缺氧相关的长链非编码RNA LINC00970在唾液腺腺样囊性癌中的表达及其作用

目的探讨唾液腺腺样囊性癌(SACC)中长链非编码RNA(lncRNA)LINC00970表达与缺氧的关系及其作用。方法低氧(1%O2)诱导48 h后,lncRNA芯片检测SACC-83细胞中缺氧诱导的lncRNA,并用实时荧光定量聚合酶链反应(PCR)验证。使用临床样本分析LINC00970在SACC组织中的表达及其与预后的关系。LINC00970敲低质粒转染SACC-LM和SACC-83细胞后,细胞增殖检测(CCK-8)和平板克隆实验检测细胞增殖能力和克隆形成能力,Transwell实验检测细胞侵袭和迁移能力,Western blot检测E-cadherin、Vimentin、Snail和N-cadherin的表达。荧光定量PCR、Western blot和Transwell等实验均重复3次所得均值为该样本测量值,使用SPSS 20.0软件进行统计学分析。SACC组织细胞与正常唾液腺组织中LINC00970相对表达量差异,采用配对t检验分析;比较沉默LINC00970对SACC-83和SACC-LM细胞增殖、克隆形成、侵袭及迁移能力的影响,以及LINC00970沉默对上皮-间充质转化(EMT)相关蛋白的影响,应用独立t检验进行统计学分析。采用Kaplan-Meier法计算LINC00970的表达与SACC患者总生存时间的相关性,并绘制生存曲线。P<0.05为差异有统计学意义。结果lncRNA芯片及实时荧光定量PCR结果显示,LINC00970是SACC-83细胞中在缺氧反应性表达最高的lncRNA(15.13±0.57 vs 1.08±0.06),差异有统计学意义(t=24.56,P<0.001)。临床样本数据分析显示,LINC00970在SACC组织表达显著高于正常唾液腺组织(0.49±0.41 vs 0.08±0.16,t=2.53,P<0.001),且LINC00970高表达患者生存率明显低于LINC00970低表达患者(HR=0.42,P=0.03)。SACC细胞敲低LINC00970后,缺氧促进的侵袭和迁移能力受抑制,且缺氧升高后的间质标志物N-cadherin表达下调,而缺氧诱导降低的上皮标志物E-cadherin恢复上调。结论缺氧可诱导SACC细胞中LINC00970的表达,其高表达与SACC患者的不良预后密切相关,LINC00970可能通过调控EMT从而促进细胞侵袭和迁移。

Depletion of Gsdma1/2/3 alleviates PMA-induced epidermal hyperplasia by inhibiting the EGFR–Stat3/Akt pathway

Homeostasis of the skin barrier is essential for maintaining normal skin function.Gasdermin A(GSDMA)is highly expressed in the skin and associated with many skin diseases,such as melanoma and psoriasis.In mice,GSDMA is encoded by three gene homologues,namely Gsdma1,Gsdma2,and Gsdma3.Although Gsdma3 gain-of-function mutations cause hair loss and skin inflammation,Gsdma3-deficient mice do not show any visible phenotypes in skin and hair structures.To explore the physiological function of GSDMA,we generated conventional Gsdma1/2/3 knockout(KO)mice.These mice showed significantly alleviated epidermal hyperplasia and inflammation induced by phorbol 12-myristate 13-acetate(PMA).Furthermore,the alleviation of epidermal hyperplasia depended on the expression of Gsdma1/2/3 specifically in keratinocytes.Mechanistically,Gsdma1/2/3 depletion downregulated epidermal growth factor receptor(EGFR)ligands,leading to the decreased EGFR–Stat3/Akt signalling.These results demonstrate that depletion of Gsdma1/2/3 alleviates PMA-induced epidermal hyperplasia partially by inhibiting the EGFR–Stat3/Akt pathway.

Mitochondrial ROS promote macrophage pyroptosis by inducing GSDMD oxidation

Disrupted mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation are often associated with macrophage pyroptosis. It remains unclear how these forms of mitochondrial dysfunction relate to inflammasome activation and gasdermin-D (Gsdmd) cleavage, two central steps of the pyroptotic process. Here, we also found MMP collapse and ROS generation induced by Nlrp3 inflammasome activation as previous studies reported. The elimination of ROS alleviated the cleavage of Gsdmd, suggesting that Gsdmd cleavage occurs downstream of ROS release. Consistent with this result, hydrogen peroxide treatment augmented the cleavage of Gsdmd by caspase-1. Indeed, four amino acid residues of Gsdmd were oxidized under oxidative stress in macrophages. The efficiency of Gsdmd cleavage by inflammatory caspase-1 was dramatically reduced when oxidative modification was blocked by mutation of these amino acid residues. These results demonstrate that Gsdmd oxidation serves as a de novo mechanism by which mitochondrial ROS promote Nlrp3 inflammasome-dependent pyroptotic cell death.

GSDMB promotes non-canonical pyroptosis by enhancing caspase-4 activity

Gasdermin B (GSDMB) has been reported to be associated with immune diseases in humans, but the detailed molecular mechanisms remain unsolved. The N-terminus of GSDMB by itself, unlike other gasdermin family proteins, does not induce cell death. Here, we show that GSDMB is highly expressed in the leukocytes of septic shock patients, which is associated with increased release of the gasdermin D (GSDMD) N-terminus. GSDMB expression and the accumulation of the N-terminal fragment of GSDMD are induced by the activation of the non-canonical pyroptosis pathway in a human monocyte cell line. The downregulation of GSDMB alleviates the cleavage of GSDMD and cell death. Consistently, the overexpression of GSDMB promotes GSDMD cleavage, accompanied by increased LDH release. We further found that GSDMB promotes caspase-4 activity, which is required for the cleavage of GSDMD in non-canonical pyroptosis, by directly binding to the CARD domain of caspase-4. Our study reveals a GSDMB-mediated novel regulatory mechanism for non-canonical pyroptosis and suggests a potential new strategy for the treatment of inflammatory diseases.

CD4+T cells memorize obesity and promote weight regain

Body weight regain often causes failure of obesity therapies while the underlying mechanism remains largely unknown.In this study,we report that immune cells,especially CD4+T cells,mediate the‘memory’of previous obese status.In a weight gain-loss-regain model,we found that C57BL/6J mice with an obesity history showed a much faster rate of body weight regain.This obesity memory could last for at least 2 months after previously obese mice were kept at the same body weight as non-obese mice.Surprisingly,such obesity memory was abrogated by dexamethasone treatment,whereas immunodeficient Rag1−/−and H2A−/−mice failed to establish such memory.Rag1−/−mice repossessed the obesity memory when immune cells or CD4+T cells isolated from previously obese mice were transferred.Furthermore,depletion of CD4+T cells led to obesity memory ablation.Taken together,we conclude that CD4+T cells mediate obesity memory and promote weight regain.

Intratumoral CD103^(+)CD8^(+)T cells predict response to neoadjuvant chemoimmunotherapy in advanced head and neck squamous cell carcinoma

Background Immune cell heterogenicity is known to determine the therapeutic response to cancer progression.Neoadjuvant chemoimmunotherapy(NACI)has shown clinical benefits in some patients with advanced head and neck squamous cell carcinoma(HNSCC),but the underlying mechanism behind this clinical response is unknown.The efficacy of NACI needs to be potentiated by identifying accurate biomarkers to predict clinical responses.Here,we attempted to identify molecules predicting NACI response in advanced HNSCC.Methods We performed combined single-cell RNA sequencing(scRNA-seq)and multiplex immunofluorescence(mIHC)staining with tumor samples derived from NACI-treated HNSCC patients to identify a new tumor-infiltrating cell(TIL)subtype,CD103^(+)CD8^(+)TILs,associated with clinical response,while both in vitro and in vivo assays were carried out to determine its antitumor efficiency.The regulatory mechanism of the CD103^(+)CD8^(+)TILs population was examined by performing cell-cell interaction analysis of the scRNA-seq data and spatial analysis of the mIHC images.Results We established intratumoral CD103^(+)CD8^(+)TILs density as a determinant of NACI efficacy in cancers.Our scRNA-seq results indicated that the population of CD103^(+)CD8^(+)TILs was dramatically increased in the responders of NACI-treated HNSCC patients,while mIHC analysis confirmed the correlation between intratumoral CD103^(+)CD8^(+)TILs density and NACI efficacy in HNSCC patients.Further receiver operating characteristic curve analysis defined this TIL subset as a potent marker to predict patient response to NACI.Functional assays showed that CD103^(+)CD8^(+)TILs were tumor-reactive T cells,while programmed cell death protein-1(PD-1)blockade enhanced CD103^(+)CD8^(+)TILs cytotoxicity against tumor growth in vivo.Mechanistically,targeting the triggering receptor expressed on myeloid cells 2-positive(TREM2^(+))macrophages might enhance the population of CD103^(+)CD8^(+)TILs and facilitate antitumor immunity during NACI treatment.Conclusions Our study highlights the impact of intratumoral CD103^(+)CD8^(+)TILs density on NACI efficacy in different cancers,while the efforts to elevate its population warrant further clinical investigation.

The role of immune cells in obesogenic memory

Obesity is a serious chronic disease and worldwide public health problem.According to data from the World Health Organization(WHO),more than 650 million adults in the world were obese and three times more individuals were overweight in 2016.Obesity is associated with type II diabetes,hepatic steatosis,cardiovascular diseases,systemic chronic inflammation,and at least 13 types of cancer.Unfortunately,few effective remedies are available for obesity.The major problem is that most patients suffer from weight regain 2–3 years after obesity treatments,such as obesity drugs,lifestyle interventions(including exercise,diet restrictions,and diet modification),and bariatric surgery.Along with weight regain,most of the initial beneficial changes in metabolomic and transcriptomic profiles dissipate,including insulin sensitivity,the level of adipokines and blood cholesterol.This phenomenon has been defined as“obesogenic memory”.1,2 Obesogenic memory causes repeated weight loss and gain along with obesity therapy,which is called“weight cycling”,leading to a worse healthy status than would result from simple obesity.3,4.