicing events but an immutability of SRSF10-regulated splicing model to produce opposite AS outcomes in vertebrates.

Quaking-5 suppresses aggressiveness of lung cancer cells through inhibiting ß-catenin signaling pathway.

Quaking-5 (QKI-5) belongs to the STAR (signal transduction and activation of RNA) family of RNA binding proteins and functions as a tumor suppressor in several human malignancies. In this study, we attempt to elucidate the role of QKI-5 in the pro-metastasis processes of lung cancer (LC) cells and the underlying mechanisms. We confirmed that QKI-5 was decreased in human LC tissues and cell lines, especially in high-metastatic cells. Moreover, QKI expression was positively correlated with LC patients' survival. Functional studies verified that QKI-5 suppressed migration, invasion and TGF-ß1-induced epithelial-mesenchymal transition (EMT) of LC cells. Mechanistically, we affirmed that QKI-5 reduced ß-catenin level in LC cells via suppressing its translation and promoting its degradation, whereas QKI-5 promoter hypermethylation suppressed QKI-5 expression. Our findings indicate that QKI-5 inhibits pro-metastasis processes of LC cells through interdicting ß-catenin signaling pathway, and that QKI-5 promoter hypermethylation is a crucial epigenetic regulation reducing QKI-5 expression in LC cells, and reveal that QKI-5 is a potential prognostic biomarker for LC patients.