小细胞肺癌药物治疗的研究进展

小细胞肺癌(small cell lung cancer,SCLC)约占肺癌患者总数的15%-20%,与吸烟关系密切,约60%-70%的患者就诊时已处于广泛期,预后很差,内科药物治疗仍然为其主要的治疗手段。由于其细胞生物学行为复杂,恶性程度高,易出现复发及耐药,通过多药联合、优化给药顺序、改变用药方法及调整药物剂量强度并没有获得预期的疗效,且毒副反应较多,其死亡率依然高居各种肿瘤之首,寻求有效的治疗方案变得迫切而棘手。近年来,随着SCLC内科治疗药物研究的不断深入,一些研究热点药物如bcl-2抑制剂、Hedgehog信号通路抑制剂、ipilimumab、贝伐单抗等已在Ⅱ期或Ⅲ期试验中显示出良好的抗肿瘤活性,为SCLC的药物治疗提供了新的方向。本文就目前针对SCLC的药物治疗进展情况作一综述。     

鸡miR-17-92基因簇靶基因ZFPM2的鉴定及功能分析

miR-17-92基因簇在哺乳动物的许多生理和病理过程中发挥重要作用。本实验室前期研究发现,miR-17-92基因簇促进鸡前脂肪细胞的增殖,但其作用机制尚不清楚。为了揭示miR-17-92基因簇促进鸡前脂肪细胞增殖的作用机制,本研究采用CCK-8细胞增殖检测方法分析干扰ZFPM2对前脂肪细胞的影响,结果发现,干扰ZFPM2能显著促进鸡前脂肪细胞的增殖(P<0.01);与CCK-8分析结果相一致,干扰ZFPM2可致使细胞增殖标志基因PCNA、Ki67、Cyclin D1的mRNA表达量明显升高(P<0.01或P<0.05)。进一步对鸡ZFPM2基因进行生物信息学分析,发现该基因mRNA的3′UTR有两个区域存在miR-17-92基因簇4个成员(miR-17-5p、miR-20a、miR-19a及miR-19b)的潜在结合位点。为验证miR-17-92基因簇是否靶作用于鸡ZFPM2基因,构建了鸡ZFPM2基因3′UTR区的荧光素酶报告基因载体(野生型)(psi-CHECK2-ZFPM2-3′UTR-WT)及其突变型的荧光素酶报告基因载体(psi-CHECK2-ZFPM2-3′UTR-MUT)。报告基因活性分析显示,过表达mi-17-92基因簇能极显著地抑制野生型ZFPM2的报告基因活性(P<0.01);转染miR-17-5p、miR-20a及miR-19a的抑制剂均能显著地提高野生型ZFPM2报告基因的活性(P<0.01或 P<0.05),但这些抑制剂对突变型ZFPM2报告基因的活性无明显影响。qRT-PCR分析发现,miR-17-5p、miR-19a及miR-20a的抑制剂能显著提高内源性ZFPM2基因mRNA的表达水平(P<0.01或P<0.05)。共转染分析发现,尽管差异不显著,但miR-17-5p和miR-19a的抑制剂均倾向于降低ZFPM2干扰片段的促细胞增殖作用。本研究结果表明：miR-17-92基因簇成员miR-17-5p、miR-20a、miR-19a及miR-19b靶作用于ZFPM2;miR-17-92基因簇至少部分通过抑制ZFPM2基因表达从而促进鸡前脂肪细胞的增殖。     

Global identification of miR-373-regulated genes in breast cancer by quantitative proteomics

Although microRNAs (miRNAs) have been reported to play an important role in carcinogenesis, their molecular mechanism remains largely unknown because of our limited understanding of miRNA target genes. miR-373 was found to be capable of promoting breast cancer invasion and metastasis, but only a target gene was experimentally identified on the basis of mRNA expression analysis. In this study, we used SILAC-based quantitative proteomics to globally identify the genes regulated by miR-373. Totally, 3666 proteins were identified, and 335 proteins were found to be regulated by miR-373. Among the 192 proteins that were downregulated by miR-373, 27 (14.1%) were predicted to have at least one potential match site at their 3'-UTR for miR-373 seed sequence. However, miR-373 did not affect the mRNA level of the five selected candidate targets, TXNIP, TRPS1, RABEP1, GRHL2 and HIP1, suggesting that the protein expressions were regulated by miR-373 via translational inhibition instead of mRNA degradation. Luciferase and mutation assays validated that TXNIP and RABEP1 were the direct target genes of miR-373. More than 30 proteins reported to be involved in cancer invasion and metastasis were found to be regulated by miR-373 in breast cancer for the first time.     

鸡miR-17-92基因簇上游调控区功能分析

miR-17-92基因簇(miR-17-92 cluster)在细胞增殖、分化、凋亡、动物发育以及肿瘤发生等过程中发挥重要作用。目前,人和小鼠等哺乳动物miR-17-92基因簇的转录调控已有深入研究,但该基因簇在鸡等鸟类中的转录调控研究还未见报道,主要原因在于鸟类miR-17-92基因簇上游的基因组序列都存在一个gap,且该基因簇启动子的位置和序列也还不清楚。为此,本研究采用染色体步移的方法获得鸡miR-17-92基因簇上游gap区序列,并采用生物信息学分析和报告基因及截短突变技术开展该gap区的功能分析。染色体步移分析发现,鸡miR-17-92基因簇上游gap区全长1704 bp,GC含量达80.11%。生物信息学分析显示,鸡miR-17-92基因簇上游gap区内1段200 bp的序列与人、牛、小鼠等9种动物miR-17-92基因簇上游序列保守性较高,且该区域为人和小鼠等哺乳动物miR-17-92基因簇宿主基因的核心启动子区。将克隆的gap区序列插入pGL3 basic荧光素酶报告基因载体,构建成启动子荧光素酶报告基因载体pGL3-cMIR17HG(-4228/-2506)。荧光素酶报告基因活性分析表明,pGL3-cMIR17HG(-4228/-2506)报告基因的活性是pGL3 basic空载体活性的417倍,证明所克隆的gap区片段是鸡miR-17-92基因簇宿主基因的启动子。为进一步分析该启动子的结构和功能,构建gap区片段的5°端缺失突变(缺失448 bp)和3°端缺失突变(缺失894 bp)的荧光素酶报告基因载体。与pGL3-cMIR17HG(-4228/-2506)相比,5°端和3°端缺失突变分别使启动子报告基因活性降低19.82%和60.14%。这些数据提示,鸡miR-17-92基因簇宿主基因启动子的重要调控区位于-3400/-2506。本研究结果为进一步开展鸡miR-17-92基因簇的转录调控奠定了基础。     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.     

Anti-tumoral Effects of miR-3189-3p in Glioblastoma

Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.     

Identification of immunodeficient molecules in neonatal mononuclear cells by proteomic differential displays

Human newborns are known to be susceptible to microbial infection. This susceptibility is generally attributed to immaturity of the newborn immune system. However, the mechanisms for impaired immunity in newborns are still incompletely defined. In this study, we sought to elucidate the protein differential display between adult and neonatal mononuclear cells (MNC) using a proteomic approach. MNC samples from cord blood and adult peripheral blood were subjected to 2-D PAGE analysis. Differential protein displays between cord blood and adult MNC were determined and validated. There were 34 differentially expressed proteins between cord blood and adult MNC identified by 2-D PAGE. The differentially displayed proteins were clustered into two major signal pathways, cellular processing and purine metabolism. After validation by Western blot, we found more abundant arginase-1 (ARG1) and Rho GDP-dissociation inhibitor 2 (RhoGDI2), while less adenosine deaminase (ADA) and β-actin in cord blood MNC. In functional validation, we found that lower ADA was proven to enhance the TNF-α production by cord blood monocytes. The results from this study discovered the proteomic displays for altered immunity between adult and neonatal MNC that support a understanding of the correction of impaired immune response in newborns.     

Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano‐LC–MS/MS following 1D SDS‐PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung‐enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.     

Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells

Certain antitumor agents have recently been extracted from the roots of Salvia miltiorrhiza Bunge. The diterpene derivative, tanshinone IIA, possesses cytotoxic activity against several human carcinoma cell lines. It also inhibits invasion and metastasis of cancer cells. In the present study, we isolated tanshinone IIA from S. miltiorrhiza, and it exhibited strong growth inhibition against human cervical cancer cells in dose‐ and time‐dependent manners with a 50% cell growth inhibition value of 2.5 μg/mL (8.49 μM). Flow cytometric analysis of cell cycle progression revealed that G₂/M arrest was initiated after a 24 h exposure to the drug. It also resulted in DNA fragmentation and degradation of poly (ADP‐ribose) polymerase indicating that tanshinone IIA may be a potential antitumor agent. Furthermore, we performed a comprehensive proteomic analysis to survey global protein changes induced by tanshinone IIA treatment on HeLa cells. Significant changes in the levels of cytoskeleton proteins as well as stress‐associated proteins were observed. Immunoblot analysis and immunofluorescence staining were used to confirm the levels of protein expression. Overexpression of the vimentin rescued these tanshinone IIA‐induced events. Computational docking methods indicated that tanshinone IIA could stably bind to the β‐subunit of the microtubule protein. An interaction network analysis of these 12 proteins using MetaCore? software suggested that tanshinone IIA treatment regulated the expressions of proteins involved in apoptotic processes, spindle assembly, and p53 activation, including vimentin, Maspin, α‐ and β‐tubulin, and GRP75. Taken together, our results suggest that tanshinone IIA strongly inhibited the growth of cervical cancer cells through interfering in the process of microtubule assembly, leading to G₂/M phase arrest and sequent apoptosis. The success of this large‐scale effort was assessed by a bioinformatics analysis of proteins through predictions of protein domains and possible functional roles. The possible contributions of these proteins to the cytotoxicity of tanshinone IIA provide potential opportunities for the development of cancer therapeutics.     

Cross‐species analysis of nicotine‐induced proteomic alterations in pancreatic cells

Toxic compounds in tobacco, such as nicotine, may adversely affect pancreatic function. We aim to determine nicotine‐induced protein alterations in pancreatic cells, thereby revealing links between nicotine exposure and pancreatic disease. We compared the proteomic alterations induced by nicotine treatment in cultured pancreatic cells (mouse, rat, and human stellate cells and human duct cells) using MS‐based techniques, specifically SDS‐PAGE (gel) coupled with LC‐MS/MS and spectral counting. We identified thousands of proteins in pancreatic cells, hundreds of which were identified exclusively or in higher abundance in either nicotine‐treated or untreated cells. Interspecies comparisons of stellate cell proteins revealed several differentially abundant proteins (in nicotine treated versus untreated cells) common among the three species. Proteins appearing in all nicotine‐treated stellate cells include amyloid beta (A4), procollagen type VI alpha 1, integral membrane protein 2B, and toll‐interacting protein. Proteins that were differentially expressed upon nicotine treatment across cell lines were enriched in certain pathways, including nicotinic acetylcholine receptor, cytokine, and integrin signaling. At this analytical depth, we conclude that similar pathways are affected by nicotine, but alterations at the protein level among stellate cells of different species vary. Further interrogation of such pathways will lead to insights into the potential effect of nicotine on pancreatic cells at the biomolecular level and the extension of this concept to the effect of nicotine on pancreatic disease.     

Honokiol inhibits HepG2 migration via down‐regulation of IQGAP1 expression discovered by a quantitative pharmaceutical proteomic analysis

Honokiol (HNK), a natural small molecular product, inhibited proliferation of HepG2 cells and exhibited anti‐tumor activity in nude mice. In this article, we applied a novel sensitive stable isotope labeling with amino acids in cell culture‐based quantitative proteomic method and a model of nude mice to investigate the correlation between HNK and the hotspot migration molecule Ras GTPase‐activating‐like protein (IQGAP1). The quantitative proteomic analysis showed that IQGAP1 was 0.53‐fold down‐regulated under 10 μg/mL HNK exposure for 24 h on HepG2 cells. Migration ability of HepG2 cells under HNK treatment was correlated with its expression level of IQGAP1. In addition, the biochemical validation on HepG2 cells and the tumor xenograft model further demonstrated that HNK decreased the expression level of IQGAP1 and its upstream proteins Cdc42/Rac1. These data supported that HNK can modulate cell adhesion and cell migration by acting on Cdc42/Rac1 signaling via IQGAP1 interactions with its upstream Cdc42/Rac1 proteins, which is a new molecular mechanism of HNK to exert its anti‐tumor activity.     

Infection of H69AR cells with retroviral particles harboring interfering RNAi significantly reduced the multidrug resistance of these small cell lung cancer cells

Incubation of the drug-sensitive H69, a small cell lung cancer cell line, with increased concentrations of adriamycin yielded multidrug resistant (MDR) H69AR cells that over-express multidrug resistance-associated protein (MRP1). MRP1 co-transports its substrate with glutathione (GSH), leading to lower intracellular GSH. In this report we tested whether depleting intracellular GSH in MRP1-expressing cells could hyper-sensitize them to anticancer drugs or not. We have found that the GSH contents in MRP1-expressing cells are significantly lower than their corresponding control cells. The treatment with MRP1 substrate verapamil or the GSH synthetase inhibitor buthionine sulfoxi-mine significantly reduced the intracellular GSH contents in MRP1-expressing cells. Interestingly, depleting intracellular GSH contents can hyper-sensitize the MRP1-cDNA transfected BHK cells to daunomycin, but not the adriamycin-selected H69AR cells. Further analyses indicated that anti-apoptotic factor Bcl2 might be a factor responsible for the fact that depleting intracellular GSH could not hyper-sensitize H69AR cells to daunomycin. We hypothesized that knocking down the expression of Bcl2 could hyper-sensitize H69AR cells to daunomycin. Interestingly, infection of H69AR cells with retroviral particles harboring Bcl2 interfering RNAi not only reduced the expression of Bcl2, but also many factors that contribute to MDR, such as Bcl-xl, MRP1 and ABCC3, etc., leading to the MDR H69AR cells more sensitive to daunomycin than the parental H69 cell. Thus, although the mechanisms of the down-regulation of the genes contributing to MDR remain to be elucidated, retroviral particles harboring Bcl2 interfering RNAi could be used as an alternative way to sensitize the MDR cancer cells to anticancer drugs.     

Identification and expression analysis of miRNAs during batch culture of HEK-293 cells

MicroRNAs (miRNAs) are small non-coding RNAs of about 20–24 nucleotides in length. They regulate gene expression negatively and have been implicated in a wide variety of biological processes. To identify potential miRNAs that may influence the growth and proliferation of mammalian cells cultured in bioreactors, we applied miRNA microarray expression profiling technology to batch cultures of HEK293 cells in protein free media. In our study, we identified miRNAs that were differentially expressed during the exponential and stationary phases, 13 of these showed distinct up regulation trends while 1 exhibited down regulation. These miRNAs have been implicated in cellular differentiation, growth arrest and apoptosis. Specifically, miR-16 and let-7b are potentially useful in the enhancement of bioreactor cell cultures.     

过表达miR-153非小细胞肺癌细胞稳转株的构建及鉴定

目的:构建过表达miR-153的非小细胞肺癌的稳转细胞株,为进一步探讨miR-153在肺癌发生发展中的作用奠定基础。方法:构建具有嘌呤霉素抗性的miR-153慢病毒载体;体外培养非小细胞肺癌细胞系SPC-A-1和A549,加入浓度梯度的嘌呤霉素溶液,筛选出最佳浓度;使用慢病毒转染细胞株,并在转染72 h后用荧光显微镜检测绿色荧光蛋白(EGFP)的表达,评价转染效率;体外实验使用PCR检测miR-153在稳转株的表达;稳转株移植裸鼠体内成瘤,取出瘤体后检测瘤体内EGFP和miR-153的表达。结果:完成过表达miR-153慢病毒载体及阴性对照载体的构建;确定嘌呤霉素最佳筛选浓度:在SPC-A-1细胞中的浓度为2.0 g/mL,A549细胞中的浓度为3.0 g/mL;目的基因miR-153被慢病毒成功导入SPC-A-1细胞和A549细胞中,荧光显微镜下能直接观察到EGFP。转染miR-153组和阴性对照组的PCR实验结果显示:在SPC-A-1细胞中,miR-153的表达量为92.9±20.6,明显高于阴性对照组(P=0.016);在A549细胞中,miR-153的表达量为2624.6±153.4,显著高于阴性对照组(P0.001)。稳转株能在裸鼠体内形成移植瘤,瘤体内明显有EGFP的表达;与阴性对照组的瘤体相比,移植瘤组miR-153的表达量为显著上调(P=0.048)。结论:成功构建过表达miR-153的非小细胞肺癌的稳转细胞株。