DNMT1-targeting remodeling global DNA hypomethylation for enhanced tumor suppression and circumvented toxicity in oral squamous cell carcinoma.

BACKGROUND: The faithful maintenance of DNA methylation homeostasis indispensably requires DNA methyltransferase 1 (DNMT1) in cancer progression. We previously identified DNMT1 as a potential candidate target for oral squamous cell carcinoma (OSCC). However, how the DNMT1- associated global DNA methylation is exploited to regulate OSCC remains unclear. METHODS: The shRNA-specific DNMT1 knockdown was employed to target DNMT1 on oral cancer cells in vitro, as was the use of DNMT1 inhibitors. A xenografted OSCC mouse model was established to determine the effect on tumor suppression. High-throughput microarrays of DNA methylation, bulk and single-cell RNA sequencing analysis, multiplex immunohistochemistry, functional sphere formation and protein immunoblotting were utilized to explore the molecular mechanism involved. Analysis of human samples revealed associations between DNMT1 expression, global DNA methylation and collaborative molecular signaling with oral malignant transformation. RESULTS: We investigated DNMT1 expression boosted steadily during oral malignant transformation in human samples, and its inhibition considerably minimized the tumorigenicity in vitro and in a xenografted OSCC model. DNMT1 overexpression was accompanied by the accumulation of cancer-specific DNA hypomethylation during oral carcinogenesis; conversely, DNMT1 knockdown caused atypically extensive genome-wide DNA hypomethylation in cancer cells and xenografted tumors. This novel DNMT1-remodeled DNA hypomethylation pattern hampered the dual activation of PI3K-AKT and CDK2-Rb and inactivated GSK3ß collaboratively. When treating OSCC mice, targeting DNMT1 achieved greater anticancer efficacy than the PI3K inhibitor, and reduced the toxicity of blood glucose changes caused by the PI3K inhibitor or combination of PI3K and CDK inhibitors as well as adverse insulin feedback. CONCLUSIONS: Targeting DNMT1 remodels a novel global DNA hypomethylation pattern to facilitate anticancer efficacy and minimize potential toxic effects via balanced signaling synergia. Our study suggests DNMT1 is a crucial gatekeeper regarding OSCC destiny and treatment outcome.

Fractionated irradiation induces radioresistant oral carcinoma cells with enhanced malignant phenotypes.

OBJECTIVE: The fact that certain oral carcinoma patients experience radiotherapy failure implies that a more radioresistant and aggressive phenotype of surviving cancer cells potentially occurs during treatment. Our study aimed to establish radioresistant oral cancer cells through a fractionated irradiation protocol that mimics clinically relevant radiotherapy dosing strategies and to investigate all-round alterations in the malignant phenotype. METHODS: Radioresistant oral carcinoma cells were generated by exposing Cal27 and Detroit 562 cells to 60 Gy radiation in 10 dose-escalating fractions and verified by cell immunofluorescence. Specific markers related to the epithelial-mesenchymal transition (EMT) process and the cancer stem cell (CSC) phenotype were assessed by Western blotting. Cell invasion and migration were evaluated using Matrigel-coated transwell and wound healing assays, respectively. Nontargeted metabolomics was used to mechanistically delineate the potential metabolic patterns linked to EMT and CSCs; the CSC phenotype was also examined by sphere formation assays and cell immunofluorescence. RESULTS: Radioresistant oral carcinoma cell lines were successfully established and validated. These cells exhibited enhanced EMT and increase in both cell invasion and migration. These radioresistant cells further demonstrated a high metabolic profile, notably marked by lipid metabolism reprogramming and functional enrichment of ATP-binding cassette (ABC) transporters. Consistently, enhanced CSC phenotype in radioresistant cells was confirmed by elevated expression of stemness markers and increased sphere-forming capacity. CONCLUSION: Radioresistant oral carcinoma cells subjected to fractionated radiation exhibit an augmented malignant phenotype. The metabolic characteristics linked to enhanced EMT and CSC phenotypes provide potential targets for improving radiotherapy in oral carcinoma.

Ionizing radiation-induced "zombie" carcinoma-associated fibroblasts with suppressed pro-radioresistance on OSCC cells.

OBJECTIVES: This study was to investigate the effect of ionizing radiation (IR) on oral carcinoma-associated fibroblasts (CAFs) and to further explore subsequent effects of IR-induced "zombie" CAFs on oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: Three primary CAFs and one primary normal-associated fibroblasts (NAFs) were separated from human OSCC and normal oral mucosa tissues, identified by immunocytochemistry. Cells were exposed to IR by X-ray irradiator under different doses. The DNA damage, proliferation, and migration of irradiated CAFs were, respectively, detected by immunofluorescence and wound healing assay, while senescence was detected by ß-galactosidase staining. Finally, the effect of irradiated CAFs on biological behavior and radioresistance of Cal-27 cells were determined via assays mentioned above. RESULTS: Oral CAFs were sensitive to IR with DNA damage increasing and proliferation decreasing. 18 Gy IR could not kill oral CAFs but induce them to "zombies," with arrested proliferation, increased senescence, and reduced migration. "Zombie" CAFs (zCAFs) could enhance proliferation, migration, and invasion of Cal-27 cells, and show suppressed pro-radioresistance by reducing DNA damage and facilitating survival. CONCLUSIONS: IR-induced zCAFs could continuously promote radioresistance of OSCC cells though being suppressed, suggesting the potential promoting effect on tumor relapse post-radiotherapy that needed further exploring.

Differential OAT methylation correlates with cell infiltration in tumor microenvironment and overall survival postradiotherapy in oral squamous cell carcinoma patient.

BACKGROUND: Given that DNA methylation and tumor microenvironment (TME) are susceptible to radiotherapy, we aimed to figure out specific differential DNA methylation to reflect oral squamous cell carcinoma (OSCC) prognosis and associated effect on TME changes postradiotherapy, performing as an efficient biomarker. MATERIALS AND METHODS: Differentially methylation analysis was performed using data from The Cancer Genome Atlas. Curves of Kaplan-Meier (K-M) survival, cumulative hazard and events, Cox proportional hazards, and linear regression model were conducted to screen and validate differential methylation genes, while multiple regression equation to analyze if ornithine aminotransferase (OAT) methylation correlates with radiotherapy. For correlation between OAT methylation and immune infiltrates, CIBERSORT and ESTIMATE algorithms were performed, following gene set enrichment analysis (GSEA) and ssGSEA analysis to evaluate biological process. RESULTS: Compared to normal tissues, only OAT in OSCC was differential significantly by K-M analysis (p = 0.0364). OAT hypermethylation was associated with increased overall survival (hazard ratio: 0.65, p = 0.0358). Radiotherapy correlated with OAT methylation (ß = -0.01, p = 0.0061); most patients with OAT hypermethylation were radiation-sensitive. Hypomethylated OAT correlated with higher cell infiltrations in TME. Neuroactive ligand-receptor interaction was most significantly related to OAT methylation (p = 9.2e-10). Sulfur metabolism was the most significantly in OAT hypermethylation group (p = 0.0041) and RIG-I-like receptor in OAT hypomethylation group (p = 0.0094). CONCLUSION: OAT methylation can serve as a predictor of OSCC prognosis postradiotherapy with potential mechanism by changing cell infiltrations in TME, but further experimental study deserves to carry out confirming the role and mechanism of OAT methylation in OSCC.

The Application Effect of Traditional Chinese Medicine Nursing on General Anesthesia Combined with Epidural Anesthesia and Electric Resection for the Treatment of Bladder Cancer and Its Influence on Tumor Markers.

OBJECTIVE: To explore the effects of traditional Chinese medicine nursing on general anesthesia combined with epidural anesthesia and electric resection to treat bladder cancer and its influence on tumor markers. METHODS: A total of 160 patients with non-muscle-invasive bladder cancer who underwent general anesthesia combined with epidural anesthesia and resection were included in this study. The patients were divided into control group (n = 80) and study group (n = 80) according to the random number table method. The control group received hydroxycamptothecin bladder perfusion therapy, and the study group received traditional Chinese medicine nursing combined with hydroxycamptothecin bladder perfusion therapy. The clinical efficacy, three-year cumulative survival rate, and postoperative recurrence rate of the two groups of patients were detected. The levels of tumor markers including vascular endothelial growth factor (VECF) and bladder tumor antigen (BTA) before and after treatment were also tested. The immune function, inflammatory factor levels, and quality of life of the two groups before and after treatment were evaluated. RESULTS: The total effective rate of the study group (83.75%) was significantly higher than that of the control group (58.75%). After treatment, the serum VEGF and BTA levels, inflammatory factors interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-α (TNF-α) levels of the two groups of patients decreased, and the decrease in the study group was more significant than that in the control group (P < 0.05). After treatment, the levels of CD3+, CD4+, and CD4+/CD8+ in the two groups increased (P < 0.05), and the increase in the study group was more significant than that in the control group (P < 0.05). After treatment, the CD8+ levels of the two groups of patients decreased (P < 0.05), and the decrease in the study group was more significant than that in the control group (P < 0.05). After treatment, the quality-of-life scores in both groups increased (P < 0.05), and the increase in the study group was even more significant (P < 0.05). CONCLUSION: Traditional Chinese medicine nursing has significant clinical effects on the treatment of bladder cancer with general anesthesia combined with epidural anesthesia and electric resection. It can more effectively prevent the risk of recurrence of bladder cancer after surgery, significantly improve the quality of life, improve immune system function, regulate the levels of VECF and BTA, effectively reduce the level of serum inflammatory factors, inhibit tumor progression, and reduce tumor viability.

Development and external validation of a dynamic nomogram for delayed cerebral ischaemia after aneurysmal subarachnoid hemorrhage: a study protocol for a multicentre retrospective cohort study.

INTRODUCTION: Delayed cerebral ischaemia (DCI) caused by aneurysmal subarachnoid haemorrhage (aSAH) is the most frequent complication and typically contributes to poor neurological outcome or deterioration of patients' condition. Therefore, early accurate and effective prediction of DCI is urgently needed. This study aims to construct a dynamic nomogram for precisely calculating the risk of DCI in patients with aSAH. Internal validation of this tool is conducted using the training cohort, and independent external validation is completed by using other medical centre datasets. METHODS AND ANALYSIS: This study is a multicentre, retrospective, observational cohort study using data from patients with aSAH. The participants include all adult patients who received surgical treatment in neurosurgery of multiple medical centres from 1 September 2019 to 1 April 2021, including Renmin Hospital of Wuhan University, Huzhou Central Hospital, First Affiliated Hospital of Harbin Medical University, General Hospital of Northern Theatre Command and Affiliated Hospital of Panzhihua University. Clinical information is collected via the electronic medical record system, including demographic data, clinical state on admission and serum laboratory tests. Modified Fisher grade at admission, admission subarachnoid clot and cerebral oedema density, and residual postoperative subarachnoid clot density are determined using the electronic imagine record software. The primary outcome is DCI. ETHICS AND DISSEMINATION: This study protocol was reviewed and approved by the Medical Ethics Committee of Renmin Hospital of Wuhan University, which is the principal affiliation of this study (approval number: WDRM2021-K022). The other Ethics Committees, including Huzhou Central Hospital (approval number: 202108005-01), First Affiliated Hospital of Harbin Medical University (approval number: H202156), General Hospital of Northern Theater Command (approval number: Y2021060) and Affiliated Hospital of Panzhihua University (approval number: 202105002), also approved the protocol. The results of this research will be published in a peer-reviewed medical journal. TRIAL REGISTRATION NUMBER: ChiCTR2100044448.

A novel 4-gene signature model simultaneously predicting malignant risk of oral potentially malignant disorders and oral squamous cell carcinoma prognosis.

OBJECTIVE: Oral squamous cell carcinoma (OSCC) is often diagnosed at late stage with a poor prognosis. The study hereunder aimed to construct a multi-gene model to simultaneously promote early diagnosis of OSCC by evaluating malignant risk of oral potentially malignant disorders (OPMDs) and predict prognosis. MATERIALS AND METHODS: 3 GEO datasets including OPMDs and OSCC samples were obtained for overlapping differentially expressed genes (DEGs) being screened. The predictive model was built with optimal DEGs by SVM algorithm, estimated by receiver operator characteristic curves and validated for double prediction via oral cancer-free survival (for malignant risk of OPMDs) and overall survival time (for OSCC) analysis respectively compared to other models. The protein expression of biomarkers in the model was validated in human samples by immunohistochemistry. RESULTS: A novel predictive model of 4-gene signature was built based on 12 common DEGs revealed from 3 GEO datasets. It could well distinguish OSCC from OPMDs and normal tissues. Both oral cancer-free survival and overall survival time analysis were significantly poorer in high-risk patients than in low-risk ones in Kaplan Meier survival curve respectively. The protein expression of biomarkers in OSCC was with significant difference compared to normal and OPMDs. CONCLUSIONS: The novel 4-gene signature model presents strong ability in simultaneous prediction of the malignant risk of OPMDs and OSCC progression, potentially benefiting both the early diagnosis and therapeutic outcomes of OSCC.

A method to calculate acoustic radiation modes based on spheroidal wave functions.

Acoustic radiation modes (ARMs) have been widely used in noise control engineering. For an arbitrarily shaped radiator, ARMs are usually obtained by using the boundary element method (BEM). For high frequencies and large structures, the BEM calculation is usually time consuming. An alternative method was recently developed in which the ARMs are obtained by using generalized singular value decomposition and spherical harmonic functions. In this paper, spheroidal harmonic functions are used instead of spherical harmonic functions. The proposed method is potentially more efficient for ARM calculations for structures with one dimension significantly larger than the other two.

Radiotherapy targeting cancer stem cells "awakens" them to induce tumour relapse and metastasis in oral cancer.

Radiotherapy is one of the most common treatments for oral cancer. However, in the clinic, recurrence and metastasis of oral cancer occur after radiotherapy, and the underlying mechanism remains unclear. Cancer stem cells (CSCs), considered the "seeds" of cancer, have been confirmed to be in a quiescent state in most established tumours, with their innate radioresistance helping them survive more easily when exposed to radiation than differentiated cancer cells. There is increasing evidence that CSCs play an important role in recurrence and metastasis post-radiotherapy in many cancers. However, little is known about how oral CSCs cause tumour recurrence and metastasis post-radiotherapy. In this review article, we will first summarise methods for the identification of oral CSCs and then focus on the characteristics of a CSC subpopulation induced by radiation, hereafter referred to as "awakened" CSCs, to highlight their response to radiotherapy and potential role in tumour recurrence and metastasis post-radiotherapy as well as potential therapeutics targeting CSCs. In addition, we explore potential therapeutic strategies targeting these "awakened" CSCs to solve the serious clinical challenges of recurrence and metastasis in oral cancer after radiotherapy.

Calculation of acoustic radiation modes by using spherical waves and generalized singular value decomposition.

Acoustic radiation modes (ARMs) have been widely used in noise control engineering owing to their convenient sound-power characteristics. However, the evaluation of ARMs for non-regular structures can be computationally intensive: it usually involves solving the boundary integral equation (BIE) by using the boundary element method in order to obtain the sound power radiation resistance matrix, followed by a singular value decomposition to obtain the radiation modes. The proposed procedure involves projecting spherical harmonics onto an enclosing surface, followed by the application of generalized singular value decomposition, with the result that the need to solve the BIE is eliminated, which potentially reduces the computational effort significantly.

miR-181a and miR-203 inhibit migration and invasion of laryngeal carcinoma cells by interacting with ATF2.

MicroRNAs (miRNAs) have been recognized to modulate the progression of tumorigenesis by serving as oncogenes or tumor suppressors. Despite the involvement of miR-181a and miR-203 in several cancers as has been substantiated, their roles in laryngeal carcinoma (LC) remain unclear. In this study, the abundances of miR-181a, miR-203 and activating transcription factor 2 (ATF2) mRNA in LC cell lines were detected by RT-qPCR. Western blot was performed to detect the protein levels of N-cadherin, E-cadherin and ATF2. Cell migration and invasion ability were assessed by Trans-well assay. The putative binding sites between miR-181a or miR-203 and ATF2 were predicted using Bioinformatics software and further validated by Dual-Luciferase reporter and RNA immunoprecipitation (RIP) assays. Results showed reduced abundances of miR-181a and miR-203 in LC cell lines. Introduction of miR-181a or miR-203 reduced cell migration and invasion, which was further confirmed by the reduction of N-cadherin and increase of E-cadherin in LC cells. ATF2 was identified to be a potential target of miR-181a and miR-203. Absence of ATF2 overturned the stimulatory effects of anti-miR-181a and anti-miR-203 on cell migration and invasion in LC cells. Our findings suggested that miR-181a and miR-203 attenuated cell migration and invasion ability by directly targeting ATF2 in LC, providing novel insight into the regulatory mechanisms of miR-181a and miR-203 in LC.

A truncating mutation in CEP55 is the likely cause of MARCH, a novel syndrome affecting neuronal mitosis.

BACKGROUND: Hydranencephaly is a congenital anomaly leading to replacement of the cerebral hemispheres with a fluid-filled cyst. The goals of this work are to describe a novel autosomal-recessive syndrome that includes hydranencephaly (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly (MARCH)); to identify its genetic cause(s) and to provide functional insight into pathomechanism. METHODS: We used homozygosity mapping and exome sequencing to identify recessive mutations in a single family with three affected fetuses. Immunohistochemistry, RT-PCR and imaging in cell lines, and zebrafish models, were used to explore the function of the gene and the effect of the mutation. RESULTS: We identified a homozygous nonsense mutation in CEP55 segregating with MARCH. Testing the effect of this allele on patient-derived cells indicated both a reduction of the overall CEP55 message and the production of a message that likely gives rise to a truncated protein. Suppression or ablation of cep55l in zebrafish embryos recapitulated key features of MARCH, most notably renal dysplasia, cerebellar hypoplasia and craniofacial abnormalities. These phenotypes could be rescued by full-length but not truncated human CEP55 message. Finally, we expressed the truncated form of CEP55 in human cells, where we observed a failure of truncated protein to localise to the midbody, leading to abscission failure and multinucleated daughter cells. CONCLUSIONS: CEP55 loss of function mutations likely underlie MARCH, a novel multiple congenital anomaly syndrome. This association expands the involvement of centrosomal proteins in human genetic disorders by highlighting a role in midbody function.

[Clinical Efficacy Comparison of Different Second Line Salvage Chemotherapy Regimens for the Treatment of Elderly Patients with Relapsed and Refractory Diffuse Large B Cell Lymphoma].

OBJECTIVE: To investigate and compare the clinical effects and safety of DICE regimen combined with rituximab and GDP regimen combined with rituximab for the treatment of elderly patients with relapsed and refractory diffuse large B cell lymphoma. METHODS: Ninety elderly patients with relapsed and refractory diffuse large B cell lymphoma were admitted in our hospital from January 2008 to June 2013 and randomly divided into 2 groups, including A group (45 patients) and B group (45 patients), the patients in A group were treated by DICL regimen combined with rituximab, while the patients in B group were treated by GDP regimen combined with rituximab; the clinical efficacy, disease-free survival time, the survival rate with follow-up and the incidence of toxic side-effects in 2 groups were compared. RESULTS: The clinical efficacy of B group was significant better than that of A group (P < 0.05). The disease-free survival time of B group was significantly longer than that of A group (P < 0.05). The survival rate with follow-up of B group was significantly higher than that of A group (P < 0.05). The difference was not significant in incidence of the toxic side effects between 2 groups (P > 0.05). CONCLUSION: Compared with DICE regimen combined with rituximab, GDP regimen combined with rituximab in treatment of elderly patients with relapsed and refractory diffuse large B cell lymphoma can efficiently reduce tumor loading, prolong the disease-free survival time, improve the long-term clinical prognosis, and not aggravate the side effects of drugs.

A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators.

Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBß accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients.

CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290.

Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.