miR-9 and let-7g enhance the sensitivity to ionizing radiation by suppression of NFκB1

The activation of nuclear factor-kappa B1 (NFkB1) in cancer cells may confer resistance to ionizing radiation (IR). To enhance the therapeutic efficiency of IR in lung cancer, we screened for microRNAs (miRNAs) that suppress NFkB1 and observed their effects on radiosensitivity in a human lung cancer cell line. From time series data of miRNA expression in γ-irradiated H1299 human lung cancer cells, we found that the expression of miR-9 was inversely correlated with that of NFκB1. Overexpression of miR-9 down-regulated the level of NFκB1 in H1299 cells, and the surviving fraction of γ-irradiated cells was decreased. Interestingly, let-7g also suppressed the expression of NFκB1, although there was no canonical target site for let-7g in the NFκB1 3' untranslated region. From these results, we conclude that the expression of miR-9 and let-7g could enhance the efficiency of radiotherapy for lung cancer treatment through the inhibition of NFκB1.     

基于亲和色谱的肺癌细胞磷酸化蛋白质组研究及其应用

磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti⁴⁺)螯合IMAC材料(Ti⁴⁺-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti⁴⁺-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti⁴⁺-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti⁴⁺-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。     

In silico prediction of human genes as potential targets for rice miRNAs

BackgroundExogenous microRNAs (miRNAs) enter the human body through food, and their effects on metabolic processes can be considerable. It is important to determine which miRNAs from plants affect the expression of human genes and the extent of their influence.MethodThe binding sites of 738Oryza sativa miRNAs (osa-miRNAs) that interact with 17 508 mRNAs of human genes were determined using the MirTarget program.ResultThe characteristics of the binding of 46 single osa-miRNAs to 86 mRNAs of human genes with a value of free energy (ΔG) interaction equal 94%–100% from maximum ΔG were established. The findings showed that osa-miR2102-5p, osa-miR5075-3p, osa-miR2097-5p, osa-miR2919 targeted the largest number of genes at 38, 36, 23, 19 sites, respectively. mRNAs of 86 human genes were identified as targets for 93 osa-miRNAs of all family osa-miRNAs with ΔG values equal 94%–98% from maximum ΔG. Each miRNA of the osa-miR156-5p, osa-miR164-5p, osa-miR168-5p, osa-miR395-3p, osa-miR396-3p, osa-miR396-5p, osa-miR444-3p, osa-miR529-3p, osa-miR1846-3p, osa-miR2907-3p families had binding sites in mRNAs of several human target genes. The binding sites of osa-miRNAs in mRNAs of the target genes for each family of osa-miRNAs were conserved when compared to flanking nucleotide sequences.ConclusionTarget mRNA human genes of osa-miRNAs are also candidate genes of cancer, cardiovascular and neurodegenerative diseases.     

Sequence-based analysis of 5′UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5′UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5′UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p’s binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5′UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5′UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.     

Preparative isolation and purification of steroidal glycoalkaloid from the ripe berries of Solanum nigrum L. by preparative HPLC–MS and UHPLC–TOF‐MS/MS and its anti‐non‐small cell lung tumors effects in vitro and in vivo

Overcoming epidermal growth factor receptor resistance is a critical problem that needs to be solved in clinical practice. Drugs that downregulate the fatty acid synthase‐epidermal growth factor receptor will become novel treatments for non‐small cell lung cancer. Solanum nigrum, extracted with water at 4°C, shows strong cytotoxic activity and inhibits tumor growth in Lewis tumor bearing‐mice in a dose‐dependent manner. A novel active compound in S. nigrum, solaoiacid, was successfully separated and purified from S. nigrum by preparative high‐performance liquid chromatography with mass spectrometry and ultra high performance liquid chromatography with time‐of‐flight tandem mass spectrometry. The IC₅₀ of solaoiacid on lung cancer cells was 2.3 µmol/L, which was significantly lower than that of the known steroidal glycoalkaloid. Label‐free proteomics and STRING Network analysis were used to identify significantly deregulated proteins in lung cancer cells that were treated with the fresh ripe fruit extracts of S. nigrum. S. nigrum regulates multiple signal pathways, including the epidermal growth factor receptor pathway. S. nigrum downregulated 24 main proteins with direct roles in fatty acid biosynthesis. Both S. nigrum and solaoiacid showed strong downregulation of the fatty acid synthase‐epidermal growth factor receptor and anti‐non‐small cell lung cancer effects, and thus will become a novel drug for the treatment of non‐small cell lung cancer.     

Synergistic Roles of Curcumin in Sensitising the Cisplatin Effect on a Cancer Stem Cell-Like Population Derived from Non-Small Cell Lung Cancer Cell Lines

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.     

Detection of non‐small cell lung cancer cells based on microfluidic polarization microscopic image analysis

In early diagnosis of lung cancer, a polarization microscopy is a powerful tool to obtain the optical information of biological tissues. In this paper, a new microfluidic polarization imaging and analysis method was proposed for the detection and classification of cancer‐associated fibroblasts and the two kinds of non‐small cell lung cancer cells, A549 and H322. A polarizing microscopy system was constructed based on a commercial microscope to obtain 3*3 Mueller matrix of cells. Based on the Muller matrix decomposition algorithm and analysis in spatial domain and frequency domain, appropriate classification parameters were selected for the characterization of different polarization characteristics of cells. Finally, the logistic regression models based on machine learning were applied to determine optimal feature parameters and classify cells. This method integrated the morphological information of the cells, and the polarization characteristics of the cells in different polarization states. It is for the first time that the polarization microscopic image analysis method has been applied to the detection and classification of non‐small cell lung cancer cells. The results show that the presented microfluidic polarization microscopic image analysis method could classify cells effectively. Compared with the Muller matrix measurement and calculation methods, the method proposed in this paper was greatly simplified in both the acquisition of polarized images and the analysis and processing of polarized images.     

Discovery,Characterization, and Analogue Synthesis of Bohemamine Dimers Generated by Non‐enzymatic Biosynthesis

Dibohemamines A–C ( 5 – 7 ), three new dimeric bohemamine analogues dimerized through a methylene group, were isolated from a marine‐derived Streptomyces spinoverrucosus. The structures determined by spectroscopic analysis were confirmed through the semi‐synthetic derivatization of monomeric bohemamines and formaldehyde. These reactions, which could occur under mild conditions, together with the detection of formaldehyde in the culture, revealed that this dimerization is a non‐enzymatic process. In addition to the unique dimerization of the dibohemamines, dibohemamines B and C were found to have nm cytotoxicity against the non‐small cell‐lung cancer cell line A549. In view of the potent cytotoxicity of compounds 6 and 7 , a small library of bohemamine analogues was generated for biological evaluation by utilizing a series of aryl and alkyl aldehydes.     

Exosomal MicroRNA as Biomarkers for Diagnosing or Monitoring the Progression of Ovarian Clear Cell Carcinoma: A Pilot Study

Ovarian cancer is the most common cause of gynecological malignancy-related mortality since early-stage disease is difficult to diagnose. Advanced clear cell carcinoma of the ovary (CCCO) has dismal prognosis, and its incidence has been increasing in Japan, emphasizing the need for highly sensitive diagnostic and prognostic CCCO biomarkers. Exosomal microRNAs (miRNAs) secreted by tumor cells are known to play a role in carcinogenesis; however, their involvement in ovarian cancer is unclear. In this study, we performed expression profiling of miRNAs from exosomes released by five cell lines representing different histological types of ovarian cancer. Exosomes isolated from culture media of cancer and normal cells were compared for miRNA composition using human miRNA microarray. We detected 143 exosomal miRNAs, whose expression was ≥1.5-fold higher in ovarian cancer cells than in the control. Among them, 28 miRNAs were upregulated in cells of all histological ovarian cancer types compared to control, and three were upregulated in CCCO cells compared to other types. Functional analyses indicated that miR-21 overexpressed in CCCO cells targeted tumor suppressor genes PTEN, TPM1, PDCD4, and MASP1. The identified miRNAs could represent novel candidate biomarkers to diagnose or monitor progression of ovarian cancer, particularly CCCO.     

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration

Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G₀/G₁ cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 μg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.     

Syntenin increases the invasiveness of small cell lung cancer cells by activating p38, AKT,focal adhesion kinase and SP1

Syntenin is a PDZ domain-containing adaptor protein that has been recently shown to regulate migration and invasion in several tumors. Small cell lung cancer (SCLC) is notorious for its invasiveness and strong potential for metastasis. We therefore studied the influence of syntenin on the invasiveness of SCLC. Immunohistochemistry in tumor tissues showed that syntenin was more frequently expressed in small cell carcinomas than other neuroendocrine tumors, such as carcinoids and neuroblastomas, suggesting that syntenin expression may be related to more aggressive forms of neuroendocrine tumors. In SCLC patients, syntenin overexpression in tumor cells was significantly associated with more extensive and advanced disease at the time of diagnosis (P=0.029). Overexpression of syntenin in SCLC cells that were intrinsically syntenin-low increased the invasiveness of cells and led to the induction of extracellular matrix (ECM)-degrading membrane type 1-matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase 2 (MMP2). In contrast, suppression of syntenin in syntenin-high cells was associated with the downregulation of MT1-MMP. Contrary to the results of previous studies using malignant melanomas and breast carcinomas, signaling cascades were shown to be further transduced through p38 MAPK and PI3K/AKT, with activation of SP1 rather than NF-κB, under circumstances not involving ECM interaction. In addition, the upstream molecule focal adhesion kinase was induced by syntenin activation, in spite of the absence of ECM interaction. These results suggest that syntenin might contribute to the invasiveness of SCLC and could be utilized as a new therapeutic target for controlling invasion and metastasis in SCLC.     

Hyaluronic Acid Modified Nanostructured Lipid Carrier for Targeting Delivery of Kaempferol to NSCLC: Preparation,Optimization, Characterization,and Performance Evaluation In Vitro

Lung cancer seriously threatens the health of human beings, with non-small cell lung cancer (NSCLC) accounting for 80%. Nowadays, the potential position of nano-delivery in treating cancer has been the subject of continuous research. The present research aimed to prepare two molecular weight hyaluronic acid (HA)-modified kaempferol (KA)-loaded nanostructured lipid carriers (HA-KA-NLCs) by the method of melting ultrasonic and electrostatic adsorption, and to assess the antitumor effect of the preparations on A549 cells. The characterization and safety evaluation of the preparations illustrated that they are acceptable for drug delivery for cancer. Subsequently, differential scanning calorimetry (DSC) curve and transmission electron microscopy (TEM) images indicated that the drug was adequately incorporated in the carrier, and the particle appeared as a sphere. Moreover, HA-KA-NLC showed predominant in vitro antitumor effects, inhibiting proliferation, migration, and invasion, promoting apoptosis and increasing cellular uptake of A549 cells. Otherwise, the Western blot assay revealed that preparations could activate epithelial-mesenchymal transition (EMT)-related signaling pathways and modulate the expression of E-cadherin, N-cadherin, and Vimentin in A549 cells. Our present findings demonstrated that HA-KA-NLC could be considered as a secure and effective carrier for targeted tumor delivery and may have potential application prospects in future clinic therapy of NSCLC.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.     

Computational Identification of MicroRNAs and Their Targets in Perennial Ryegrass (Lolium perenne)

MicroRNAs (miRNAs) are small non-coding RNA molecules of 22 nucleotides in length that have been characterized as regulators of messenger RNA (mRNA) regulating a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. miRNAs have been extensively studied in various plant species; however, few information are available about miRNAs in perennial ryegrass, animal feed, and industrial raw materials. In this study, the 12 potential perennial ryegrass miRNAs were identified for the first time by computational approach. Using the newly identified miRNA sequences, the perennial ryegrass mRNA database was further used for BLAST search and detected 33 potential targets of miRNAs. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. Our result should be useful for further investigation into the biological functions of miRNAs in perennial ryegrass. The selected miRNAs representing four families were verified by RT-PCR experiment, indicating that the prediction method that we used to identify the miRNAs was effective.     

Urinary metabolomic study of non‐small cell lung carcinoma based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Metabolic profiles from human urine reveal the significant difference of carnitine and acylcarnitines levels between non‐small cell lung carcinoma patients and healthy controls. Urine samples from cancer patients and healthy individuals were assayed in this metabolomic study using ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. The data were normalized by the sum of all intensities and creatinine calibration, respectively, before orthogonal partial least squares discriminant analysis. Twenty differential metabolites were identified based on standard compounds or tandem mass spectrometry fragments. Among them, some medium‐/long‐chain acylcarnitines, for example, cis‐3,4‐methylene heptanoylcarnitine, were found to be downregulated while carnitine was upregulated in urine samples from the cancer group compared to the control group. Receiver operating characteristic analysis of the two groups showed that the area under curve for the combination of carnitine and 11 selected acylcarnitines was 0.958. This study suggests that the developed carnitine and acylcarnitines profiling method has the potential to be used for screening non‐small cell lung carcinoma.     

Antitumor effect of guava leaves on lung cancer: A network pharmacology study

Guava is known for its hypoglycemic, antivirus, antibacterial, anti-inflammatory, antioxidant, and antitumor properties. In this study, triterpenoids, sesquiterpenes, and flavonoids were examined as potential targets of constituents of guava leaves. Our study was aimed to reveal the antitumor mechanism and construct the network pharmacology network of guava leaf constituents and lung cancer. The potential targets of guava leaf constituents were searched in target databases, while the disease genes were searched in the GeneCards database. The common targets of drugs and diseases were screened out. A network map was constructed by the Cytoscape software, and the GO and KEGG pathways were analyzed. The existing cases were studied by SystemsDock molecular docking and cBioPortal tumor database study. Among the 66 chemical constituents of guava leaves, 153 of their targets were the lung cancer genes involved in many signaling pathways, such as the PI3K-Akt signaling pathway, in small cell lung cancer and non-small cell lung cancer. There was a binding activity between ligand compounds and receptor proteins. Guava leaves inhibited tumor through a gene regulatory network, and may play an important role in gene-targeting therapy. Through network pharmacology, we found that guava leaves had potential targets that interacted with various tumors, regulating the signaling pathways of cancers. This study preliminarily verified the pharmacological basis and the mechanism of the antitumor effect of guava leaves, providing a foundation for further research.