ZC3H13 reduced DUOX1-mediated ferroptosis in laryngeal squamous cell carcinoma cells through m6A-dependent modification.

Laryngeal squamous cell carcinoma (LSCC) is the second most common head and neck cancer. To identify the link between ferroptosis and LSCC, we targeted the dual oxidase 1 (DUOX1) gene. This study aimed to reveal the intrinsic mechanism by which the DUOX1-zinc-finger CCCH domain-containing protein 13 (ZC3H13) ferroptosis axis affected the LSCC process. GEPIA was used to investigate the expression of DUOX1 in LSCC, and the expression levels of DUOX1 and ZC3H13 were manipulated by overexpression and RNA interference. MTT assay was used to detect cell proliferation. Chromatin immunoprecipitation (CHIP) detected the binding of DUOX1 and ZC3H13, and ROS assessment and intracellular Fe2+ content determination were performed to examine the ferroptosis. MeRIP was used to analyze the m6A methylation of DUOX1. Ferroptosis-related proteins were detected by qRT-PCR. DUOX1 was found to be poorly expressed in LSCC cells, low DUOX1 level promoted LSCC cell proliferation, and low ZC3H13 level decreased LSCC cell proliferation. Besides, there was an interaction between DUOX1 and ZC3H13. DUOX1 could inhibit the expression levels of ferroptosis-related genes GPX4 and F1H1 in LSCC cells DUOX1 inhibited the expression levels of ROS and ferroptosis-related genes GPX4 and F1H1 and increased intracellular iron content in LSCC cells, but ZC3H13 reversed this phenomenon by inhibiting DUOX1 gene through m6A methylation modification. ZC3H13 reduced DUOX1-mediated ferroptosis in LSCC cells through m6A-dependent modification. The regulatory pathway of DUOX1 and ferroptosis are potential targets for designing diagnostic and combination therapeutic strategies for LSCC patients.

Treatment of Intracranial Pseudoaneurysms With a Novel Covered Stent: A Series of 19 Patients With Midterm Follow-Up.

Background: The optimal treatment for intracranial pseudoaneurysm is unclear. This study aims to analyze the outcome of treating intracranial pseudoaneurysm with a novel covered stent. Materials and Methods: The institutional imaging and clinical databases were retrospectively reviewed for patients with intracranial pseudoaneurysms treated with Willis covered stent from January 2017 to December 2019. The clinical presentations, etiology, intraoperative complications, and immediate and follow-up outcomes were analyzed. Results: A total of 19 patients with 20 pseudoaneurysms were enrolled for analysis. Seventeen patients presented with vision loss and two with epistaxis. Nineteen Willis covered stents were used with one for each patient without technical failure. Intraoperative thrombosis was encountered in one patient (5.3%), which was recanalized by tirofiban. During clinical follow-up, no further epistaxis occurred, and visual acuity improved in three (17.6%) patients. Endoleak occurred in seven (36.8%) patients after the initial balloon inflation and persisted in one (5.3%) patient after balloon re-inflation. This endoleak disappeared at 8 month follow-up. Finally, during angiographic follow-up (median 13 months), parent artery occlusion and in-stent stenosis occurred in one (5.3%) patient. No stent-related ischemic event was encountered. Conclusions: The Willis covered stent is feasible, safe, and efficient in treating intracranial pseudoaneurysms.

Identification and assessment of pathogenicity of a naturally reassorted infectious bursal disease virus from Henan, China.

Infectious bursal disease virus (IBDV) is still a vital etiological agent in poultry farms. IBDV outbreaks occasionally occur due to the presence of very virulent, reassortment or variant strains. Vaccine immunization has played crucial roles in IBD control for decades. However, survival pressure of IBDV from the vaccine immunization also increases the reassortments of circulating viruses. In this study, an IBDV strain was isolated from several broiler farms in Henan Province, central part of China, and named IBDV HN strain. Based on the results of RT-PCR, sequencing and phylogenic analyses of VP1 and VP2 genes, the IBDV HN strain is a novel reassortment strain in the Henan region. Segment A of this strain appears to originate from the very virulent IBDV strain, while segment B comes from the other field reassortment strains. This may be the result of natural reassortant of virus circulating in the field. About 60% (6/10) of experimentally infected specific pathogen-free chickens died after 3 to 5 d post-infection with typical symptom and pathological lesions. The IBDV HN strain was prone to horizontal transmission, which poses a serious threat to the chicken industry. Further investigation on the prevalence, virulence, and evolution of HN strain IBDV will provide a foundation for the prevention and control of the disease in this region.

[Clinical application of internal carotid artery occlusion].

OBJECTIVE: To evaluate the efficiency of the internal carotid artery (ICA) occlusion in treatment of head and neck diseases. METHODS: To retrospectively analyze 28 cases related with the ICA. According to the result of balloon test occlusion (BTO) and cerebral angiogram, the balloon, spring coil or glue were used to block the fistula or haemorrhage, sometimes even to embolism the related ICA. RESULTS: Twenty-six cases and 2 cases presented negative and positive in total 28 cases in BTO. Of the 26 patients, 22 were succeed in ICA occlusion. The clinical symptoms disappeared in some patients, and the others had the related tumor or foreign body being resected. Two cases (glomus jugulare tumors and intractable epistaxis after Juvenile nasopharyngeal angiofibroma surgery in 1 case respectively) presented delayed cerebral infarction. With the thrombolysis anticoagulant therapy, the clinical symptoms in the former disappeared and in the later reduced. The balloon was let out in 1 case with the embolism the distal middle cerebral artery branches. However, the patient did not show any symptoms during the operation and observation period. One case with intractable epistaxis in nasopharygeal cancer after radiotherapy was dead. In emergency, the left ICA was blocked, then bleeding reduced with the left hemisphere presented extensive cerebral embolism. However, he died in nasal bleeding in three days. CONCLUSIONS: ICA occlusion was an efficient method to save the life. Moreover, the patient's tolerance and collateral circulation should be evaluated in detail before the operation.

[Features of clinical presentation, pathology and magnetic resonance or computed tomography imaging for ocular lymphoma].

OBJECTIVE: To explore the distribution and magnetic resonance imaging or computed tomography (MRI/CT) of lymphoma in orbit. METHODS: Retrospective analyses were conducted for 73 cases with ocular complaints at Beijing Tong'ren Hospital from June 2006 to September 2012. They were confirmed pathologically as lymphoma with complete MRI/CT records. RESULTS: Ocular adnexal lymphoma occurred predominantly in adults. There were 47 males and 26 females with a median age of (60 ± 12.0) years. The lesions were bilateral (n = 24), right side (n = 23) and left side (n = 26) respectively. The pathological types included mucosa-associated lymphoid tissue (MALT) lymphoma (n = 59), diffuse large B-cell lymphoma (DLBCL, n = 7), mantle cell lymphoma (MCL, n = 3), lymphoplasmacytic lymphoma (LPL, n = 2) and follicular lymphoma and natural killer/T-cell lymphoma (FL and NKT, n = 1) respectively. They had well-defined margins in 64 sides, less well-defined margins in 17 sides and ill-defined margins in 16 sides. And 29 sides of lesions presented as a regional mass within conjunctiva in 16/97 sides (16.5%), lacrimal gland in 5/97 sides (5.2%), eyelids in 3/97 sides (3.1%), ocular muscle in 2/97 sides (2.1%) and lacrimal sac in 3/97 sides (3.1%). Moreover, 68 sides of lesion were diffusely distributed. The invaded sites included preseptal region in 38/97 sides (39.2%), extraconal compartment in 69/97 sides (71.1%), intraconal compartment in 54/97 sides (55.7%), eyeball region in 45/97 sides (46.4%), subperiosteum in 14/97 sides (14.4%), cone compartment in 73/97 sides (75.3%), lacrimal gland in 38/97 sides (39.2%) and sheath of optic nerve in 33/97 sides (34.0%). And, on MRI scan, 70 cases (94 sides) had isointensity in 88/94 sides, hypointensity in 6/94 sides on T1WI and isointensity in 52/94 sides, mild hypointensity in 40/94 sides or mixed signal in 2/94 sides on T2WI. In addition, the signals were homogeneous in 67/94 sides and heterogeneous in 27/94 sides. After contrasting, these lesions showed mild contrast-enhancement (CE) in 27/94 sides, moderate CE in 67/94 sides with homogeneous CE in 52/94 sides and heterogeneous CE in 42/94 sides. On CT scan, 21 cases (24 sides) had isodensity in 18/24 sides and mild hyperdensity in 6/24 sides with homogeneous in 7/24 sides and heterogeneous in 17/24 sides. Moreover, most adjacent bone show no change (16/24 sides). Only 16/24 sides and 2/24 sides showed reformation or destruction respectively. At the same time, the lesions also involved other para-orbit structures, such as orbital apex (27/97 sides, 27.8%), fissure orbitalis inferior (14/97 sides, 14.4%), fossa pterygopalatina (10/97 sides, 10.3%), supraorbital nerve (8/97 sides, 8.2%), cavernous sinus (8/97 sides, 8.2%) and facial soft tissue (6/97 sides, 6.2%), et al. CONCLUSION: Ocular adnexal lymphoma occurs predominantly in male adults. And multiple strictures and sites are often involved. There is iso-/mild hyperintensity with mild-to-moderate homo-/heterogeneous CE and little change of adjacent bone on T1WI and T2WI.

[Growth inhibition effection of perlecan anti-sense cDNA on human laryngeal carcinoma xnograft in nude mice].

OBJECTIVE: To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. METHOD: Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. RESULT: Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phßApr-neol cells (P < 0.01). CONCLUSION: These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.

[Complications and the management of fully covered retrievable metal stent placement in benign tracheal stenosis].

OBJECTIVE: To study complications and the management of the use of covered retrievable expandable metallic stents in the treatment of benign tracheal stenosis. METHODS: Fully covered retrievable metal stents were placed in 21 patients with benign tracheal stenosis. Stent-related complications and the management were reviewed and analysed. RESULTS: Twenty-eight fully covered retrievable metallic stents were successfully placed fluoroscopically in 21 patients. Stents were removed 4 - 12 months [mean (5.5 ± 2.2) mouths] after placement in all patients. Stent-related complications included granulation tissue (n = 18), stent migration (n = 4), stent expectoration (n = 2), halitosis (n = 8), mucous retention (n = 21) and mucus plugging (n = 1). Granulation tissue was removed with a carbon dioxide laser in 2 patients. Stents were replaced for 2 times and 3 times respectively in 2 patients after stent migration and stent expectoration. An additional stent was placed in 2 patients after stent migration. Symptom of halitosis was relieved after prolonged course of systemic antibiotics treatment in 8 patients. Symptom of mucous retention was relieved with nebulized saline and N-acetylcysteine saline inhalation. Mucous plug was expelled after severe coughing after suctioning using an aspirator in one patient. There were statistically significant differences in stricture diameter, rank of tachypnea and pulmonary function (FEV(1)) in all 21 patients before stent insertion and after stents removal. No patient has experienced recurrence during the follow-up period of 1 - 36 months [mean (23.2 ± 8.0) months]. CONCLUSION: Fully covered retrievable metallic stent may be a safe and effective in benign tracheal stenosis. Stent-related complications may be effectively managed.

Effect of DRB on the biological characteristics of human laryngeal carcinoma Hep-2 cell line.

In order to study the effect of 5, 6-Dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry (FCM) and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose-and time-dependent manner. After being treated with 0, 10, 20, 40, 80 microm mol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was (0.68+/-0.19)%, (1.95+/-0.12)%, (8.51+/-0.26)%, (11.26+/-0.17)% and (14.99+/-0.32)%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 microm mol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.

[Neurofibromatosis type 2].

OBJECTIVE: To recognise the predisposing factors, clinical manifestations, diagnosis and treatment of neurofibromatosis type 2 (NF2). METHOD: The clinical data of one NF2 case was reported and the literatures were also reviewed. RESULT: The patient was diagnosed at a much later stage than onset. Progressive hearing loss and tinnitus were the initial symptoms. MRI scan indicated space-occupying lesions in the bilateral cerebellopontine angles, bilateral cavernous sinuses and bilateral cervical parts of the patient. The patient was diagnosed as NF2 according to the National Institutes of Health (NIH) criteria, and received operation on the left acoustic tumor. The tumor was proved to be schwannomas by pathological test. The hearing loss and the facial nerve paralysis (House-Brackmann II) had appeared after operation. CONCLUSION: NF2 is an autosomal dominant, highly penetrant disease which is characterized by bilateral vestibular schwannomas. Early diagnosis and management for tumor is very important for survival and hearing preservation. The "golden standard" in terms of diagnostic precision is the magnetic resonance imaging (MRI) scan with gadolinium enhancement.

[Inhibitory effects of small interfering RNA specific to protein kinase CK2a on the growth of laryngeal carcinoma cells].

OBJECTIVE: To assess the effect of small interfering RNA (siRNA) specific to protein kinase CK2a on proliferation and apoptosis of Hep-2 cell line. METHODS: siRNA expression plasmid psiRNA-hH1neo-CK2 specific to protein kinase CK2a and non-specific siRNA expression plasmid psiRNA-hH1neo-cont were constructed respectively, and then were transfected into Hep-2 cells by lipofectamine methods. Protein kinase CK2a mRNA and protein of the transfected cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. RESULTS: Protein kinase CK2a mRNA and protein expressions were significantly decreased in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05). The Hep-2 cells grew slowly after transfected with psiRNA-hH1neo-CK2(P < 0.05). Obvious subdiploid peaks were found in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05). CONCLUSIONS: siRNA expression plasmid specific to protein kinase CK2a suppressed the protein kinase CK2a expression and the proliferation of Hep-2, and induced apoptosis of Hep-2 cells.

[Application of rat tail collagen in the primary culture of rats marginal cells of stria vascularis].

OBJECTIVE: To study the feasibility of application of rat-tail collagen in the primary culture of rats marginal cells of stria vascularis. METHOD: The effect of self-made rat-tail collagen on the primary culture of rats marginal cells of stria vascularis was observed and estimated. RESULT: When cochlear stria vascularis fragment was isolated and cultured for 24 h, a few culture cells appeared around the fragments. About 48 to approximately 72 h later, clusters of culture cells could be seen,these cells showed the cobblestone shape under the microscope. The immunohistochemistry and the transmission electron microscopy showed the epithelial origination of these cells. CONCLUSION: It is feasible to the primarily culture the rats marginal cells of stria vascularis with self-made rat-tail collagen.

Expression of vascular endothelial growth factor and cyclooxygenase-2 in laryngeal squamous cell carcinoma and its significance.

In order to study the expressions of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in human laryngeal squamous cell carcinoma (LSCC) and its significance, the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues (both P < 0.01), and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ill + IV tissues of LSCC as compared with the stage I + II tissues of LSCC (P < 0.01). There was a high positive correlation between VEGF and COX-2 expression in LSCC (r = 0.756, P < 0.01). These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.

[Expression of perlecan in laryngeal carcinoma cell and its significance].

OBJECTIVE: To study the expression of perlecan in human laryngeal carcinoma cells and its significance. METHOD: The expression of perlecan mRNA in human laryngeal carcinoma cells, Hep-2 cells were investigated by using the techniques of semi-quantify RT-PCR. The expression of perlecan in the Hep-2 cells was investigated by using the techniques of immunohistochemistry. RESULT: It showed that the expression level of perlecan and perlecan mRNA significantly increased in Hep-2 cells as compared with the normal cells, Hacat cells (P < 0.01). CONCLUSION: These data raise the possibility that perlecan many play key roles in the growth,invasion and metastasis of human laryngeal carcinoma cells through either a paracrine or an autocrine mechanism.

[Suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA].

OBJECTIVE: To study the suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA. METHOD: In this study, the plasmids of recombination eukaryotic expression vector perlecan anti-sense cDNA (pAP) were transfected into Hep-2 cells by using cationic liposome (lipofectamine 2000) and divided into three groups: non-transfected group, WT group; transfection with no load carrier, neo group; and transfection with the pAP plasmid, pAP group. Semi quantify RT-PCR, western blot assay and MTT assay were used to detected the expression of perlecan mRNA and protein in the three groups; the level of cell proliferation; and the responsivity of basic fibroblast growth factor (bFGF). RESULT: It was showed that the expression of perlecan mRNA and protein were significantly reduced in the pAP group compared with WT group and ph beta Apr-neol transfected group ( P < 0.01). In the presence of 1 microg/L of bFGF in low serum (0.1% FCS), the pAP transfected cells showed a reduced proliferation rate (MTT assay) while the wild type cells and ph beta Apr-neol transfected cells grew rapidly. CONCLUSION: The growth of Hep-2 cells could be inhibited significantly by perlecan anti-sense cDNA plasmids transfection.

[Expression of vascular endothelial growth factor and its receptors in laryngeal carcinoma cell and its significance].

OBJECTIVE: To study the expression of vascular endothelial growth factor (VEGF) and both the Flt-1 and KDR high affinity VEGF receptors in human laryngeal carcinoma cells and its significance. METHOD: In this study, we investigated the expression of VEGF mRNA and both the Fit-1 mRNA and KDR mRNA high affinity VEGF receptors in human laryngeal carcinoma cells using techniques of semi-quantify RT-PCR. RESULT: It was showed that the expression level of VEGF mRNA was significantly increased in human laryngeal carcinoma cells as compared with the normal cells, Hacat cells (P <0.05), and there was the high expression level of Flt-1 mRNA in the Hep-2 cells, but not KDR mRNA. CONCLUSION: These data raise the possibility that VEGF and its receptors many play key roles in the growth, invasion and metastasis of human laryngeal carcinoma cells through either a paracrine or an autocrine mechanism.

Effects of neuroglobin gene transfer in vivo on hearing response properties of neurons in the inferior colliculus in mice after administration of sodium salicylate.

The effects of neuroglobin (NGB) gene transfer in vivo mediated by GeneJamer on the hearing response properties of the inferior colliculus (IC) neurons in mice after administration of sodium salicylate were studied. Forty-eight Kunming mice were divided into 4 groups (n=12 in each group): Group A1 (negative control);Group A2 (positive control);Group B, sodium salicylate (450 mg/kg every day) + pEGFP-C1;Group C, sodium salicylate (450 mg/kg every day) + pEGFP-NGB. The GeneJamer and pEGFP-NGB were mixed and injected into IC neurons in mice. The expression of NGB mRNA and protein of IC neurons in mice was detected by using RT-PCR and Western blot methods. The intensity-rate functions, intensity-latency functions and frequency-turning curves in IC neurons were recorded by extracellular electrophysiological recording techniques and the effects of pEGFP-NGB transfer following injection of sodium salicylate on them were studied. It was found that: (1) The GeneJamer-mediated pEGFP-NGB could be effectively transferred into the IC brain tissues in mice and NGB could be expressed intensively. (2) The intensity-rate functions of IC neurons were raised after administration of sodium salicylate. The non-monotonic styles of intensity-rate functions in groups A1, A2 and C were accounted for 74.6%, 72.2 %, 59.3 %, respectively, and the function in group B for 47%. There were significant differences between group B and groups A1, A2 or C (P<0.01, P<0.01, P<0.05). (3) The intensity-latency functions in IC neurons were reduced after administration of sodium salicylate. The non-monotonic styles of intensity-latency functions in groups A1, A2 and C were accounted for 3.2 %, 5.1 %and 21 %, respectively, and that in group B for 45.5 %. There were significant differences between group B and groups A1, A2 or C (P<0.01, P<0.01, P<0.05, respectively). (4) The frequency-turning curves in groups A1 and A2 were sharpened. In 72 acoustic neurons recorded in the group B, the frequency-turning curves from 53 neurons were broadened while those of the rest were sharpened. In group C the frequency-turning curves recorded from 12 of 67 acoustic neurons were broadened while those of the remaining were sharpened. These results suggest that in vivo transfer of NGB gene is highly expressed in IC neurons in mice. In vivo transfer of NGB gene reverses the change of intensity-rate functions, intensity-latency functions and the code styles after administration of sodium salicylate in IC neurons in mice.

Study on remodeling of astrocytes in facial neuclus after peripheral injury.

To observe the glial reactions surrounding facial motor neurons following facial nerve anastomosis. At 1, 7, 21 and 60 d following facial nerve anastomosis, the recovery process of facial movement was observed, the glial fibrillary acidic protein (GFAP) immunoreactivity was analyzed by a combined method of fluorescent retrograde tracing and immunofluorescent histochemical staining, and the ultrastructure of astrocytes were observed under a transmission electron microscope (TEM), respectively. Postoperatively the function of facial muscles could not return to normal, often accompanied with hyperkinetic syndromes such as synkinesis at the late stage. Motor neurons in every facial subnucleus could be retrogradely labeled by fluoro-gold (FG), and displayed an evident somatotopic organization. Normally there was a considerable number of GFAP-positive cells in nonnucleus regions but few inside the facial nucleus region. Postoperatively the GFAP immunoreactivity in the anastomotic side increased significantly, but gradually decreased at the late stage. The ultrastructure of astrocytes in our experiment showed that the sheet-like process of astrocytes invested and protected the injured facial motor neurons. The present study shows that reactive astrocytes undergo some characteristic changes during the process of facial nerve injury and regeneration. The plastic change at the late stage may be involved in the mechanism of synkinesis.