Nampt及高糖高胰岛素微环境对人顺铂耐药肺癌细胞株增殖和转移的影响

摘要 目的：探究烟酰胺磷酸核糖转移酶（Nampt）及高糖高胰岛素（HG+HI）微环境对人顺铂耐药肺癌细胞增殖和转移的影响。方法：将人顺铂耐药细胞株A549/DDP分为6组（n=6）：对照组（control）、高糖高胰岛素干预组（HH，使用添加30 mmol/L的葡萄糖和500 mU/L的胰岛素的培养基培养72 h）、分别转染sh-NC和sh-Nampt组（sh-NC和sh-Nampt，使用Lipofectamine 2000将sh-NC和sh-Nampt分别转染到细胞中，转染时间为48 h）、HH干预sh-NC和sh-Nampt组（HH+sh-NC和HH+sh-Nampt）。每组6个重复样本。qRT-PCR检测转染效率，MTT法检测细胞增殖，流式细胞仪检测细胞凋亡，Transwell检测细胞迁移和侵袭，qRT-PCR检测Nampt mRNA，Western blot检测Nampt、Bcl-2、Bax、MMP-2、MMP-9、p-PI3K、PI3K、p-AKT和AKT蛋白表达。结果：与对照组和sh-NC组比较，sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，而细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。与对照组和sh-NC组比较，HH组和HH+sh-NC组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平升高，Bax蛋白表达水平降低（P<0.005）。与HH组和HH+sh-NC组比较，HH+sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低，细胞凋亡率升高，Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低，Bax蛋白表达水平升高（P<0.005）。结论：高糖高胰岛素微环境可能通过上调Nampt/PI3K/AKT信号通路诱导人顺铂耐药肺癌细胞的增殖和转移。     

Differential effects of sPLA<Subscript>2</Subscript>-GV and GX on cellular proliferation and lipid accumulation in HT29 colon cancer cells

The purpose of the article is to investigate the role of IARS2 in proliferation, apoptosis, and cell cycle of gastric cancer (GC) cells in vitro. The IARS2-shRNA lentiviral vector was established and used to infect the GC cell line AGS. qRT-PCR and Western blot were employed to determine the efficiency of IARS2 knockdown. The effects of IARS2 knockdown on cell proliferation, cell clone formation, and cell cycle were assessed by MTT assay, colony formation assay, and flow cytometer analysis, respectively. Finally, a PathScan Antibody Array Kit was used to detect the expression levels of cell cycle-related proteins after IARS2 knockdown in AGS cells to elucidate the underlying mechanisms. Compared with negative control group, IARS2 was significantly knocked down by transfection with lentivirus encoding shRNA of IARS2 in AGS cells. IARS2 knockdown significantly inhibited the proliferation and colony formation ability and induced cycle arrest at G2/M phase of AGS cells. IARS2 knockdown significantly decreased the expression levels of phosphorylation of (p-Smad2), p-SAPK/JUK, cleavage-Caspase-7, and p-TAK1, but increased the expression levels of p-53 and cleavage-PARP in AGS cells compared to shCtrl group. We demonstrated that IARS2 knockdown inhibits proliferation, suppresses colony formation, and causes cell cycle arrest in AGS cells. We also found that IARS2 regulates key molecules of cell apoptosis-related signaling pathway.     

ANKRD33 is overexpressed in gastric adenocarcinoma and predictive for poor prognosis

ABSTRACT

The aim of the current study was to investigate and discuss the function of ANKRD33 gene in the pathogenesis of gastric adenocarcinoma. The marked up-regulated expression of ANKRD33 gene in gastric adenocarcinoma tissues compared to normal tissues was found by bioinformatics analysis. Kaplan-Meier analysis revealed that high expression of ANKRD33 is correlated with lower overall survival of gastric adenocarcinoma patients. The results of qPCR revealed that mRNA expression level of ANKRD33 was dramatically higher in AGS, SGC7901, and BGC823 cell lines than that in the GES1 cells. Knockdown of ANKRD33 remarkably inhibited the viability, invasion, and migration of AGS and BGC823 cells. Furthermore, the ratio of p-AKT/AKT and p-mTOR/mTOR was significantly decreased in AGS cells which transfected with si- ANKRD33. All the above results illustrated that ANKRD33 would act as a tumor forwarder in gastric adenocarcinoma development and have a high potential to be a marker molecule in the diagnosis and treatment of gastric tumors.     

Regulatory mechanism of ZNF139 in multi-drug resistance of gastric cancer cells

Our previous study found increased zinc finger protein 139 (ZNF139) expression in gastric cancer (GC) cells. Purpose of the study is to further clarify the role and mechanism of ZNF139 in multi-drug resistance (MDR) of GC cells. MTT assay, RT-PCR, Western blotting were employed to detect susceptibility of GC cells to chemotherapeutic agents (5-FU, L-OHP) in vitro, and expressions of ZNF139 and MDR associated genes MDR1/P-gp, MRP1, Bcl-2, Bax were also detected. siRNA specific to ZNF139 was transfected into MKN28 cells, then chemosensitivity of GC cells as well as changes of ZNF139 and MDR associated genes were detected. It’s found the inhibition rate of 5-FU, L-OHP to well-differentiated GC tissues and cell line was lower than that in the poorly differentiated tissues and cell line; expressions of ZNF139 and MDR1/P-gp, MRP1 and Bcl-2 in well-differentiated GC tissues and cell line MKN28 were higher, while Bax expression was lower. After ZNF139-siRNA was transfected into MKN28, ZNF139 expression in GC cells was inhibited by 90 %; inhibition rate of 5-FU, L-OHP to tumor cells increased, and expressions of MDR1/P-gp, MRP1 and Bcl-2 were down-regulated, while Bax was up-regulated. ZNF139 was involved in GC MDR by promoting expressions of MDR1/P-gp, MRP1 and Bcl-2 and inhibiting Bax simultaneously.     

Association between Epstein-Barr virus infection and chemoresistance to docetaxel in gastric carcinoma

Epstein-Barr virus (EBV) is associated with human cancers such as nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s disease, and gastric carcinoma (GC). EBV is associated with about 10% of all GC cases globally. EBV-associated GC has distinct features from EBV-negative GC. However, it is still unclear if EBV infection has any effect on GC chemoresistance. Cell proliferation assay, cell cycle analysis, and active caspase Western blot revealed that the EBV-positive GC cell line (AGS-EBV) showed chemoresistance to docetaxel compared to the EBV-negative GC cell line (AGS). Docetaxel treatment increased expression of Bax similarly in AGS and AGS-EBV cell lines. However, Bcl-2 induction was markedly higher in AGS-EBV cells, after docetaxel treatment. Although docetaxel increased the expression of p53 to a similar extent in both cell lines, induction of p21 in AGS-EBV cells was lower than in AGS cells. Furthermore, expression of survivin was higher in AGS-EBV cells than in AGS cells following docetaxel treatment as well as at basal state. EBVlytic gene expression was induced by docetaxel treatment in AGS-EBV cells. The results suggest that EBV infection and lytic induction confers chemoresistance to GC, possibly by regulating cellular and EBV latent and lytic gene expression.     

Knockdown of LncRNAZFAS1 suppresses cell proliferation and metastasis in non-small cell lung cancer

ABSTRACT

To evaluate the effects of LncRNAZFAS1 on cell proliferation and tumor metastasis in non-small cell lung cancer (NSCLC), we detected the expression level of LncRNAZFAS1 in NSCLC-related tissues and cells. qRT-PCR results revealed that LncRNAZFAS1 in tumor tissues was significantly higher than that in normal lung tissue, especially significantly up-regulated in stage III / IV and in metastatic NSCLC tissues. LncRNAZFAS1 expression was dramatically up-regulated in 4 NSCLC-related cells (A549, SPC-A1, SK-MES-1, and NCI-H1299), with having the highest expression level in A549 cells. Furthermore, we implemented a knockdown of LncRNAZFAS1 in A549 cells, and the results of CCK8 and Transwell assays suggested that knockdown of LncRNAZFAS1 significantly inhibited NSCLC cell proliferation and metastasis. Next, we constructed a tumor xenograft model to evaluate the effect of LncRNAZFAS1 on the NSCLC cell proliferation in vivo. The results indicated that knockdown of LncRNAZFAS1 dramatically inhibited A549 cells proliferation and repressed tumor growth. Additionally, knockdown of LncRNAZFAS1 drastically weakened the expressions of MMP2, MMP9 and Bcl-2 proteins, whereas noticeably strengthened the expression of BAX protein. Our results altogether suggest that knockdown of LncRNAZFAS1 has a negative effect on the proliferation and metastasis of NSCLC cell, which implying LncRNAZFAS1 is a potential unfavorable biomarker in patients with NSCLC.     

MicroRNA-630 suppresses epithelial-to-mesenchymal transition by regulating FoxM1 in gastric cancer cells

In the present study, we investigated the functional role of microRNA (miR)-630 in epithelial-to-mesenchymal transition (EMT) of gastric cancer (GC) cells, as well as the regulatory mechanism. Cells of human GC cell line SGC 7901 were transfected with miR-630 mimic or miR-630 inhibitor. The transfection efficiency was confirmed by qRT-PCR. Cell migration and invasion were determined by Transwell assay. Protein expression of E-cadherin, vimentin, and Forkhead box protein M1 (FoxM1) was tested by Western blot. Moreover, the expression of FoxM1 was elevated or suppressed, and then the effects of miR-630 abnormal expression on EMT and properties of migration and invasion were examined again, as well as protein expression of Ras/phosphoinositide 3-kinase (PI3K)/AKT related factors. The results showed that (i) the EMT and properties of migration and invasion were statistically decreased by overexpression of miR-630 compared to the control group but markedly increased by suppression of miR-630. However, (ii) abnormal expression of FoxM1 reversed these effects in GC cells. Moreover, (iii) expression of GTP-Rac1, p-PI3K, and p-AKT was decreased by miR-630 overexpression but increased by FoxM1 overexpression. (iv) The decreased levels of GTP-Rac1, p-PI3K, and p-AKT induced by miR-630 overexpression were dramatically elevated by simultaneous overexpression of FoxM1. In conclusion, our results suggest that miR-630 might be a tumor suppressor in GC cells. MiR-630 suppresses EMT by regulating FoxM1 in GC cells, supposedly via inactivation of the Ras/PI3K/AKT pathway.     

miR-125b-5p对人血管瘤内皮细胞HemES增殖和凋亡的影响及其机制

目的:研究miR-125b-5p对人血管瘤内皮细胞HemECs增殖、凋亡的影响.方法:RT-qPCR检测人血管瘤内皮细胞HemECs及其旁系组织细胞中miR-125b-5p与MCL-1 mRNA的表达;选取HemECs细胞分为对照组、miR-NC 组、miR-125b-5p mimic 组、miR-125b-5p in...     

Silencing of S-phase kinase-associated protein 2 enhances radiosensitivity of esophageal cancer cells through inhibition of PI3K/AKT signaling pathway

We investigated the effect of S-phase kinase-associated protein 2 (SKP2) on radiosensitivity of esophageal cancer (EC) cells. Expression of SKP2, PI3K, AKT, Bcl-2 and Bax were assayed in EC. EC cells were transfected with SKP2-siRNA/IGF-1 to detect expression of SKP2, PI3K, AKT, Bcl-2 and Bax. At last, the radiosensitivity of cells in different doses of X (0, 2, 4, 6, 8 Gy) irradiation and cell apoptosis were also detected. EC cells displayed a higher positive expression rate of SKP2, elevated mRNA and protein expression of SKP2, PI3K, AKT, Bcl-2 and Bax, as well as higher extent of PI3K and AKT phosphorylation. SKP2 silencing downregulated mRNA and protein expression of PI3K, AKT and Bcl-2 but increased p27 protein expression, and inhibited the cell survival rate while promoting cell apoptosis. Taken together, silencing SKP2 can inhibit the PI3K/AKT signaling pathway, thereby increasing the radiosensitivity of EC cells.     

蜂毒素通过下调F2RL1表达从而遏制胶质瘤细胞荷瘤小鼠肿瘤增殖的机制

摘要 目的：探讨蜂毒素通过下调F2RL1表达从而遏制胶质瘤细胞荷瘤小鼠肿瘤增殖的机制。方法：40只雄性 NOD/SCID小鼠（5周龄,15-18 g）购自北京维塔河实验动物技术公司。实验前,让小鼠适应环境一周。NOD/SCID 小鼠在右侧海马体中注射了2×10⁵ U87-MG细胞建立异种移植模型。当肿瘤体积增长到100 mm³时,将小鼠随机分为模型组（空腹注射生理盐水）和蜂毒素组（腹腔注射5 mg/kg 蜂毒素）,每组20只小鼠。在第5天(注射的第5天)、第10天、第15天每次处死5只小鼠,通过实时PCR分析小鼠肿瘤组织中F2RL1、Bcl-2、Bax和Capase-3的mRNA表达。使用卡尺测量荷瘤小鼠肿瘤体积,使用电子天平对肿瘤组织进行称重测量。通过蛋白印迹分析肿瘤组织中p-PI3K、p-AKT 和p-mTOR的蛋白表达。通过TUNEL染色检测人脑肿瘤中凋亡细胞的百分比。结果：蜂毒素组F2RL1 mRNA表达较模型组降低（P<0.05）,蜂毒素抑制F2RL1 mRNA表达。第5 d、10 d和15 d测得肿瘤体积发现蜂毒素肿瘤体积较模型组减小（P<0.05）。第5 d、10 d和15 d测得肿瘤体积发现蜂毒素肿瘤重量较模型组减轻（P<0.05）。蜂毒素组p-PI3K、p-AKT 和p-mTOR的蛋白表达较模型组降低（P<0.05）。蜂毒素组Bax和Capase-3的mRNA表达较模型组降低（P<0.05）,蜂毒素组Bcl-2 mRNA表达较模型组升高（P<0.05）。蜂毒素组TUNEL阳性细胞的百分比较较模型组升高（P<0.05）。结论：蜂毒素通过下调肿瘤小鼠体内F2RL1表达抑制PI3K/AKT信号通路激活,促进了体内肿瘤细胞凋亡,从而有效抑制经胶质瘤细胞的生长、增殖。     

MCM7 silencing promotes cutaneous melanoma cell autophagy and apoptosis by inactivating the AKT1/mTOR signaling pathway

Cutaneous melanoma (CM) has become a major public health concern. Studies illustrate that minichromosome maintenance protein 7 (MCM7) participate in various diseases including skin disease. Our study aimed to study the effects of MCM7 silencing on CM cell autophagy and apoptosis by modulating the AKT threonine kinase 1 (AKT1)/mechanistic target of rapamycin kinase (mTOR) signaling pathway. Initially, microarray analysis was used to screen the CM-related gene expression data as well as differentially expressed genes. Subsequently, MCM7 expression vector and lentivirus RNA used for MCM7 silencing (LV-shRNA-MCM7) were constructed, and these vectors, dimethyl sulfoxide (DMSO) and AKT activator SC79 were then introduced into CM cell line SK-MEL-2 to validate the role of MCM7 in cell autophagy, viability, apoptosis, cell cycle, migration, and invasion. To further investigate the regulatory mechanisms of MCM7 in CM progress, the expression of MCM7, AKT1, mTOR, cyclin D1, as well as autophagy and apoptosis relative factors, such as LC3B, SOD2, DJ-1, p62, Bcl-2, Bax, and caspase-3 in melanoma cells was determined. MCM7 might mediate the AKT1/mTOR signaling pathway to influence the progress of melanoma. MCM7 silencing contributed to the increased expression of Bax, capase-3, and autophagy-related genes (LC3B, SOD2, and DJ-1), but decreased the expression of Bcl-2, which suggested that MCM7 silencing promoted autophagy and cell apoptosis. At the same time, MCM7 silencing also attenuated cell viability, invasion, and migration, and reduced the cyclin D1 expression and protein levels of p-AKT1 and p-mTOR. Taken together, MCM7 silencing inhibited CM via inactivation of the AKT1/mTOR signaling pathway.     

Anticancer effects of echinacoside in hepatocellular carcinoma mouse model and HepG2 cells

Echinacoside (ECH) is a phenylethanoid glycoside extracted from a Chinese herbal medicine, Cistanches salsa. ECH possesses many biological properties, including anti-inflammation, neural protection, liver protection, and antitumor. In the current study, we aimed to explore the effects of ECH on hepatocellular carcinoma (HCC) and the underlying mechanisms. The results showed that ECH could attenuate diethylnitrosamine (DEN)-induced HCC in mice, and exerted antiproliferative and proapoptotic functions on HepG2 HCC cell line. ECH exposure in HepG2 cells dose-dependently reduced the phosphorylation of AKT (p-AKT) and enhanced the expression of p21 (a cell cycle inhibitor) and Bax (a proapoptotic protein). Furthermore, ECH significantly suppressed insulin-like growth factor-1-induced p-AKT and cell proliferation. These data indicated that phosphoinositide 3-kinase (PI3K)/AKT signaling was involved in the anti-HCC activity of ECH. Gene set enrichment analysis results revealed a positive correlation between the PI3K pathway and triggering receptors expressed on myeloid cells 2 (TREM2) expression in HCC tissues. ECH exposure significantly decreased TREM2 protein levels in HepG2 cells and DEN-induced HCC. Furthermore, ECH-mediated proliferation inhibition and AKT signaling inactivation were notably attenuated by TREM2 overexpression. In conclusion, ECH exerted its antitumor activity via decreasing TREM2 expression and PI3K/AKT signaling.     

双氢青蒿素对Raji细胞放射敏感性的影响

目的：研究双氢青蒿素（DHA）对Raji细胞放射敏感性的影响并探讨其作用机制。方法：CCK8测定DHA对Raji细胞活力的影响,流式细胞术检测细胞凋亡、胞内ROS及线粒体膜电位,Western blot检测AKT、p-AKT、Bcl-2、Bax和Cleaved-Caspase-3蛋白表达量。结果：实验分为对照组、DHA组（5 μmol/L DHA）、放射组（4 Gy γ射线）、联合放射组（5 μmol/L DHA和4 Gy γ射线）,与其他3组相比,联合放射组Raji细胞的线粒体膜电位显著降低（P<0.01）,胞内ROS含量和凋亡率显著升高（P<0.01）;此外,Raji细胞AKT表达量与其他3组相比无明显差异,但AKT的磷酸化受到抑制;Bcl-2表达量显著降低,而Bax、Cleaved-Caspase-3表达量显著升高。结论：DHA可能通过抑制磷酸肌醇3-激酶（PI3K-AKT）信号通路及激活了Raji细胞的线粒体凋亡途径,引起氧化应激反应,从而增加Raji细胞对放射的敏感性。     

CEACAM5 targeted by miR-498 promotes cell proliferation,migration and epithelial to mesenchymal transition in gastric cancer

ObjectiveRecent studies have shown that carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) may serve as an independent predictor of advanced gastric cancer (GC). The purpose of this research is to explore the patterns of expression, functions, and upstream regulatory pathway of CEACAM5 in GC.MethodsThe levels of miR-498 and CEACAM5 expression in GC cells and tissues were measured via qRT-PCR. Wound-healing, CCK-8, and western blotting experiments were conducted for the evaluation of GC cell migration, proliferation, and epithelial-mesenchymal transition (EMT), respectively. The targeting relationship between miR-498 and CEACAM5 was validated via pull-down and luciferase reporter assays. Xenograft tumor mouse models were established to observe CEACAM5’s influence on the growth of tumors in vivo.ResultsElevated levels of CEACAM5 were detected among the GC cells and tissues. The results of the in vitro experiments revealed that the knockdown of CEACAM5 in GC cells significantly inhibited their proliferation, migration, and EMT. Moreover, CEACAM5 inhibition effectively hampered GC cell growth within the nude mice. Moreover, miR-498 directly targeted CEACAM5. MiR-498 downregulation had been observed among the cells and tissues of GC. The stimulation of GC cell proliferation, migration, and EMT, which had been engendered by CEACAM5 overexpression, was reversible through the overexpression of miR-498.ConclusionThe outcomes of this research suggest that miR-498 is capable of repressing the proliferation, migration, and EMT of GC cells through CEACAM5 downregulation.     

ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS enhanced the expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function.     

MT1G inhibits the growth and epithelial-mesenchymal transition of gastric cancer cells by regulating the PI3K/AKT signaling pathway

Gastric carcinoma (GC) is a malignant tumor that has high mortality and morbidity worldwide. Although many efforts have been focused on the development and progression of GC, the underlying functional regulatory mechanism of GC needs more clarification. Metallothionein 1G (MT1G) is a member of the metallothionein family (MTs), and hypermethylation of MT1G occurred in a variety of cancers, including gastric cancer. However, the functional mechanism of MT1G in GC remains unclear. Here, we demonstrated that MT1G was down-regulated in GC tissues and cells. Overexpression of MT1G inhibited cell proliferation, foci formation and cell invasion, while knockdown of MT1G increased cell proliferation, foci formation and cell invasion. In addition, MT1G overexpression inhibited cell cycle progression and MT1G deficiency exerted opposite phenotype. p-AKT was negatively regulated by MT1G. In summary, our study reveals that MT1G exerts crucial role in regulating of cell proliferation and migration of gastric cancer, providing new insights for MT1G-related pathogenesis and a basis for developing new strategies for treatment of GC. Keywords: MT1G, GC, PI3K/AKT signaling pathway, cell growth, EMT