MYG1 promotes proliferation and inhibits autophagy in lung adenocarcinoma cells via the AMPK/mTOR complex 1 signaling pathway

Melanocyte proliferating gene 1 (MYG1) is an exonuclease that participates in RNA processing and is required for normal mitochondrial function. However, its role in tumorigenesis remains unknown. The present study aimed to investigate the role of MYG1 and its underlying mechanisms in human lung adenocarcinoma (LUAD). The expression levels of MYG1 in tumor tissues of patients with LUAD were obtained from public cancer databases and analyzed using the UALCAN online software. The association between MYG1 expression levels and the prognosis of patients with LUAD was analyzed using the Kaplan-Meier plotter. In addition, the role of MYG1 in the LUAD A549 and H1993 cell lines was determined by knocking down MYG1 expression with a specific small interfering RNA or by overexpressing it with a MYG1-containing plasmid. The results demonstrated that MYG1 expression levels were upregulated in LUAD tissues compared with those in normal lung tissues from healthy subjects, and high MYG1 expression levels were associated with an unfavorable prognosis. MYG1 promoted the proliferation, migration and invasion of A549 and H1993 cells. In addition, MYG1 inhibited autophagy via the AMP-activated protein kinase/mTOR complex 1 signaling pathway. Collectively, the present results suggested that MYG1 may serve an oncogenic role in LUAD and may be a potential therapeutic target for LUAD.     

Flotilin-1 promotes the tumorigenicity and progression of malignant phenotype in human lung adenocarcinoma

Lung adenocarcinoma (LUAD) accounts for the most common histological subtype of lung cancer which remains the leading cause of cancer death worldwide. The discovery of more sensitive and specific novel target biomarkers for predicting the development and progression of LUAD is imperative. Flotillin-1 (Flot-1) has been reported to have important roles in the progression of several tumor types but not been reported in the progression of LUAD. Here, we demonstrated that the expression of flotillin-1 was upregulated in 5 LUAD cells. Moreover, multiple approaches were used to explore the tumorigenicity of flotillin-1 in LUAD cell lines. The expression levels of flotillin-1 were analyzed by immunoblotting after overexpression and siRNA-based knockdown. Cell proliferation, scratch wound healing, transwell migration and matrigel invasion and xenograft tumor growth assays were used to determine the role of flotillin-1 in LUAD progression. Downregulation of flotillin-1 reversed, whereas upregulation of flotillin-1 enhanced, the malignant phenotype of LUAD cells in vitro. Consistently, cells with flotillin-1 knockdown formed smaller tumors in nude mice than cells transfected with the empty vector. Furthermore, the control group demonstrated significantly more tumorigenic effects compared to the flotillin-1-silenced group in the xenograft model of LUAD. In all, there draws a conclusion that flotillin-1 is a tumorigenic protein that plays an important role in promoting the proliferation and tumorigenicity of LUAD, suggesting that flotillin-1 may represent a novel the therapeutic target to LUAD.     

MiR-200b regulates autophagy associated with chemoresistance in human lung adenocarcinoma

Chemoresistance remains a major clinical problem in combating human lung adenocarcinoma (LAD), and abnormal autophagy is closely associated with this phenomenon. In the present study, an inverse correlation between miR-200b and autophagy-associated gene 12 (ATG12) expressions was observed in docetaxel-resistant (SPC-A1/DTX and H1299/DTX) and sensitive (SPC-A1 and H1299) LAD cells as well as in tissue samples. Further study showed that miR-200b directly targeted ATG12 in LAD. Moreover, miR-200b-dependent ATG12 downregulation inhibited autophagy and enhanced the chemosensitivity of SPC-A1/DTX and H1299/DTX cells both in vivo and in vitro. LAD chemoresistance is therefore closely related to downregulation of miR-200b and the corresponding upregulation of ATG12. These results provide new evidence for the mechanisms governing the microRNA (miRNA)-ATG12 network and their possible contribution to autophagy modulation and LAD chemoresistance.     

Knockdown of FOXP1 promotes the development of lung adenocarcinoma

Lung cancer is one of the most common cancers in the world, which accounts for about 27% of all cancer deaths. However, the mechanisms underlying the pathogenesis of lung cancer cells remain largely elusive. In this study, we examined the role of the Forkhead box protein P1 (FOXP1) in lung cancer development. Our Oncomine analysis shows that FOXP1 is downregulated in lung adenocarcinoma compared with normal lung tissue. Knockdown of FOXP1 promotes the growth and invasion of PC9 and A549 cells by regulating genes of chemokine signaling molecules, including CCR1, ADCY5, GNG7, VAV3, and PLCB1. Simultaneous knockdown of CCR1 and FOXP1 attenuated FOXP1 knockdown-induced increase of lung cancer cell growth. Finally, knockdown of FOXP1 in PC9 cells promotes the tumorigenesis via CCR1 signaling in xenograft mouse model. Taken together, our data suggest that FOXP1 plays important roles in preventing lung adenocarcinoma development via suppressing chemokine signaling pathways.     

Galectin-1 is overexpressed in CD133+ human lung adenocarcinoma cells and promotes their growth and invasiveness

Previous studies demonstrated that a subpopulation of cancer cells, which are CD133 positive (CD133⁺) feature higher invasive and metastatic abilities, are called cancer stem cells (CSCs). By using tumor cells derived from patients with lung adenocarcinoma, we found that galectin-1 is highly overexpressed in the CD133⁺ cancer cells as compared to the normal cancer cells (CD133⁻) from the same patients. We overexpressed galectin-1 in CD133⁻ cancer cells and downregulated it in CSCs. We found that overexpression of galectin-1 promoted invasiveness of CD133⁻ cells, while knockdown of galectin-1 suppressed proliferation, colony formation and invasiveness of CSCs. Furthermore, tumor growth was significantly inhibited in CSCs xenografts with knockdown of galectin-1 as compared to CSCs treated with scramble siRNAs. Biochemical studies revealed that galectin-1 knockdown led to the suppression of COX-2/PGE2 and AKT/mTOR pathways, indicating galectin-1 might control the phenotypes of CSCs by regulating these signaling pathways. Finally, a retrospective study revealed that galectin-1 levels in blood circulation negatively correlates with overall survival and positively correlates with lymph node metastasis of the patients. Taken together, these findings suggested that galectin-1 plays a major role on the tumorigenesis and invasiveness of CD133⁺ cancer cells and might serve as a potential therapeutic target for treatment of human patients with lung adenocarcinoma.     

内皮素-1对人肺腺癌细胞SPC-A1生长的影响

背景与目的内皮素-1（endothelin-1,ET-1）是强大的细胞促有丝分裂素,在一些肿瘤细胞生长和肿瘤血管新生中起重要作用。本研究的目的是探讨ET-1对人肺腺癌细胞SPC-A1生长的影响。方法采用细胞体外培养技术,四甲基偶氮唑盐[3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide,MTT]显色分光光度法测细胞增殖,流式细胞仪测细胞周期。结果ET-1（1×10^-15-1×10^-9mol/L）具有促进SPC-A1细胞增殖作用,作用呈浓度依赖性,在1×10-11mol/L时作用呈饱和状态;ET-1（1×10^-10mol/L）促SPC-A1细胞增殖作用可被内皮素受体A（endothelin receptor A,ETA）拮抗剂BQ123（1×10^-7mol/L）阻断,但不受内皮素受体B（endothelin receptor B,ETB）拮抗剂BQ788（1×10^-7mol/L）影响;用乙二胺四乙酸（ethylene diamine tetraacetic acid,EDTA）（0.4mmol/L）去除细胞外钙离子及电压依赖型Ca2＋通道阻滞剂硝苯地平（nifedipine,1μmol/L）能抑制ET-1诱导的肺腺癌细胞增殖。ET-1（1×10^-10mol/L）对SPC-A1细胞周期没有显著影响。结论ET-1为促SPC-A1细胞生长因子,ET-1促肺腺癌细胞生长的作用主要通过SPC-A1细胞表面ETA受体实现,细胞外钙离子通过电压依赖型Ca^2＋通道内流在ET-1促肺腺癌细胞生长中起重要作用。     

GOLPH3 promotes glioma progression by enhancing PHB2-mediated autophagy

Due to the hypoxia and nutrient deficiency microenvironment, malignant glioma exhibits high autophagy activity and autophagy plays a significant role in the occurrence and development of glioma. However, the potential molecular mechanism of autophagy in glioma remains unknown. In this study, we demonstrated that Golgi phosphorylation protein 3 (GOLPH3), a highly conserved protein basically concentrates in the trans-Golgi network, promoted glioma autophagy. Inhibiting autophagy by using chloroquine suppressed the stimulating effect of GOLPH3 on glioma malignant development both in vitro and in vivo. Mechanistically, GOLPH3 interacted with and recruited prohibitin-2 (PHB2), an autophagy receptor of mitochondrion, and LC3-II. PHB2 promoted cell autophagy and down-regulation of PHB2 abolished the effect of GOLPH3 on autophagy. On the side, the relative mRNA and protein levels of GOLPH3 and PHB2 were positively associated with each other and both also correlated with autophagy in glioma tissues. Together, our results revealed that GOLPH3 promotes glioma progression by enhancing PHB2-mediated autophagy and inhibiting autophagy may benefit glioma patients with GOLPH3 high level. The novel GOLPH3-PHB2-autophagy axis maybe a potential and prospective therapeutic target for gliomas.     

硫氧还蛋白相互作用蛋白通过抑制自噬途径促进结肠腺癌细胞的凋亡

目的:探究过表达硫氧还蛋白相互作用蛋白（thioredoxin interacting protein,TXNIP）对结肠腺癌细胞凋亡的影响及其机制。方法:收集我院30例结肠腺癌患者的结肠腺癌组织及癌旁组织,实时荧光定量PCR检测组织中TXNIP mRNA表达,免疫组织化学染色检测组织中自噬标志蛋白LC3-Ⅱ与凋亡标志蛋白Cleaved Caspase-3表达;将人结肠腺癌 LoVo细胞随机分为对照组、阴性空载体组（LV-GFP）和 TXNIP 过表达组（LV-GFP-TXNIP）,倒置荧光显微镜下观察细胞转染效率,收集转染72 h后的LoVo细胞,通过流式细胞术检测细胞凋亡,透射电子显微镜观察细胞自噬情况,免疫荧光染色检测细胞中LC3表达,Western blot 检测细胞中自噬相关蛋白与凋亡相关蛋白表达变化。结果:与癌旁组织比较,结肠腺癌组织中TXNIP mRNA相对表达量显著降低（P＜0.01）,LC3-Ⅱ阳性表达率显著升高（P＜0.05）,Cleaved Caspase-3阳性表达率显著降低（P＜0.05）;慢病毒转染72 h后转染效率较高（P＜0.05）;与对照组比较,LV-GFP-TXNIP组LoVo细胞的凋亡率显著增加（P＜0.05）,细胞质较为致密,自噬体数目明显减少,LC3荧光染色减弱,Beclin-1、LC3-Ⅱ蛋白表达显著下降（P＜0.05）,而LC3-I、p62蛋白表达显著增加（P＜0.05）,同时,Bax、Cleaved Caspase-3蛋白表达显著增加（P＜0.05）,Bcl-2蛋白表达显著下降（P＜0.05）。结论:在结肠腺癌细胞中过表达TXNIP可通过抑制自噬来促进细胞凋亡。     

肺腺癌患者淋巴转移的分子指纹鉴定

背景与目的：远处转移是肺癌患者的重要死因,癌转移可能与细胞内基因表达模式改变有关。急需运用新技术来筛选和分析这些基因,以便进一步阐明癌转移的机制并寻找新的治疗靶点。本研究旨在筛选肺腺癌患者淋巴转移差异表达基因。方法：原发癌组织及区域淋巴结取自22例接受根治性手术的肺腺癌患者。根据组织来源将标本分为三组：不伴淋巴转移的肺腺癌组织（TxN-,n=11）、伴有淋巴转移的肺腺癌组织（TxN＋,n=11）及相应转移淋巴结中的肺腺癌细胞（N＋,n=11）。对各组进行激光显微切割以获得纯净癌细胞,T7RNA线性扩增获取足够量的RNA,实验通道和参照通道分别标记以后与含6000个已知人类基因或表达序列标签的cDNA基因芯片杂交,扫描荧光信号以后进行数据分析。结果：TxN＋组与TxN-组共有17个差异表达基因,其中12个基因表达上调,5个基因表达下调。有53个基因可将N＋组与TxN＋组区分开,其中在N＋组高表达的基因有25个,在TxN＋组高表达的有28个。结论：早期癌形成中的遗传学变化和后期癌进展中的获得性分子学变异共同决定肺腺癌的淋巴转移。     

lncRNA NEAT1 通过抑制DNA损伤促进肺腺癌PC-9 细胞的增殖

[摘要] 目的：研究长链非编码RNA核富集转录体1（lncRNA NEAT1）对肺腺癌PC-9 细胞增殖能力的影响,并初步探讨其作用机制。方法：qPCR 检测人肺腺癌PC-9 细胞与人胚肺二倍体2BS 细胞中lncRNA NEAT1 的表达水平;设计并合成lncRNA NEAT1 的小干扰RNA（siRNA）序列,采用脂质体法转染PC-9 细胞,通过qPCR 检测转染前后PC-9 细胞NEAT1 的表达水平。MTT法、流式细胞术分别检测lncRNA NEAT1 敲低对PC-9 细胞增殖及细胞周期的影响;WB检测转染前后DNA损伤相关蛋白,即共济失调毛细血管扩张突变蛋白（ATM）和双链DNA损伤标志物γ-H2AX的表达水平。结果：与2BS细胞相比,PC-9 细胞中lncRNA NEAT1 呈高表达（P<0.05）。成功建立NEAT1 敲低的PC-9 细胞,转染siRNA 12 h 后siNEAT1-1 及siNEAT1-2 干扰组细胞增殖能力较空白对照组及空转染组明显下降（P<0.05）;干扰组细胞周期被阻滞在G1 期［（88.97±2.64）%,（88.15±1.48）% vs（84.5±1.72）%,P<0.05］和G2/M期［（8.35±2.02）%（, 8.11±1.36）% vs（4.28±1.28）%,P<0.05］;干扰组细胞中DNA损伤相关蛋白ATM和γ-H2AX表达水平显著升高（均P<0.05）。结论：lncRNA NETA1 在肺腺癌PC-9 细胞呈高表达,其可通过抑制DNA损伤导致细胞周期G1/M期转变促进肺腺癌细胞PC-9 的增殖能力。     

槲寄生碱抑制肺癌细胞增殖作用初步观察

目的：研究槲寄生碱对人肺腺癌细胞株SPC-A1及肺鳞癌细胞株SK—MES-1的抑制作用。方法：采用四甲基噻唑氮蓝（methy lthiazolyl tetrazolium,MTT）比色法检测不同浓度槲寄生碱作用24和48h对细胞增殖的影响,以含不同浓度的5-氟尿嘧啶（5-FU）细胞组为阳性对照,不舍药物的细胞组为阴性对照,计算半数抑制浓度（IC50）。流式细胞术（flow cytometry,FCM）检测高、中和低3组浓度的槲寄生碱作用24h后的细胞的凋亡率。结果：不同浓度槲寄生碱作用24h,对SPC-A1细胞的IC50为（14．43±0．53）／μg／mL,对SK—MES-1细胞的IC50为（8．09±0．40）μg／mL。其作用48h,对SPC—A1细胞的IC50为（12．11±0．25）μg／mL,对SK—MES-1细胞的ICs0为（6．43±0．33）μg／mL。并且抑制作用随碱浓度的升高和作用时间的延长而增加,P〈0．05。阳性对照组5-FU作用24h,对SPC-A1细胞的IC50为（4．79±0．45）〉g／mL,对SK—MES-1细胞的IC50为（5．15士0．23）μg／mL。作用48h,对SPC—A1细胞的IC50为（4．35±0．41）μg／mL,对SK—MES-1细胞的IC50为（4．11±0．38）／lg／mL。FCM检测SPc_A1细胞未加入药物的对照组凋亡率为（0．43±0．01）％,高剂量组药物浓度28．86μg／mL,凋亡率为（31．09±0．05）%,中剂量组药物浓度14．43〉g／mL,凋亡率为（18．19士0．02）％,低剂量组药物浓度7．22μg／mL,凋亡率为（7．99±0．01）％。SK—MES-1细胞未加入药物的对照组凋亡率为（O．57士0．02）％,高剂量组药物浓度16．18ug／mL,凋亡率为（38．24±0．03）％,中剂量组药物浓度为8．09μg／mL,凋亡率为（21．81±0．01）%,低剂量组药物浓度4．05μg／mL,凋亡率为（9．11±0．01）％,与没有加入槲寄生碱的细胞相比,加入槲寄生碱后SPC—A1细胞与SK—MES-1细胞凋亡率均明显增加,P〈0．05,并且凋亡率随槲寄生碱剂量的增加而升高,P〈0．05。结论：槲寄生碱能够抑制人肺腺癌细胞株SPC-A1及肺鳞癌细胞株SK-MES-1的增殖,并能促进细胞的凋亡,为槲寄生碱应用于肺癌的临床治疗提供了实验依据。     

UHRF1通过调控细胞自噬抑制肺腺癌细胞增殖的分子机制研究

目的 探讨泛素样含PHD和环指结构域1(Ubiquitin like with PHD and ring finger domains 1, UHRF1)在肺腺癌(Lung adenocarcinoma)细胞的增殖、自噬中的作用及其潜在机制。方法 通过生物信息学网站(TCGA)检测UHRF1在肺腺癌组织中的表达。qRT-PCR和Western blot检测肺腺癌细胞系(PC-9、A549和H1299)以及人支气管上皮细胞(16HBE)中UHRF1的表达。转染UHRF1-shRNA后, 采用CCK-8、克隆形成以及Ki-67检测肺腺癌A549细胞活性和增殖能力的改变;蛋白免疫印记检测自噬相关LC3-I/II、Beclin-1蛋白以及增殖相关蛋白CDK6、Rb和PCNA蛋白的变化;透射电镜观察敲除UHRF1后A549细胞中对于自噬小体的影响。结果 在肺腺癌患者组织中UHRF1表达明显高于癌旁组织, 而相对于正常的支气管上皮细胞16HBE, 肺腺癌细胞系A549与H1299中UHRF1的mRNA和蛋白水平均明显升高。此外, CCK-8法和克隆形成实验表明沉默UHRF1能够降低肺腺癌细胞系A549的生长效率。Ki-67免疫荧光法检测结果显示敲除UHRF1后A549细胞增殖能力相对于正常对照组明显降低。此外我们发现, 敲除UHRF1导致CKD6和PCNA蛋白水平相对于Control-siRNA组表达升高, 而Rb蛋白表达下调。我们同时还发现, 沉默UHRF1后能够提高LC3-II/LC3-I的比例, 诱导Beclin-1的表达上调。沉默UHRF1促进A549细胞中自噬小体的形成。结论 UHRF1在肺腺癌中高表达, 沉默UHRF1能够发挥抑制增殖的作用。而这种作用可能是通过促进细胞自噬产生的。     

FAM111B enhances proliferation of KRAS‐driven lung adenocarcinoma by degrading p16

Lung cancer is a common type of cancer that represents a health problem worldwide; lung adenocarcinoma (LUAD) is a major subtype of lung cancer. Although several treatments for LUAD have been developed, the mortality rate remains high because of uncontrollable progression. Further biological and clinicopathological studies are therefore needed. Here, we investigated the role of family with sequence similarity 111 member B (FAM111B), which is highly expressed in papillary‐predominant LUAD; however, its role in cancer is unclear. An immunohistochemical analysis confirmed that papillary‐predominant adenocarcinomas exhibited higher expression of FAM111B, compared with lepidic‐predominant adenocarcinomas. Additionally, FAM111B expression was significantly correlated with clinical progression. In vitro functional analyses using FAM111B‐knockout cells demonstrated that FAM111B plays an important role in proliferation and cell cycle progression of KRAS‐driven LUAD under serum‐starvation conditions. Furthermore, FAM111B regulated cyclin D1‐CDK4‐dependent cell cycle progression by degradation of p16. In summary, we revealed the clinical importance of FAM111B in human tumor tissues, as well as its function as a degradative enzyme. Therefore, FAM111B has potential as a clinicopathological prognostic marker for LUAD.     

RNAi沉默肺腺癌A549细胞系PD-L1基因表达对其增殖和凋亡的影响

目的:沉默肺癌细胞系A549的程序性死亡配体1(programmed death ligand 1,PD-L1)基因,观察其对A549细胞增殖和凋亡的影响。方法:构建PD-L1 shRNA重组质粒p GPU6/PD-L1,并应用脂质体转染法将其转染入A549细胞。RT-PCR法检测PD-L1基因的表达;Western blotting法检测PD-L1蛋白的表达;MTT法检测p GPU6/PD-L1对A549细胞增殖的影响;AnnexinⅤ-FITC/PI双染法检测p GPU6/PD-L1对A549细胞凋亡的影响。结果:成功将p GPU6/PD-L1转染入A549细胞;RT-PCR结果显示p GPU6/PD-L1使A549细胞PD-L1基因表达水平降低;Western blotting结果显示p GPU6/PD-L1转染A549细胞使PDL1蛋白表达减少;MTT结果显示p GPU6/PD-L1能够抑制A549细胞增殖;AnnexinⅤ-FITC/PI双染法检测结果显示p GPU6/PD-L1能够诱导A549细胞凋亡。结论:p GPU6/PD-L1能够下调肺癌细胞A549 PD-L1的表达,抑制细胞增殖,并诱导细胞凋亡。     

MCT4-dependent lactate secretion suppresses antitumor immunity in LKB1-deficient lung adenocarcinoma

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肺浸润性腺癌中GASP-1的表达及意义

目的:观察肺浸润性腺癌中G蛋白偶联受体相关分选蛋白1(GASP-1)的表达,探讨其在术后组织中表达的临床意义,分析血清及术后组织中表达的一致性.方法:选择2014年09月至2015年03月确诊为肺浸润性腺癌并行根治手术的患者59例作为研究对象,选择肿瘤组织作为观察组,选择肺原位腺癌59例作为对照组,选择正常肺组织59例...