FBXO11 variants are associated with intellectual disability and variable clinical manifestation in Chinese affected individuals.

F-box protein 11 (FBXO11) is a member of F-Box protein family, which has recently been proved to be associated with intellectual developmental disorder with dysmorphic facies and behavioral abnormalities (IDDFBA, OMIM: 618089). In this study, 12 intellectual disability individuals from 5 Chinese ID families were collected, and whole exome sequencing (WES), sanger sequencing, and RNA sequencing (RNA-seq) were conducted. Almost all the affected individuals presented with mild to severe intellectual disability (12/12), global developmental delay (10/12), speech and language development delay (8/12) associated with a range of alternate features including increased body weight (7/12), short stature (6/12), seizures (3/12), reduced visual acuity (4/12), hypotonia (1/12), and auditory hallucinations and hallucinations (1/12). Distinguishingly, malformation was not observed in all the affected individuals. WES analysis showed 5 novel FBXO11 variants, which include an inframe deletion variant, a missense variant, two frameshift variants, and a partial deletion of FBXO11 (exon 22-23). RNA-seq indicated that exon 22-23 deletion of FBXO11 results in a new mRNA structure. Conservation and protein structure prediction demonstrated deleterious effect of these variants. The DEGs analysis revealed 148 differentially expressed genes shared among 6 affected individuals, which were mainly associated with genes of muscle and immune system. Our research is the first report of FBXO11-associated IDDFBA in Chinese individuals, which expands the genetic and clinical spectrum of this newly identified NDD/ID syndrome.

Chromosome microarray analysis combined with karyotype analysis is a powerful tool for the detection in pregnant women with high-risk indicators.

BACKGROUND: Karyotype analysis and fluorescence in situ hybridization (FISH) are commonly used for prenatal diagnosis, however they have many disadvantages. Chromosome microarray analysis (CMA) has the potential to overcome these disadvantages. This study aimed to evaluate the clinical value of CMA in the diagnosis of fetal chromosomal anomalies in southwest of China. METHODS: A total of 3336 samples of amniotic fluid or umbilical cord blood from pregnant women with high-risk indicators at our center in southwest of China from June 2018 to January 2023 were included in the retrospective analysis. 3222 cases tested by CMA and karyotyping, 114 cases only tested by CMA. RESULTS: 3336 samples divided into 2911 cases with single and 425 cases with multiple high-risk indicators. The aneuploidy and pathogenic/likely pathogenic copy number variations (CNVs) of 2911 cases with single high-risk indicator were 4.43% (129/2911) and 2.44% (71/2911) respectively; the aneuploidy and pathogenic/likely pathogenic CNVs of 425 cases with multiple high-risk indicators were 6.82% (29/425) and 2.12% (9/425) respectively. The rate of aneuploidy increased significantly with pregnancy age or NT value. The detection rate of aneuploidy on cases with AMA combined NT ≥ 2.5 mm was significantly higher than that in cases only with AMA (p < 0.001); the detection rate of aneuploidy and pathogenic/likely pathogenic CNVs in cases with AMA combined NIPT high-risk were higher than that in cases only with AMA (p < 0.001, p < 0.05). CONCLUSIONS: The combined application of CMA and karyotyping were recommended in prenatal diagnosis for providing a scientific and accurate genetic diagnosis and improving the quality of prenatal genetic counseling.

Expanding the mutational and clinical spectrum of Chinese intellectual disability patients with two novel CTCF variants.

CCCTC-Binding Factor (CTCF) is a protein-coding gene involved in transcriptional regulation, insulator activity, and regulation of chromatin structure, and is closely associated with intellectual developmental disorders. In this study, we report two unrelated Chinese patients with intellectual disability (ID). According to variant interpretation results from exome sequencing data and RNA-seq data, we present two novel heterozygous CTCF variants, NM_006565.3:c.1519_2184del (p. Glu507_Arg727delins47) and NM_006565.3:c.1838_1852del (p.Glu613_Pro617del), found in two distinct unrelated patients, respectively. Moreover, RNA-seq data of patient 1 indicated the absence of the mutant transcript, while in patient 2, the RNA-seq data revealed a CTCF mRNA transcript with a deletion of 15 nucleotides. Notably, the RNA sequencing data revealed 507 differentially expressed genes shared between these two patients. Specifically, among them, 194 were down-regulated, and 313 were up-regulated, primarily involved in gene regulation and cellular response. Our study expands the genetic and clinical spectrum of CTCF and advances our understanding of the pathogenesis of CTCF in vivo.

Whole exome sequencing and transcriptome analysis in two unrelated patients with novel SET mutations.

The human SET nuclear proto-oncogene (SET) gene is a protein-coding gene that encodes proteins that affects chromatin remodeling and gene transcription. Mutations in the SET gene have been reported to cause intellectual disability (ID) and epilepsy. In this study, we collected and analyzed clinical, genetic, and transcript features of two unrelated Chinese patients with ID. Both patients were characterized by moderate intellectual disability. Whole-exome sequencing identified two novel heterozygous mutations in the SET gene: NM_001122821.1:c.532-3 T > A and NM_001122821.1:c.3 G > C (p.0?). Additionally, RNA sequencing revealed widespread dysregulation of genes involved in NF-kB signaling and neuronal system in these two patients. To our knowledge, this is the first report of SET mutations causing ID in the Chinese population, broadening the genetic and ethnic spectrum of SET-related disorders and highlighting the importance of screening for SET gene variants.

Thioridazine Enhances Cisplatin-Induced DNA Damage in Cisplatin-Resistant Human Lung Cancer Cells.

Thioridazine was used to sensitize cisplatin against cisplatin-resistant human lung cancer cells. Cells received thioridazine, cisplatin, or both drugs (the combination). Thioridazine synergized cisplatin to increase percentages of dead and apoptotic cells. DNA damage was detected using the comet assays; the combination led to the highest alkaline- and neutral-comet percentages, demonstrating exacerbation of both single- and double-strand breaks. After thioridazine treatment, levels of glutathione, and BRCA2, RAD51, and ERCC1 proteins were decreased. These data manifested that thioridazine decreased the capacities of detoxification and DNA repair, thereby enhancing cisplatin-induced DNA damage in resistant cells.

HBx promotes hepatocarcinogenesis by enhancing phosphorylation and blocking ubiquitinylation of UHRF2.

BACKGROUND AND AIMS: The major cause of Hepatocellular carcinoma (HCC) is acute or chronic infection caused by hepatotropic viruses and HBV infection is the main cause. UHRF2, a ubiquitin-protein ligase E3, is associated with cancer development. This study aimed to investigate the connection and mechanism between UHRF2 and HBV-associated HCC. METHODS: The expression of UHRF2 in human HBV-positive HCC tissues and paracancerous tissues was detected by western blot and tissue microarray. The effects of UHRF2 on invasion, migration and proliferation were detected in HBV-positive hepatoma cell lines. Furthermore, western blot, immunofluorescence, Co-immunoprecipitation and ubiquitination assays were used to explore the relationship and mechanism between UHRF2 and HBV-associated HCC. RESULTS: HBV-positive HCC tissues had higher UHRF2 expression levels than adjacent non-tumor tissues. The HBV-positive HCC patients with a low UHRF2 level in cancer tissues had longer overall and recurrence-free survival compared with those with a high UHRF2 level. UHRF2 induced invasion, migration and proliferation in human HBV-positive HCC cell lines HepG2.2.15 and Hep AD38(-). HBx, an encoding protein of HBV, maintained the stability of UHRF2 by blocking the ubiquitination of UHRF2. HBx up-regulated CDK2 expression through ETS1. UHRF2 bound to CDK2 directly and enhanced UHRF2 phosphorylation at serine 643. CONCLUSIONS: These results suggest that HBx-ETS1-CDK2-UHRF2 pathway plays an important role in the pathogenesis of HBV-associated HCC and represents new therapeutic targets for human HCC. CLINICAL TRIALS REGISTRATION: ChiCTR2000041416.

Ultrasound Enhances ZD2767P-Carboxypeptidase G2 against Chemoresistant Ovarian Cancer Cells by Altering the Intracellular Pharmacokinetics of ZD2767D.

Prodrug-carboxypeptidase G2 (e.g., ZD2767P+CPG2) can realize a targeted treatment where the specific advantage is a lack of CPG2 analogues in humans, but it is limited by low efficacy. Here ultrasound was employed to enhance ZD2767P+CPG2 (i.e., ZD2767P+CPG2+US) against chemoresistant human ovarian cancer cells. The release dynamics of ZD2767D (activated drug) by CPG2 were investigated. The in vitro efficacy was explored in SKOV3 and SKOV3/DDP (cisplatin-resistant subline) cells; spectrophotometry was established to quantify ZD2767P and ZD2767D, and then intracellular pharmacokinetics were evaluated. The in vivo efficacy was validated in both subcutaneous and orthotopic tumors. With insonation, the ZD2767D concentration was increased during an early period. Insonation synergized ZD2767P+CPG2 to enhance cell death and apoptosis, and efficacies in SKOV3 and SKOV3/DDP cells were similar. Intracellular pharmacokinetics of ZD2767D were nonproportional, and insonation increased the peak level, area under the level vs time curve, and mean residence time. In subcutaneous xenografts, ZD2767P+CPG2 and ZD2767P+CPG2+US resulted in volume-inhibitory rates of 20.4% and 26.5% in SKOV3 tumors and 36.8% and 81.6% in SKOV3/DDP tumors, respectively. In the orthotopic tumor model, the survival time in group ZD2767P+CPG2 or ZD2767P+CPG2+US was prolonged compared with group control, in SKOV3 (33.0 ± 3.5 or 39.2 ± 1.8 vs 25.0 ± 1.6 days, p < 0.0001) and SKOV3/DDP (16.2 ± 4.8 or 22.3 ± 7.3 vs 8.7 ± 3.9 days, p = 0.0015) tumors. These data indicated that ZD2767P+CPG2+US was effective against resistant ovarian cancer cells.

Thioridazine Sensitizes Cisplatin Against Chemoresistant Human Lung and Ovary Cancer Cells.

Human lung cancer cell lines A549 and A549/DDP, and ovarian cancer cell lines SKOV3 and SKOV3/DDP were subjected to thioridazine (Thio), cisplatin, or the combination; A549/DDP and SKOV3/DDP were the cisplatin-resistant sublines. Cell viability, apoptosis, and cell cycle were detected; the mitochondrial membrane potential and proteins related to mitochondrial apoptosis were determined. Thio induced cell death, and the combination of Thio and cisplatin led to the highest percentage of dead cells in four cells lines. Thio and the combined modality led to cell apoptosis by inducing G0/G1 arrest. The collapse of mitochondrial membrane potential, activation of caspase 9, upregulation of Bax protein, and downregulation of Bcl-2 protein demonstrated that apoptosis was mitochondria dependent. These data indicated that Thio could be used to modulate cisplatin-based chemotherapeutic regimen in lung and ovary cancers.

Nanosecond Electric Pulses Induce Early and Late Phases of DNA Damage and Cell Death in Cisplatin-Resistant Human Ovarian Cancer Cells.

Chemoresistance is a challenge for management of ovarian cancer, and therefore the response of resistant cells to nanosecond electric pulses (nsEP) was explored. Human ovarian cancer cell line COC1 and the cisplatin-resistant subline COC1/DDP were subjected to nsEP (32 ns, 10 kV/cm, 10 Hz pulse repletion frequency, and 10 min exposure duration), and then the cellular responses were followed. The percentages of dead cells and of comet-formed cells in the alkaline assay displayed two peak levels (i.e., 2 and 8 h after nsEP exposure), with the highest value noted at 8 h; the percentage of comet-formed cells in the neutral assay was increased at 8 h; the apoptotic percentage was increased at 8 h, with collapse of the mitochondrial membrane potential and the activation of caspase-3 and caspase-9. The comet assay demonstrated DNA single-strand break at 2 h and double-strand break at 8 h. nsEP resulted in lower cytotoxicity in COC1/DDP cells compared with COC1 cells. These findings indicated that nsEP induced early and late phases of DNA damage and cell death, and these two types of cell death may have distinct applications to treatments of chemoresistant ovarian cancers.

Insulin Treatment Cannot Promote Lipogenesis in Rat Fetal Lung in Gestational Diabetes Mellitus Because of Failure to Redress the Imbalance Among SREBP-1, SCAP, and INSIG-1.

Gestational diabetes mellitus (GDM) has a higher incidence of neonatal respiratory distress syndrome, and lipogenesis is required for the synthesis of pulmonary surfactants. The aim of this study was to determine the effect of insulin treatment in GDM on the production of lipids in the lungs of fetal rats. GDM was induced by streptozotocin, and insulin was used to manage diabetes. Type II alveolar epithelial cells (AEC II), bronchoalveolar lavage fluid (BALF), and lung tissues of the neonatal rats were sampled for analyses. Insulin treatment could not decrease plasma glucose to normal level at a later gestational stage. Lipids/phospholipids in AEC II, BALF, and lung tissues decreased in GDM, and insulin treatment could not increase the levels; quantitative PCR and western blotting demonstrated a lower level of sterol regulator element-binding protein 1 (SREBP-1), SREBP cleavage-activating protein (SCAP), and insulin-induced gene 1 (INSIG-1) in GDM, but insulin treatment upregulated only SREBP-1. Nuclear translocation of the SREBP-1 protein in AEC II was impaired in GDM, which could not be ameliorated by insulin treatment. These findings indicated that insulin treatment in GDM cannot promote lipogenesis in the fetal lung because of failure to redress the imbalance among SREBP-1, SCAP, and INSIG-1.

Knockdown of BRCA2 enhances cisplatin and cisplatin-induced autophagy in ovarian cancer cells.

Clinical implications of the BRCA2 expression level on treatments of ovarian cancer are controversial. Here, we demonstrated that platinum-resistant cancer had a higher percentage of high BRCA2 level (87.5% vs 43.6%, P = 0.001), and that patients with a low BRCA2 level in cancer tissues had longer progression-free survival (with a median time of 28.0 vs 12.0 months, P < 0.001) and platinum-free duration (with a median time of 19.0 vs 5.0 months, P < 0.001) compared with those with a high BRCA2 level. In human ovarian cancer cell lines CAOV-3 and ES-2, cisplatin induced an upregulation of the RAD51 protein, which was inhibited after silencing BRCA2; silencing BRCA2 enhanced the action of cisplatin in vitro and in vivo Knockdown of BRCA2 promoted cisplatin-induced autophagy. Interestingly, the autophagy blocker chloroquine enhanced cisplatin in BRCA2-silenced cells accompanied by an increase in apoptotic cells, which did not occur in BRCA2-intact cells; chloroquine enhanced the efficacy of cisplatin against BRCA2-silenced CAOV-3 tumors in vivo, with an increase in LC3-II level in tumor tissues. Sensitization of cisplatin was also observed in BRCA2-silenced CAOV-3 cells after inhibiting ATG7, confirming that chloroquine modulated the sensitivity via the autophagy pathway. These data suggest that a low BRCA2 level can predict better platinum sensitivity and prognosis, and that the modulation of autophagy can be a chemosensitizer for certain cancers.

Deactivation of cisplatin-resistant human lung/ovary cancer cells with pyropheophorbide-α methyl ester-photodynamic therapy.

The aim of this study was to determine whether photodynamic therapy (PDT) alone or combined with cisplatin (DDP), can deactivate cisplatin-resistant cancer cells. Human cancer cell lines A549 and SKOV3, and chemoresistant sublines A549/DDP and SKOV3/DDP, were subjected to PDT, DDP, or PDT combined with DDP. Cell viability and apoptosis were analyzed, and then intracellular reactive oxygen species (ROS) and proteins related to apoptosis were determined. PDT caused cell death, and PDT combined with DDP led to the highest percentage of dead cells in 4 cell lines; similar results were detected in ROS; a quantification evaluation manifested that the combined effect was addition. DDP increased the percentage of apoptotic cells, and the ROS level in A549 and SKOV3 cells, which was not observed in A549/DDP and SKOV3/DDP cells. Western blot revealed an increase of caspase 3 and Bax, and a decrease of Bcl-2, demonstrating the occurrence of apoptosis. The data suggest that PDT can efficiently deactivate resistant cells and enhance the action of DDP against resistant cancer cells.

NIRF, a Novel Ubiquitin Ligase, Inhibits Hepatitis B Virus Replication Through Effect on HBV Core Protein and H3 Histones.

Np95/ICBP90-like RING finger protein (NIRF), a novel E3 ubiquitin ligase, has been shown to interact with HBc and promote its degradation. This study investigated the effects of NIRF on replication of hepatitis B virus (HBV) and the mechanisms. We have shown that NIRF inhibits replication of HBV DNA and secretion of HBsAg and HBeAg in HepG2 cells transfected with pAAV-HBV1.3. NIRF also inhibits the replication and secretion of HBV in a mouse model that expressed HBV. NIRF reduces acetylation of HBV cccDNA-bound H3 histones. These results showed that NIRF is involved in the HBV replication cycle not only through direct interaction with HBc but also reduces acetylation of HBV cccDNA-bound H3 histones.

UHRF2, another E3 ubiquitin ligase for p53.

UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.

NIRF, a novel ubiquitin ligase, interacts with hepatitis B virus core protein and promotes its degradation.

Hepatitis B virus (HBV) core protein (HBc) is a major component of viral nucleocapsid and a multifunctional protein involved in viral maturation and release. It is unstable and present in cells at low level because of K96 lysine residue, which is a ubiquitin acceptor site. Np95/ICBP90-like RING finger protein (NIRF) has auto-ubiquitination activity which is the hallmark of a ubiquitin ligase. In the present study, ubiquitin ligase, NIRF, binds to HBc and leads to the proteasome-mediated degradation of HBc in vivo. NIRF down-regulates HBc protein level, resulting in the decrease of the amount of HBV particles in supernatant of HepG2.2.15 cells. However knockdown of NIRF significantly increases endogenous HBc protein level, leading to HBV release. The results reveal that NIRF interacts with HBc and promotes the degradation of HBc in vivo. The pathway of NIRF-mediated ubiquitin-proteasome affects the release of HBV particles by controlling the amounts of HBc. It indicates that NIRF may participate in the maturation of HBV.