RNA干扰下调不同肿瘤细胞表皮生长因子受体表达及其意义

背景与目的：表皮生长因子受体在多种上皮源性肿瘤中过表达，与肿瘤的发生、发展密切相关，是肿瘤治疗的重要靶点。本研究采用针对EGFR的小干扰RNA（siRNA），探讨其在不同类型肿瘤细胞A431、HeLa、SPC-A-1中的RNA干扰效应。方法：化学合成针对EGFR的siRNA，转染A431、HeLa、SPC—A-1细胞，通过定量RT-PCR、免疫荧光染色和流式细胞仪检测EGFR表达；通过集落形成实验观察细胞集落形成能力，分析不同类型肿瘤细胞的RNA干扰效应。结果：转染siRNA-EGFR后，3种肿瘤细胞的EGFR表达均明显下调。在A431细胞，其mRNA水平下调73．9％，蛋白水平下调77．0％。在HeLa和SPC-A-1细胞，mRNA水平的下调分别为44．6％和57．7％。蛋白水平下调61．3％和65．2％。EGFR下调后细胞集落形成能力均出现抑制，A431、HeLa和SPC-A-1的集落形成抑制率分别为27．2％、53．9％和59．1％。结论：siRNA-EGFR可在不同类型肿瘤细胞中产生RNA干扰效应。抑制细胞集落形成能力。RNA干扰技术在开发广谱抗肿瘤靶向治疗药物中具有应用价值。     

Androgen‐induced lncRNA POTEF‐AS1 regulates apoptosis‐related pathway to facilitate cell survival in prostate cancer cells

Although long non‐coding RNAs (lncRNAs) have been associated with a variety of cancers, the interplay between lncRNAs and androgen receptor signaling in prostate cancer is still unclear. We identified an androgen‐dependent lncRNA, POTEF‐AS1, whose expression was regulated by androgen receptor in two androgen‐dependent cells by using directional RNA sequencing analysis. POTEF‐AS1 promoted cell growth, repressed genes related to the Toll‐like receptor signaling and apoptosis pathways, and inhibited apoptosis in docetaxel‐treated LNCaP cells. These findings suggest that POTEF‐AS1 would play a key role in the progression of prostate cancer by repressing Toll‐like receptor signaling.     

大麻素受体2在胫骨癌痛大鼠脊髓背角细胞中的表达

目的：观察胫骨癌痛大鼠脊髓大麻素受体2（CB2）表达的变化,探讨CB2在骨癌痛中作用及其可能的脊髓机制。方法：雌性SD大鼠56只,体重160-180g,随机分为7组（n=8）：对照组、假手术组、骨癌痛组,假手术组和骨癌痛组又各分为3个亚组（术后7d、14d和21d组）,分别用免疫印迹法检测各组大鼠脊髓水平CB2表达的变化,激光共聚焦技术观察CB2的表达部位。结果：免疫印迹结果显示对照组基本没有CB2的表达,与对照组相比,假手术组及骨癌痛组脊髓CB2表达水平升高（P〈0.05）;与假手术组相比,骨癌痛7d组脊髓CB2蛋白表达明显上调（P〈0.05）。激光共聚焦技术显示CB2主要表达于脊髓小胶质细胞和星形胶质细胞。结论：胫骨癌痛大鼠脊髓背角有CB2表达,早期为其表达的高峰,且主要表达于小胶质细胞和星形胶质细胞。     

大麻素受体2在胫骨癌痛大鼠脊髓背角细胞中的表达

目的:观察胫骨癌痛大鼠脊髓大麻素受体2(CB2)表达的变化,探讨CB2在骨癌痛中作用及其可能的脊髓机制。方法:雌性SD大鼠56只,体重160-180g,随机分为7组(n=8):对照组、假手术组、骨癌痛组,假手术组和骨癌痛组又各分为3个亚组(术后7d、14d和21d组),分别用免疫印迹法检测各组大鼠脊髓水平CB2表达的变化,激光共聚焦技术观察CB2的表达部位。结果:免疫印迹结果显示对照组基本没有CB2的表达,与对照组相比,假手术组及骨癌痛组脊髓CB2表达水平升高(P<0.05);与假手术组相比,骨癌痛7d组脊髓CB2蛋白表达明显上调(P<0.05)。激光共聚焦技术显示CB2主要表达于脊髓小胶质细胞和星形胶质细胞。结论:胫骨癌痛大鼠脊髓背角有CB2表达,早期为其表达的高峰,且主要表达于小胶质细胞和星形胶质细胞。     

Silencing of DLEU2 suppresses pancreatic cancer cell proliferation and invasion by upregulating microRNA‐455

Long noncoding RNA (lncRNA) DLEU2 has been shown to be dysregulated in several type of tumor. However, the potential biological roles and molecular mechanisms of DLEU2 in pancreatic cancer (PC) progression are poorly understood. In this study, we found that the DLEU2 level was substantially upregulated in PC tissues and PC cell lines, and significantly associated with poor clinical outcomes in PC patients. Overexpression of DLEU2 significantly induced PC cell proliferation and invasion, whereas knockdown of DLEU2 impaired cell proliferation and invasion in vitro. Furthermore, bioinformatics analysis, luciferase reporter assay, and RNA immunoprecipitation assay revealed that DLEU2 directly bond to microRNA‐455 (miR‐455) and functioned as an endogenous sponge for miR‐455, which could remarkably suppress cell growth and invasion. We also determined that SMAD2 was a direct target of miR‐455, and the restoration of SMAD2 rescued cell growth and invasion that were reduced by DLEU2 knockdown or miR‐455 overexpression. In addition, low miR‐455 expression and high SMAD2 expression was correlated with poor patient survival. These results indicate that DLEU2 is an important promoter of PC development, and targeting the DLEU2/miR‐455/SMAD2 pathway could be a promising therapeutic approach in the treatment of PC.     

Establishment and characterization of CRISPR/Cas9‐mediated NF2−/− human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2‐knockout human mesothelial cell line, MeT‐5A (NF2‐KO). In NF2‐KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2‐WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2‐WT and NF2‐KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c‐Jun, and retinoblastoma (Rb) in NF2‐KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2‐KO clone. In addition, the disruption of FGFR2 in the NF2‐KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c‐Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2‐negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2‐positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.     

Effects of SMYD2‐mediated EML4‐ALK methylation on the signaling pathway and growth in non‐small‐cell lung cancer cells

A specific subtype of non‐small‐cell lung cancer (NSCLC) characterized with an EML4‐ALK fusion gene, which drives constitutive oncogenic activation of anaplastic lymphoma kinase (ALK), shows a good clinical response to ALK inhibitors. We have reported multiple examples implying the biological significance of methylation on non‐histone proteins including oncogenic kinases in human carcinogenesis. Through the process to search substrates for various methyltransferases using an in vitro methyltransferase assay, we found that a lysine methyltransferase, SET and MYND domain‐containing 2 (SMYD2), could methylate lysine residues 1451, 1455, and 1610 in ALK protein. Knockdown of SMYD2 as well as treatment with a SMYD2 inhibitor in two NSCLC cell lines with an EML4‐ALK gene significantly attenuated the phosphorylation levels of the EML4‐ALK protein. Substitutions of each of these three lysine residues to an alanine partially or almost completely diminished in vitro methylation of ALK. In addition, we found that exogenous introduction of EML4‐ALK protein with the substitution of lysine 1610 to an alanine in these two cell lines reduced the phosphorylation levels of AKT, one of the downstream oncogenic molecules in the EML4‐ALK pathway, and suppressed the growth of the two cell lines. We further showed that the combination of a SMYD2 inhibitor and an ALK inhibitor additively suppressed the growth of these two NSCLC cells, compared with single‐agent treatment. Our results shed light on a novel mechanism that modulates the kinase activity of the ALK fused gene product and imply that SMYD2‐mediated ALK methylation might be a promising target for development of a novel class of treatment for tumors with the ALK fused gene.     

MGr1‐Ag/37LRP induces cell adhesion‐mediated drug resistance through FAK/PI3K and MAPK pathway in gastric cancer

It is well known that tumor microenvironment plays a vital role in drug resistance and cell adhesion‐mediated drug resistance (CAM‐DR), a form of de novo drug resistance. In our previous study, we reported that MGr1‐Ag/37LRP ligation‐induced adhesion participated in protecting gastric cancer cells from a number of apoptotic stimuli caused by chemotherapeutic drugs. Further study suggested that MGr1‐Ag could prompt CAM‐DR through interaction with laminin. However, the MGr1‐Ag‐initiated intracellular signal transduction pathway is still unknown. In this study, our experimental results showed that gastric cancer MDR cell lines mediated CAM‐DR through upregulation of Bcl‐2 by MGr1‐Ag interaction with laminin. Further study found that, as a receptor of ECM components, MGr1‐Ag/37LRP may activate the downstream signal pathway PI3K/AKT and MAPK/ERK through interaction with phosphorylated FAK. Moreover, the sensitivity to chemotherapeutic drugs could be significantly enhanced by inhibiting MGr1‐Ag/37LRP expression through mAbs, siRNA, and antisense oligonucleotide. According to these results, we concluded that the FAK/PI3K and MAPK signal pathway plays an important role in MGr1‐Ag‐mediated CAM‐DR in gastric cancer. MGr1‐Ag/37LRP might be a potential effective reversal target to MDR in gastric cancer.     

Frequent homozygous deletion of Cdkn2a/2b in tremolite‐induced malignant mesothelioma in rats

The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2‐day time‐lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array‐based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed.     

New metastatic model of human small‐cell lung cancer by orthotopic transplantation in mice

Small‐cell lung cancer (SCLC) is an aggressive cancer with high metastatic ability and novel strategies against the metastasis are urgently needed to improve SCLC treatment. However, the mechanism of metastasis of SCLC remains largely to be elucidated. For further studies of SCLC metastasis, we developed a new orthotopic transplantation model in mice. We established a GFP‐labeled subline from the human SCLC cell line DMS273 and transplanted them orthotopically into the lung of nude mice with Matrigel. The GFP‐labeled cells showed significant metastatic activity and formed metastatic foci in distant tissues such as bone, kidney, and brain, as observed in SCLC patients. From a bone metastasis focus of the mouse, we isolated another subline, termed G3H, with enhanced metastatic potential and higher hepatocyte growth factor (HGF) expression than the parental line. Further studies indicated that the HGF/MET signaling pathway was involved in in vitro motility and invasion activities of the G3H cells and treatments with MET inhibitors decreased formation of distant metastases in our orthotopic model using G3H cells. These data indicated that our model mimics the clinical aspect of SCLC such as metastatic tropism and autocrine of HGF/MET signaling. Compared with other orthotopic SCLC models, our model has a superior ability to form distant metastases. Therefore, our model will provide a valuable tool for the study of SCLC metastasis.     

TGF‐β induced PAR‐1 expression promotes tumor progression and osteoclast differentiation in giant cell tumor of bone

Although protease activated receptor‐1 (PAR‐1) has been confirmed as an oncogene in many cancers, the role of PAR‐1 in giant cell tumor (GCT) of bone has been rarely reported. The mechanism of PAR‐1 in tumor‐induced osteoclastogenesis still remains unclear. In the present study, we detected that PAR‐1 was significantly upregulated in GCT of bone compared to normal tissues, while TGF‐β was also overexpressed in GCT tissues and could promote the expression of PAR‐1 in a dose and time dependent manner. Using the luciferase reporter assay, we found that two downstreams of TGF‐β, Smad3 and Smad4, could activate the promoter of PAR‐1, which might explain the mechanism of TGF‐β induced PAR‐1 expression. Meanwhile, PAR‐1 was also overexpressed in microvesicles from stromal cells of GCT (GCTSCs), and might be transported from GCTSCs to monocytes through microvesicles. In addition, knockout of PAR‐1 by TALENs in GCTSCs inhibited tumor growth, angiogenesis and osteoclastogenesis in GCT in vitro. Using the chick CAM models, we further showed that inhibition of PAR‐1 suppressed tumor growth and giant cell formation in vivo. Using microarray assay, we detected a number of genes involved in osteoclastogenesis as the possible downstreams of PAR‐1, which may partly explain the mechanism of PAR‐1 in GCT. In brief, for the first time, these results reveal an upstream regulatory role of TGF‐β in PAR‐1 expression, and PAR‐1 expression promotes tumor growth, angiogenesis and osteoclast differentiation in GCT of bone. Hence, PAR‐1 represents a novel potential therapeutic target for GCT of bone.     

Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor β‐induced extracellular matrix production in lung cancer cells

Activation of transforming growth factor β (TGF‐β) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial‐mesenchymal transition (EMT). Hypoxia‐inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF‐1α expression by protein phosphatase activity on dissociation of the E‐cadherin/β‐catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF‐β. By using lung cancer cells treated with HIF‐1α stabilizers or carrying doxycycline‐dependent HIF‐1α deletion or point mutants, we investigated the role of stabilized HIF‐1α expression on TGF‐β‐induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF‐β and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF‐1α expression was inhibited. Stabilized HIF‐1α protein expression inhibited the TGF‐β‐stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF‐1α‐induced repression of the TGF‐β‐stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF‐1α expression on inhibition of TGF‐β‐induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.     

Prospective evaluation of the SeptiFAST multiplex real‐time PCR assay for surveillance and diagnosis of infections in haematological patients after allogeneic stem cell transplantation compared to routine microbiological assays and an in‐house real‐time PCR method

We prospectively evaluated a multiplex real‐time PCR assay (SeptiFast, SF) in a cohort of patients undergoing allo‐BMT in comparison to an in‐house PCR method (IH‐PCR). Overall 847 blood samples (mean 8 samples/patient) from 104 patients with haematological malignancies were analysed. The majority of patients had acute leukaemia (62%) with a mean age of 52 years (54% female). Pathogens could be detected in 91 of 847 (11%) samples by SF compared to 38 of 205 (18.5%) samples by BC, and 57 of 847 (6.7%) samples by IH‐PCR. Coagulase‐negative staphylococci (n=41 in SF, n=29 in BC) were the most frequently detected bacteria followed by Escherichia coli (n=9 in SF, n=6 in BC). Candida albicans (n=17 in SF, n=0 in BC, n=24 in IH‐PCR) was the most frequently detected fungal pathogen. SF gave positive results in 5% of samples during surveillance vs in 26% of samples during fever episodes. Overall, the majority of blood samples gave negative results in both PCR methods resulting in 93% overall agreement resulting in a negative predictive value of 0.96 (95% CI: 0.95‐0.97), and a positive predictive value of 0.10 (95% CI: ?0.01 to 0.21). SeptiFast appeared to be superior over BC and the IH‐PCR method.