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.

[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.

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.

[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.

[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.

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.