Non-small cell lung cancer (NSCLC), the main histological type of lung cancer, poses a serious threat to human health. Increasing evidence has shown that long non-coding RNA (lncRNA) MNX1-AS1 is involved in the development and progression of cancers, including lung cancer. Apoptosis and ferroptosis, which are two forms of regulated cell death, can be induced by anti-cancer drugs. However, the roles of MNX1-AS1 in apoptosis and ferroptosis are still unclear. Here, we found that knockdown of MNX1-AS1 promoted the ferroptosis induced by RSL3 in NSCLC cells, with a decrease in cell viability and increases in reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Meanwhile, acridine orange/ethidium bromide (AO/EB) double staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and Annexin V/PI double staining revealed that knockdown of MNX1-AS1 promoted the apoptosis caused by paclitaxel in NSCLC cells. In addition, knockdown of MNX1-AS1 resulted in increased expression of pro-apoptotic protein BAX as well as the cleaved caspase-3 and PARP1, and decreased expression of anti-apoptotic protein Bcl-2. RNA sequencing and quantitative real-time PCR assay identified that the expression of ACSL4 was increased, while the expression of ABCG2 was reduced when MNX1-AS1 was knocked down. Rescue assay showed that ACSL4 and ABCG2 were involved in MNX1-AS1-mediated ferroptosis and apoptosis, respectively. Furthermore, knockdown of MNX1-AS1 increased the sensitivity of NSCLC cells to the combination of RSL3 and paclitaxel. Taken together, our data suggest that MNX1-AS1 might be a potential therapeutic target for lung cancer, especially in combination of ferroptosis and/or apoptosis-inducing drugs.

Eukaryotic elongation factor 2 (EF2), is a critical enzyme solely responsible for catalyzing the translocation of the elongated peptidyl-tRNA from the A to P sites of the ribosome during the process of protein synthesis. EF2 is found to be highly expressed in a variety of malignant tumors and is correlated with cancer cell progression and recurrence. The present study was designed to uncover the function of EF2 on lung squamous cell carcinoma (LSCC) cancer cell growth and progression. Our results from clinical tissue studies showed that EF2 protein was significantly overexpressed in LSCC tissues, compared with the adjacent normal lung tissues, which was confirmed by western blotting and tissue microarray. Forced expression of EF2 resulted in the enhancement of lung squamous carcinoma NCI-H520 cells growth through promotion of G2/M progression in cell cycle, activating Akt and Cdc2/Cyclin B1. In nude mice cancer xenograft model, overexpression of EF2 significantly facilitated cell proliferation in vivo. Furthermore, forced expression of EF2 in the cells increased the capabilities of migration and invasion by changing the expressions of EMT-related proteins and genes. These results provided novel insights into the role of EF2 in tumorigenesis and progression in LSCC. EF2-targeted therapy could become a good strategy for the clinical treatment of LSCC.

Background Lung cancer is a serious threat to human health and is the first leading cause of cancer death. Ferroptosis, a newly discovered form of programmed cell death associated with redox homeostasis, is of particular interest in the lung cancer, given the high oxygen environment of lung cancer. NADPH has reducing properties and therefore holds the potential to resist ferroptosis. Resistance to ferroptosis exists in lung cancer, but the role of NADK in regulating ferroptosis in lung cancer has not been reported yet.Methods Immunohistochemistry (IHC) was used to analyse the expression of NADK in 86 cases of lung adenocarcinoma(LUAD) and adjacent tissues, and a IHC score was assigned to each sample. Chi-square and kaplan-meier curve was performed to analyse the differences in metastasis and five-year survival between the two groups with NADK high or low scores. Proliferation of NADK-knockdown LUAD cell lines was detected in vivo and vitro. Furthermore, leves of ROS, MDA and Fe2+ were measured to validate the effect and mechanism of NADK on ferroptosis in LUAD.Results The expression of NADK was significantly evaluated in LUAD tissues as compared to adjacent non-cancerous tissues. The proliferation of NADK-knockdown cells was inhibited both in vivo and vitro, and increasing levels of intracellular ROS, Fe2+ and lipid peroxide products (MDA) were observed. Furthermore, NADK-knockdown promoted the ferroptosis of LUAD cells induced by Erastin/RSL3 by regulating the level of NADPH and the expression of FSP1. Knockdown of NADK enhanced the sensitivities of LUAD cells to Erastin/RSL3-induced ferroptosis by regulating NADPH level and FSP1 expression.Conclusions NADK is over-expressed in LUAD patients. Knockdown of NADK inhibited the proliferation of LUAD cells both in vitro and in vivo and promotes the Erastin/RSL3-induced ferroptosis of LUAD cells by down-regulating the NADPH/FSP1 axis.

Protein disulfide isomerase family 6 (PDIA6) belongs to the protein disulfide isomerase (PDI) family, which function as isomerases and molecular chaperones. PDIA6 has recently been shown to promote the proliferation and growth of various types of human cancer cells; however the underlying molecular mechanism remains elusive. Here, we report that PDIA6 enhances the proliferation of HeLa cells through activation of the Wnt/β-catenin signaling pathway. Ectopic overexpression of PDIA6 in HeLa cells led to increased cell proliferation accompanied with accelerated cell cycle progression. Further mechanistic investigation demonstrated that overexpression of PDIA6 resulted in decreased phosphorylation of β-catenin at Ser45 and Ser33/Ser37/Thr41, while increased β-catenin nuclear accumulation, and upregulation of Wnt/ β-catenin signaling target genes cyclinD1 and c-myc, which was abolished by ubiquitin-proteasome inhibitor MG132. These results demonstrated that PDIA6 overexpression promoted the proliferation of HeLa cells by suppressing the phosphorylation of β-catenin, thereby inhibiting the degradation of β-catenin through the ubiquitin-proteasome pathway.

Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA) is a kind of pentacyclic triterpene, which widely distributes in nature. OA possesses a powerful anti-cancer effect; however, its low solubility limits its bioavailability and application. In this study, a new OA derivative, K73-03, was used to determine its effect on liver cancer cells and detailed molecular mechanisms. Here, we show that K73-03 may lead to the disorder of mitochondria in HepG2 cells, leading to excessive ROS production and apoptosis in cells. Meanwhile, K73-03 could induce cell apoptosis by inhibiting JAK2/STAT3 pathway and NF-κB/P65 pathway. Collectively, this study may provide a preliminary basis for further cancer treatment of hepatocellular carcinoma.

It contains supplementary materials and methods, supplementary table S1-S3, supplementary figure S1-S11, and uncut images of western blotting

Phosphocreatine (PCr) has been shown to have a cardio-protective effect during cardiopulmonary resuscitation (CPR). However, little is known about its impact on atherosclerosis. In this study, we first evaluated the pharmacological effects of PCr on antioxidative defenses and mitochondrial protection against hydrogen peroxide (H2O2) induced human umbilical vascular endothelial cells (HUVECs) damage. Then we investigated the hypolipidemic and antioxidative effects of PCr on hyperlipidemic rat model. Via in vitro studies, H2O2 significantly reduced cell viability and increased apoptosis rate of HUVECs, while pretreatment with PCr abolished its apoptotic effect. PCr could reduce the generation of ROS induced by H2O2. Moreover, PCr could increase the activity of SOD and the content of NO, as well as decrease the activity of LDH and the content of MDA. PCr could also antagonize H2O2-induced up-regulation of Bax, cleaved-caspase3, cleaved-caspase9, and H2O2-induced down-regulation of Bcl-2 and p-Akt/Akt ratio. In addition, PCr reduced U937 cells' adhesion to H2O2-stimulated HUVECs. Via in vivo study, PCr could decrease MDA, TC, TG and LDL-C levels in hyperlipidemic rats. Finally, different-concentration PCr could increase the leaching of TC, HDL, and TG from fresh human atherosclerotic plaques. In conclusion, PCr could suppress H2O2-induced apoptosis in HUVECs and reduce hyperlipidemia through inhibiting ROS generation and modulating dysfunctional mitochondrial system, which might be an effective new therapeutic strategy to further prevent atherosclerosis.

Abstract Lung cancer, with non‐small cell lung cancer (NSCLC) being the main subtype, is the leading cause of cancer death worldwide, which is mainly due to the cancer metastasis. Glutathione peroxidase 2 (GPX2), an antioxidant enzyme, is involved in tumor progression and metastasis. Nevertheless, the role of GPX2 in NSCLC metastasis has not been clarified. In this study, we found that GPX2 expression was elevated in NSCLC tissues and high GPX2 expression was correlated with poor prognosis in patients with NSCLC. In addtion, GPX2 expression was related to the patient's clinicopathological features, including lymph node metastasis, tumor size, and TNM stage. Overexpression of GPX2 promoted epithelial–mesenchymal transition (EMT), migration, and invasion of NSCLC cells in vitro. Knockdown of GPX2 showed the opposite effects in vitro and inhibited the metastasis of NSCLC cells in nude mice. Furthermore, GPX2 reduced reactive oxygen species (ROS) accumulation and activated the PI3K/AKT/mTOR/Snail signaling axis. Therefore, our results indicate that GPX2 promotes EMT and metastasis of NSCLC cells by activating the PI3K/AKT/mTOR/Snail signaling axis via the removal of ROS. GPX2 may be an effective diagnostic and prognostic biomarker for NSCLC.

Abstract Purpose Ferroptosis is a newly discovered programmed cell death, but its molecular mechanism remains largely unknown. AMPD2 is a protein, which related to oxygen metabolism in non-small cell lung cancer (NSCLC). In this study, we aimed to clarify whether AMPD2 is a regulatory factor of ferroptosis, which may provide a new theoretical basis for inducing ferroptosis to treat tumors. Methods we constructed AMPD2 overexpression and knockdown cells lines using lentivirus packaging technique. Immunohistochemistry was used to detect the expression of AMPD2 in cancerous tissue and adjacent normal tissue. DCFH-DA probe was used to detect the changes of reactive oxygen species(ROS). MDA assay was used to detect the changes of lipid peroxidation. CCK-8 assay was used to detect cell viability. Correlations of AMPD2 with Ferroptosis-related signaling pathways were analyzed using GEPIA. Western blot assay was used to detect changes in Ferroptosis-related molecular markers and signaling pathway. Results AMPD2 is higher expressed in NSCLC patient’s tissues. The Overexpression of AMPD2 significantly reduces intracellular ROS and MDA in NSCLC cells, inhibits ferroptosis induced by RSL3 (ferroptosis activator), enhances the expression of p38, p-p38 and GPX4, whereas knockdown of AMPD2 reverse the results. Moreover, treatment with AMP will increase ferroptosis sensitivity medicated by AMPD2. Conclusions AMPD2 regulates cellular ferroptosis by modulating the level of AMP affecting the p38 pathway and GPX4.

目的 检测正常甲状腺细胞系、中分化甲状腺癌细胞系和低分化甲状腺癌细胞系蛋白质表达谱,寻找与甲状腺癌分化相关的生物标志物.方法 应用液相色谱(liquid chromatogram,LC)与质谱(Mass Spectrum,MS)联用分析方法,找出正常甲状腺细胞与中、低分化甲状腺癌细胞系间的差异蛋白.利用生物信息学方法对差异蛋白进行分析,并利用Western blot检测THJ-16T、FTC133、Nthy-ori3-1等3个细胞系中I型胶原α1(collagen type Iα1,COL1A1)、I型胶原α2(collagen type Iα2,COL1A2)、内凹陷蛋白(caveolin-1,Cav-1)、纤维束蛋白同源物1(fascin 1,FSCN1)、硫氧化还原蛋白(thioredoxin,TRX)、高迁移率族蛋白A1(high mobility group protein A1,HMGA1)、超氧化物歧化酶2(superoxide dismutase,SOD2)、Rho关联含卷曲螺旋结合蛋白激酶1(Rho-associated protein kinase 1,Rock-1)、角蛋白7(cytoskeletal 7,KRT7)、角蛋白8(cytoskeletal 8,KRT8)的表达,利用免疫细胞化学方法进一步验证,从而筛选出分化相关蛋白.结果 通过分析获得中分化与低分化甲状腺癌细胞细胞系差异表达蛋白质178个.生物信息学研究表明,这些差异蛋白质主要参与细胞內吞信号、T-淋巴细胞介导的细胞凋亡、维生素C运输等通路,且通过Western blot检测发现COL1A1、Cav-1、TRX、Rock-1的表达与细胞的分化能力呈正相关,部分蛋白表达水平使用免疫细胞化学验证,与Western blot结果相符.结论 COL1A1、Cav-1、TRX、Rock-1可能是分化相关的标记物,此为进行大样本的临床检测提供了线索.

Background and Objective: Metastasis is the leading cause of death in patients with advanced non-small cell lung cancer (NSCLC), and epithelial-mesenchymal transition (EMT) is a crucial event in the metastasis of NSCLC. Our previous works demonstrated that NgBR promoted EMT in NSCLC. However, the molecular mechanism was unclear. Methods: TGF-β1 was used to induce EMT process of NSCLC cells. The biological functions of NgBR in promoting TGF-β1-induced NSCLC metastasis were studied by gain- and loss-of-function assays both in vitro and in vivo. The underlying mechanisms were studied using molecular biology assays. Results: We found that knockdown of NgBR inhibited TGF-β1-induced cell migration and invasion in NSCLC cells. In contrast, NgBR overexpression promoted TGF-β1-induced EMT of A549 cells. Mechanically, we found that knockdown of NgBR facilitated ubiquitination and degradation of TβRI, leading to downregulation of TβRI expression in NSCLC cells. Moreover, we confirmed a positive correlation between NgBR and TβRI in NSCLC tissues. Conclusions: Our findings provide a novel role of NgBR in modulating TGF-β1-induced EMT and propose NgBR as a new therapeutic target for treating NSCLC patients.

Abstract Lymph node metastasis is one of the most malignant clinical features in patients with esophageal squamous cell carcinoma (ESCC). Understanding the mechanism of lymph node metastasis will provide treatment strategies for patients with ESCC. Long noncoding RNAs (lncRNA) play a critical role in the development and progression of human cancers. However, the role and mechanism of lncRNAs in lymph node metastasis remain largely unknown. Here we show that VEGFC mRNA stability–associated long noncoding RNA (VESTAR) is involved in lymph node metastasis of ESCC. VESTAR was overexpressed in ESCC tissues and was predictive of poor prognosis in patients with ESCC. In ESCC, NXF1 and SRSF3 facilitated nuclear export of VESTAR to the cytoplasm, which was associated with lymph node metastasis. Depletion of VESTAR inhibited ESCC-associated lymphangiogenesis and lymphatic metastasis. Mechanistically, VESTAR directly bound and stabilized VEGFC mRNA. VESTAR also interacted with HuR, a positive regulator of VEGFC mRNA stability, and increased HuR binding to VEGFC mRNA. Our study reveals a novel lncRNA-guided mechanism of lymph node metastasis in ESCC and may provide a potential target for treatment of ESCC lymphatic metastasis. Significance: These findings illustrate the lncRNA-guided regulation of VEGFC mRNA stability via direct RNA–RNA interactions, highlighting a therapeutic target for patients with ESCC with lymphatic metastasis.

Background/Aims Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers with poor prognosis. Metastasis is the leading cause of cancer-related deaths. The growth arrest and DNA damage-inducible 45 gamma (GADD45G) has been reported to correlate with survival, invasion, and metastasis of ESCC. This study was aimed to investigate the role and mechanism of GADD45G in ESCC cell migration and invasion. Methods Both the effects of GADD45G and its need for E-cadherin to function on ESCC cell migration and invasion were determined through loss- and gain-of-function approaches via Transwell assays. The interaction between GADD45G and E-cadherin was detected by GST-pull down and IP assays. The expression of E-cadherin upon GADD45G overexpression was evaluated by RT-qPCR and western blot. The level of E-cadherin in cytoplasmic, nuclear, and membrane fractions was examined by western blot following subcellular fractionation. Results Knockdown of GADD45G increased the migration and invasion abilities of KYSE150 cells, while overexpression of GADD45G showed the opposite effects on YES2 and KYSE30 cells. GADD45G could interact with E-cadherin and enhanced its membrane level. Knockdown of E-cadherin abolished the inhibitory effects of GADD45G on ESCC cell migration and invasion. Intriguingly, dimer-dissociating mutant of GADD45G could not interact with E-cadherin and almost lost its ability to suppress the ESCC cell migration and invasion. Conclusions This study reveals a novel role for GADD45G in inhibiting the ESCC cell migration and invasion, which will provide a new insight in understanding the ESCC metastatic mechanism.

OBJECTIVES:LOC100133669 is a lncRNA whose function during tumorigenesis remains unclear now. Thus, we aimed to explore its clinical significance and function in oesophageal squamous cell carcinoma (ESCC).MATERIALS AND METHODS:ISH was used to detect LOC100133669 expression in ESCC tissues. The full-length LOC100133669 was identified by using RACE assay. Subcellular distribution of LOC100133669 was examined by nuclear/cytoplasmic RNA fractionation and qPCR. The role of LOC100133669 in ESCC cell growth was determined by colony formation, MTT and flow cytometry experiments in vitro, as well as xenograft tumour experiment in vivo. RNA pull-down assay was performed to find LOC100133669-interacted protein, which was further examined by RIP, IP, Western blot and rescue experiments.RESULTS:LOC100133669 was upregulated in ESCC tissues compared with adjacent non-tumour tissues. High LOC100133669 expression was associated with poor prognosis of patients with ESCC. We defined LOC100133669 to be 831 nt in length and mainly localized in the cytoplasm of ESCC cells. Knockdown of LOC100133669 inhibited ESCC cell proliferation and cell cycle progression, while overexpression of LOC100133669 showed the opposite effects. Furthermore, LOC100133669 could bind to Tim50 and upregulated its protein level through inhibiting ubiquitination. Overexpression of Tim50 in part abolished the LOC100133669 depletion-caused inhibitory effect on ESCC cell proliferation.CONCLUSIONS:LOC100133669 plays an oncogenic role in ESCC and may serve as a promising diagnostic marker and therapeutic target for ESCC patients.

以我校2018级临床医学专业本科生为研究对象,8个班为实验组,采用线上线下混合式教学模式,其余8个班为对照组,采用传统教学模式.通过期末考试、问卷调查等方式,分析混合式教学模式在胚胎学中的教学效果.期末成绩分析表明,实验组胚胎学成绩明显高于对照组.调查问卷结果显示实验组学生对混合式教学模式普遍感到满意.混合式教学模式对激发学生学习兴趣,增强学生的自主学习能力、发现问题和解决问题等方面有明显的促进作用,并能促进师生互动,同时也提高教师教学水平.本研究为混合式教学模式的推广应用奠定了实践基础.

目的 研究1-酰基甘油-3-磷酸酰基转移酶4(1-acyl-sn-glycerol-3-phosphate acyltransferaseδ,AGPAT4)对人非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞株A549及H1299细胞体外侵袭与迁移能力的影响及潜在的机制.方法 通过RNAi技术构建A549及H1299瞬时敲降AGPAT4细胞模型,通过Lipofactamine 2000脂质体转染法瞬时干扰肺腺癌细胞A549以及H1299中AGPAT4的表达,应用Real time PCR及Western blot检测A549细胞及H1299细胞中AGPAT4的敲降效率,检测上皮间叶转化(epithelial-mesenchymal transition,EMT)标志物E-cadherin及N-cadherin水平,检测p65和IKBα及其磷酸化水平,Transwell侵袭迁移实验检测敲降AGPAT4对A549及H1299细胞侵袭能力的影响.结果 敲降AGPAT4可以提高p65和IKBα的磷酸化水平,下调E-cadherin蛋白表达水平,且上调N-cadherin蛋白表达水平,同时敲降AGPAT4可增强NSCLC细胞的侵袭和迁移能力.结论 敲降AGPAT4激活p65、IKBα 磷酸化、促进NSCLC细胞的侵袭迁移能力.因此,AGPAT4可能是非小细胞肺癌潜在的治疗靶点.

Fachen Zhou* Jin Wang * Xinming Chi Xin Zhou Zhou Wang 1 1Department of Thoracic Surgery, Shandong Provincial Hospital Affiliated with Shandong University, Jinan, Shandong, People’s Republic of China; 2Department of Thoracic Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, People’s Republic of China; 3Department of Histology and Embryology, Dalian Medical University, Dalian, Liaoning, People’s Republic of China

PURPOSE:Metastasis is a crucial cause of the high mortality in patients with lung cancer. Long non-coding RNAs (lncRNAs) are emerging as important players in the development and progression of human cancers. Here, we aimed to identify metastasis-associated lncRNA and to study its roles in the migration and invasion of lung cancer cells.MATERIALS AND METHODS:We screened differentially expressed lncRNAs between high- and low-metastatic lung cancer cell lines by using microarray and identified the target lncRNA TM4SF1-AS1. The effect of the TM4SF1-AS1 on the invasion and migration was evaluated through the wound healing experiment and transwell assay. The expression of related genes was assessed by RNA sequence and Western blotting.RESULTS:TM4SF1-AS1 was highly expressed in high metastatic lung cancer cell line, and it was also significantly up-regulated in lymph node metastatic lung cancer and was associated with lymph node metastasis. Overexpression of TM4SF1-AS1 promoted the migration and invasion of lung cancer cells. Overexpression of TM4SF1-AS1 decreased the expression of E-Cadherin and increased the expression of Vimentin, Snail and Twist, while knockdown of TM4SF1-AS1 exhibited the opposite trend. Furthermore, RNA sequence analysis revealed that some signaling pathways, including PI3K/AKT signaling pathway, were enriched upon TM4SF1-AS1 overexpression. Western blotting further confirmed that the PI3K/AKT signaling pathway was activated by TM4SF1-AS1.CONCLUSION:This study illustrates that TM4SF1-AS1 promotes the migration and invasion of lung cancer cells by activating the PI3K/AKT signaling pathway. TM4SF1-AS1 might be a novel target of molecular treatment for lung cancer.

Background: Protein phosphorylation plays a pivotal role in cancer metastasis. We first clarified the relation of Serine 27(Ser27) site phosphorylation of minichromosome maintenance protein 2 (pMCM2- Ser27) to tumor metastasis and here aimed to clear the clinical significance of the site phosphorylation in ovarian cancer and the role of the site phosphorylation in the process of ovarian cancer (OC) metastasis. Methods: Immunohistochemistry (IHC) was undertaken to investigate pMCM2- Ser27 expression in ovarian cancer tissues and normal adjacent tissues. Wound-healing and Transwell assays were used to uncover the relationship between the dephosphorylation of MCM2 at Ser27 site (depMCM2-Ser27) and cell migration, invasion. Lung metastasis model (n=5) was established to detect whether depMCM2-Ser27 affects OC cells metastasis in vivo. SB216763 was used to inactivate glycogen synthase kinase-3β (GSK3β). Western blot, Immunofluorescence (IF), GST pull-down, Coimmunoprecipitation (CoIP) and Immunoprecipitation (IP) analyses were conducted to explore underlying mechanism of depMCM2-Ser27 in metastasis suppression. Licl, Wnt3α, SKL2001 and TOP/FOP assays were used to investigate the correlation of depMCM2-Ser27 to Wnt/β-catenin pathway. Findings: depMCM2-Ser27 lead to a marked attenuation in OC cells metastasis in vivo and vitro. In addition, GSK3β could interact with MCM2 and phosphorylate MCM2 at Ser27 site. depMCM2-Ser27 augmented the interaction of GSK3β with β-catenin and decreased β-catenin stability, accompanied by the up-regulation of β-catenin phosphorylation and ubiquitination, the attenuation of β-catenin nuclear translocation and the inactivation of Wnt/β-catenin pathway. depMCM2-Ser27 suppressed OC cells metastasis by inactivating Wnt/β-catenin pathway. Interpretation: These findings elucidate the vital role of pMCM2-Ser27 in the Wnt/β-catenin pathway mediated-metastasis process and provides insights for the development of novel therapies to treat advanced OC. Funding Statement: This work was supported by the National Natural Science Foundation of China (81470367).Declaration of Interests: The authors do not have a commercial or other association with pharmaceutical companies or other parties that might pose a conflict of interest. Ethics Approval Statement: All laboratory animals operations were conducted in accordance with the rules of the Laboratory Animal Ethics Committee (Number of the using of Laboratory Animal: SCXK (jing) 2014-004). The research was approved by the Ethics Committee of Dalian Medical University.