Ferroptosis in laryngeal squamous cell carcinoma and its regulation by M2 macrophage-derived exosomes / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate ferroptosis in laryngeal squamous cell carcinoma (LSCC) and its regulation by M2 macrophage-derived exosomes. Methods: LSCC and adjacent noncancerous tissue samples were collected from 32 patients treated in the Department of Otorhinolaryngology, Head and Neck Surgery of the Second Affiliated Hospital of Harbin between September 2018 and April 2021, including 26 males and 6 females, aged 43-79 years. The expressions of ferroptosis marker glutathione peroxidase 4(GPX4) in LSCC and adjacent noncancerous tissues were detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction(RT-PCR). The correlations between GPX4 expression and clinicopathological factors in LSCC were analyzed. Biological changes of TU212 cells after treated with ferroptosis-induced agent erastin were detected by transmission electron microscope, cell counting kit-8(CCK-8), clone test, reactive oxygen species(ROS), malondialdehyde(MDA), glutathione(GSH), JC-1, RT-PCR and western blot. Exosomes were isolated from the supernatant of M0/M2 macrophages (M0-exos/M2-exos) and co-incubated with erastin-treated TU212 cells to detect the change of ferroptosis in cells of each group. The data were analyzed by SPSS software of version19.0. Results: GPX4 expression in LSCC tissues was significantly higher than that in adjacent noncancerous tissues (2.04±0.65 vs. 0.99±0.09, F=30.36, P<0.001), and was closely related to T stage and clinical stage (Ⅰ-Ⅱvs.Ⅲ-Ⅳ: 1.75±0.39 vs. 2.18±0.71, F=2.25, P<0.05; T1-2 vs. T3-4: 1.71±0.42 vs. 2.20±0.69, F=2.06, P<0.05). In TU212 cells treated with erastin, mitochondrial crest became smaller, membrane density increased, proliferation rate decreased, intracellular ROS level increased, mitochondrial membrane potential depolarized, GSH content decreased, intracellular MDA level increased and expressions of GPX4 mRNA and protein decreased. Change of M0 into M2 macrophages was induced by IL-4 stimulation. When erastin-treated TU212 cells were incubated with M2-exos, cell proliferation was partially restored and GPX4 expression was enhanced, and also with the recoveries of levels of ROS, MDA and GSH (all P<0.05). Conclusions: Ferroptosis is one of the cell death ways of LSCC. M2-exos may inhibit ferroptosis of LSCC cells.

The function and mechanism of long non-coding RNA RP11-159K7.2 in sinonasal squamous cell carcinoma / 中华耳鼻咽喉头颈外科杂志

Objective: To explore the role and mechanism of long non-coding RNA RP11-159K7.2 in the progression of sinonasal squamous cell carcinoma (SNSCC). Methods: Sixty-five cases of SNSCC tissues and adjacent tissues were selected from the Department of Otorhinolaryngology Head and Neck Surgery, the Second Affiliated Hospital of Harbin Medical University from 2009 to 2014. The expression of RP11-159K7.2 in SNSCC and adjacent tissues was detected by RNAscope in situ hybridization to observe its association with prognosis. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins 9 (CRISPR/Cas9) was used to knockout the expression of RP11-159K7.2 in RPMI-2650 cells (SNSCC cell line). Cell counting kit-8 (CCK-8), wound healing and Transwell were performed to observe the changes of proliferation, migration and invasion of SNSCC cells in vitro after down-regulation of RP11-159K7.2. Moreover, the growth of xenograft in nude mice after down-regulation of RP11-159K7.2 was examined in vivo. Mechanically, the protein chip, Western blot and RNA immunoprecipitation were performed to identify the proteins bound by RP11-159K7.2. SPSS 17.0 was used for statistical analysis. Results: The expression of RP11-159K7.2 in SNSCC tissue was significantly higher than that in adjacent tissues. RP11-159K7.2 expression was closely related with T grade, nodal metastasis and differentiation of SNSCC (χ2 value was 4.697, 4.235 and 10.753, respectively, all P<0.05). The five-year survival rate of RP11-159K7.2 high expression patients was significantly lower than that of RP11-159K7.2 low expression ones (P=0.013 7). After the down-regulation of RP11-159K7.2, the proliferation, migration and invasion ability of SNSCC cells decreased significantly, and the growth of SNSCC xenograft was significantly inhibited. There were 31 candidate proteins that may bind to RP11-159K7.2. RP11-159K7.2 directly bound to nuclear factor-κB (NF-κB) in SNSCC cells, and the regulation of RP11-159K7.2 on the proliferation and invasion of SNSCC cells depended on NF-κB. Conclusion: The increased expression of RP11-159K7.2 in SNSCC may serve as a potential molecular marker for SNSCC prognosis assessment. It is currently considered that the carcinogenic mechanism of RP11-159K7.2 in SNSCC is related to the regulation of NF-κB protein.

Transoral CO2 laser epiglottectomy for early epiglottic carcinomas / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To compare the functional results of transoral CO2 laser epiglottectomy with transcervical supraglottic laryngectomy and to find the landmarks for CO2 laser surgery.</p><p><b>METHODS</b>Forty-nine preoperatively untreated patients diagnosed as clinical T1-2N0M0 epiglottic carcinomas enrolled from June 1, 2006, to November 1, 2009 in the Second Affiliated Hospital of Harbin Medical University were studied retrospectively. Seventeen cases were treated by CO2 laser whereas 32 by open surgeries. The upper edge of thyroid cartilage and mucosal recess infra epiglottic tubercle could be used as intraoperation landmarks for transoral CO2 laser surgery. Optional neck dissections (II, III) were performed for 34 patients with tumors on laryngeal face, but not for 15 with tumors on the edges of epiglottis or small laryngeal face tumors.</p><p><b>RESULTS</b>The follow-up for this study covers a period ranging from 3 to 6 years postoperatively. Four local recurrences were found for this study, 2 for laser surgery who underwent additional repeated exisions and 2 for conventional techniques, one received radiotherapy (60 Gy) and another with total laryngectomy. The incidence of lymph node metastasis was 10.2% (5/49) in all cases. Time of naso-feeding or hospitalisation was significantly shorter for CO2 laser treated patients than that for open techniques. Overall 3 year's survival rates were 100% and 90.6% for CO2 laser and the open techniques respectively.</p><p><b>CONCLUSIONS</b>Compared to the open technique, transoral CO2 laser epiglottectomy is a well-tolerated and promising resection technique with low morbidity for early epiglottic carcinomas, and the identification of landmarks is useful for entire resection of epiglottis.</p>

Role of siRNA mediated matrix metalloproteinase-2 gene silencing in the inhibition of invasion and growth of laryngeal cancer cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the inhibitory effect of matrix metalloproteinase-2 (MMP-2) gene silencing in vitro and in vivo on the invasion and growth of laryngeal cancer cells.</p><p><b>METHODS</b>siRNA recombinant lentivirus targeting MMP-2 gene was transfected into Hep-2 cells, and MMP-2 protein expression was analyzed consequently by using western-blot. Invasive properties of transfectants were evaluated by Boyden assay. In addition, the lentivirus was intratumorally injected in a model of the grafted nude mouse and the morphological changes of transfectants were examined by transmission electron microscope. Finally, cell proliferation in xenografts was measured by immunolabeling of proliferating cell nuclear antigen (PCNA).</p><p><b>RESULTS</b>Over 90% of target cancer cells were found to be transfected by MMP-2-RNAi-Lentivirus. Western-blot analysis revealed that none of transfectants expressed MMP-2 protein whereas most untreated cancer cells exhibited positive protein expression. Significant differences were found between the treated and untreated groups regarding the number of transfectants penetrating through an artificial basement in a Boyden chamber (12 +/- 4 vs 35 +/- 6, x +/- s, t = 14.492, P < 0.01), and the average value of weight [(1.186 +/- 0.225) g vs [(2.127 +/- 0.344) g] and volume [(0.974 +/- 0.216) cm3 vs (1.618 +/- 0.272) cm3] of the grafted tumors (t was 7.094 and 5.684, P < 0.01). The overall tumor inhibitive rate was about 44.2%. Transmission electron microscope showed an obviously decreased invasive feature of transfectants. Finally, the percentages of transfectants immunolabeled for PCNA were significantly lower in the treated group (49.588 +/- 6.995) than those (71.434 +/- 7. 043) in control one (t = 9. 573, P < 0.01).</p><p><b>CONCLUSIONS</b>The invasion, growth and proliferation of laryngeal cancer can be inhibited by siRNA mediated MMP-2 gene silencing. These data strongly suggest that MMP-2 gene silencing by siRNA technology could be a promising approach to cancer therapy.</p>