miR‐134 inhibits non‐small cell lung cancer growth by targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently activated in a wide range of solid tumours and represents an important therapeutic target. MicroRNAs (miRNAs) have recently been recognized as a rational and potential modality for anti‐EGFR therapies. However, more EGFR‐targeting miRNAs need to be explored. In this study, we identified a novel EGFR‐targeting miRNA, miRNA‐134 (miR‐134), in non‐small‐cell lung cancer (NSCLC) cell lines. Luciferase assays confirmed that EGFR is a direct target of miR‐134. In addition, the overexpression of miR‐134 inhibited EGFR‐related signaling and suppressed NSCLC cells proliferation by inducing cell cycle arrest and/or apoptosis, suggesting that miR‐134 functions as a tumour suppressor in NSCLC. Further mechanistic investigation including RNAi and rescue experiments suggested that the down‐regulation of EGFR by miR‐134 partially contributes to the antiproliferative role of miR‐134. Last, in vivo experiments demonstrated that miR‐134 suppressed tumour growth of A549 xenograft in nude mice. Taken together, our findings suggest that miR‐134 inhibits non‐small cell lung cancer growth by targeting the EGFR.     

Cytotoxic activity of gypenosides and gynogenin against non-small cell lung carcinoma A549 cells

The activity of gypenosides and gynogenin of Gynostemma pentaphyllum against non-small cell lung carcinoma (NSCLC) A549 cells was investigated to identify the structural characteristics of gypenosides and gynogenin to have anti-NSCLC activity. Of the tested dammarane-type compounds, 20S-dammar-24-en-2α,3β,12β,20-tetrol showed the strongest activity against A549 cells. Based on the structure and cytotoxic activity relationships of gypenosides and gynogenin, the OH group in C-2, the connected sugar number and the configuration in C-20 were important for cytotoxic activity against A549 cells.     

Significance of tumour cell HLA‐G5/‐G6 isoform expression in discrimination for adenocarcinoma from squamous cell carcinoma in lung cancer patients

Human leucocyte antigen (HLA)‐G has seven isoforms, of which HLA‐G1‐G4 are membrane‐bound and HLA‐G5‐G7 are soluble. Previous studies reinforced HLA‐G expression was strongly related to poor prognosis in different types of cancers. Among these studies, the monoclonal antibody (mAb) 4H84 was used which detects all HLA‐G isoform heavy chain; unfortunately, leaves the specific types of isoforms expressed in lesions undistinguished and its clinical significance needs to be clarified. To explore clinical significance of lesion soluble HLA‐G (sHLA‐G) in non‐small‐cell lung cancer (NSCLC), mAb 5A6G7 recognizing HLA‐G5/‐G6 molecules was used. Tumour cell sHLA‐G expression in 131 primary NSCLC lesions (66 squamous cell carcinoma, 55 adenocarcinoma and 10 adenosquamous carcinoma) were analysed with immunohistochemistry. Data showed that sHLA‐G expression was observed in 34.0% (45/131) of the NSCLC lesions, which was unrelated to patient age, sex, lymph nodal status, tumour–node–metastasis stage and patient survival. However, tumour cell sHLA‐G expression in lesions was predominately observed in adenocarcinoma lesions (73.0%, 40/55) which was significantly higher than that in squamous cell carcinoma (6.0%, 4/66) and adenosquamous carcinoma lesions (10.0%, 1/10, P < 0.001). The area under the receiver operating characteristic curve for lesion sHLA‐G was 0.833 (95% CI: 0.754–0.912, P < 0.001) for adenocarcinoma versus squamous cell carcinoma. Our findings for the first time showed that tumour cell sHLA‐G was predominately expressed in lung adenocarcinoma, which could be a useful biomarker to discriminate adenocarcinoma from squamous cell carcinoma in NSCLC patients.     

Phospholipase C ε‐1 inhibits p53 expression in lung cancer

The pathogenesis of lung cancer is to be further investigated. Recent reports indicate that phospholipase C ε‐1 (PLCE1) is a critical molecule involved in tumour growth. This study aims to investigate the role of PLCE1 in the regulation of apoptosis in lung cancer cells. In this study, the surgically removed non‐small‐cell lung cancer (NSCLC) tissue was collected from 36 patients. Single NSCLC cells were prepared from the tissue, in which immune cells of CD3⁺, CD11c⁺, CD19⁺, CD68⁺ and CD14⁺ were eliminated by magnetic cell sorting. The expression of PLCE1 and p53 was assessed by quantitative real‐time polymerase chain reaction and Western blotting. Apoptosis of NSCLC cells was analysed by flow cytometry. The results showed that, in cultured NSCLC cells, high levels of PLCE1 and low levels p53 were detected; the two molecules showed a negative correlation (p < 0.01). The addition of anti‐PLCE1 antibody increased the expression of p53 in NSCLC cells, which increased the frequency of apoptotic NSCLC cells. We conclude that NSCLC cells express high levels of PLCE1, which suppresses the expression of p53 in NSCLC cells. PLCE1 can be a therapeutic target of NSCLC. Copyright © 2013 John Wiley & Sons, Ltd.     

MiR‐138 inhibits cell proliferation and reverses epithelial‐mesenchymal transition in non‐small cell lung cancer cells by targeting GIT1 and SEMA4C

Non‐small‐cell lung cancer (NSCLC) is one of the most common and lethal malignant tumours worldwide with a poor 5‐year survival rate. Recent studies indicated that miRNAs have been involved in the tumorigenic driver pathways in NSCLC, but the relevant molecular mechanisms are not well‐understood. In this study, we investigated the biological functions and molecular mechanisms of miR‐138 in human NSCLC. The effects of miR‐138 on the NSCLC cell growth and epithelial‐mesenchymal transition (EMT) were first examined. Then the targeting connections of miR‐138 with G‐protein‐coupled receptor kinase‐interacting protein 1 (GIT1) and semaphorin 4C (SEMA4C) were confirmed by dual luciferase reporter assays. Finally, the effects of GIT1 and SEMA4C on the NSCLC cell growth and EMT were investigated respectively. We found that the ectopic expression of miR‐138 resulted in a significant inhibition of NSCLC growth and reversion of EMT. GIT1 and SEMA4C were identified as two novel targets of miR‐138. Furthermore, GIT1 and SEMA4C knockdown inhibited the cell growth and reversed EMT, just like the effects of miR‐138 overexpression on NSCLC cells, whereas ectopic expression of GIT1 and SEMA4C partly rescued the suppressive effects of miR‐138 in NSCLC cells. These data represent a crucial step towards the understanding of the novel roles and molecular mechanism of miR‐138, GIT1 and SEMA4C in NSCLC progression, which may provide some new targets or prognostic biomarkers for NSCLC treatment, thus having implications in translational oncology.     

Δ133p53 decreases the chemosensitivity of carcinoma cell line H1299

The research evaluated the effect of Δ133p53 on the chemosensitivity of lung adenocarcinoma cell line H1299. By this study, the drug‐resistant molecular marker and a new target for cancer therapy could be provided. Δ133p53 or negative control plasmid were transferred into H1299 cells by lentivirus vector. The expression of Δ133p53 in transfected cells was examined using immunofluorescence. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) method and colony formation test were applied to detect drug sensitivity after cisplatin or 5‐fluorouracil (5‐FU) treatment. After cisplatin (CDDP)/FU treatment, MTT assay demonstrated that the inhibition rate of H1299/Δ133p53 cell was reduced compared with that of the H1299 and H1299/NEG cells at the same concentration of drug. The 50% inhibitory concentrations (IC ₅₀) of CDDP and 5‐FU rose by 36.1 and 30.2%, respectively (P < 0.05). The colony formation assay suggested that the cell proliferation ability of H1299/Δ133p53 cell was prominently increased when compared with that of control group H1299 and H1299 /NEG cells (P < 0.05). The present study demonstrated that the transfection of the Δ133p53 gene in H1299 cells led to the reduction of chemosensitivity.     

MicroRNA‐30e‐5p promotes cell growth by targeting PTPN13 and indicates poor survival and recurrence in lung adenocarcinoma

Aberrant microRNA expression is involved in the regulation of various cellular processes, such as proliferation and metastasis in multiple diseases including cancers. MicroRNA‐30e‐5p (miR‐30e) was previously reported as an oncogenic or tumour suppressing miRNA in some malignancies, but its function in lung adenocarcinoma (LAC) remains largely undefined. In this study, we found that the expression of miR‐30e was increased in LAC tissues and cell lines, associated with tumour size and represented an independent prognostic factor for overall survival and recurrence of LAC patients. Further functional experiments showed that knockdown of miR‐30e suppressed cell growth while its overexpression promoted growth of LAC cells and xenografts in vitro and in vivo. Mechanistically, PTPN13 was identified as the direct target of miR‐30e in LAC, in which PTPN13 expression was down‐regulated in LAC tissues and showed the inverse correlation with miR‐30e expression. Overexpression of PTPN13 inhibited cell growth and rescued the proliferation‐promoting effect of miR‐30e through inhibition of the EGFR signalling. Altogether, our findings suggest that miR‐30e could function as an oncogene in LAC via targeting PTPN13 and act as a potential therapeutic target for treating LAC.     

A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

A dual role for integrin‐linked kinase and β1‐integrin in modulating cardiac aging

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin‐linked kinase (ilk) and β1‐integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z‐bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1‐integrin protein levels in old compared with young wild‐type flies, and cardiac‐specific overexpression of mys in young flies causes aging‐like heart dysfunction. Moreover, moderate cardiac‐specific knockdown of integrin‐linked kinase (ILK)/integrin pathway‐associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK‐associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine‐tuning of this pathway can retard the age‐dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin‐associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age‐dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.     

Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

Senescence of alveolar type 2 (ATII) cells, progenitors of the alveolar epithelium, is implicated in the pathogeneses of idiopathic pulmonary fibrosis (IPF), an aging‐related progressive fatal lung disorder with unknown etiology. The mechanism underlying ATII cell senescence in fibrotic lung diseases, however, remains poorly understood. In this study, we report that ATII cells in IPF lungs express higher levels of serpine 1, also known as plasminogen activator inhibitor 1 (PAI‐1), and cell senescence markers p21 and p16, compared to ATII cells in control lungs. Silencing PAI‐1 or inhibition of PAI‐1 activity in cultured rat ATII (L2) cells leads to decreases in p53 serine 18 phosphorylation (p53^S18P), p53 and p21 protein expressions; an increase in retinoblastoma protein phosphorylation (ppRb); and a reduction in the sensitivity to bleomycin‐ and doxorubicin‐induced senescence. Silencing p53, on the other hand, abrogates PAI‐1 protein‐stimulated p21 expression and cell senescence. In vivo studies, using ATII cell‐specific PAI‐1 conditional knockout mouse model generated recently in this laboratory, further support the role of PAI‐1 in the activation of p53‐p21‐Rb cell cycle repression pathway, ATII cell senescence, and lung fibrosis induced by bleomycin. This study reveals a novel function of PAI‐1 in regulation of cell cycle and suggests that elevation of PAI‐1 contributes importantly to ATII cell senescence in fibrotic lung diseases.     

Tumour‐derived exosomal miR‐3473b promotes lung tumour cell intrapulmonary colonization by activating the nuclear factor‐κB of local fibroblasts

Tumour‐derived exosomes have been shown to induce pre‐metastatic niche formation, favoring metastatic colonization of tumour cells, but the underlying molecular mechanism is still not fully understood. In this study, we showed that exosomes derived from the LLC cells could indeed significantly enhance their intrapulmonary colonization. Circulating LLC‐derived exosomes were mainly engulfed by lung fibroblasts and led to the NF‐κB signalling activation. Further studies indicated that the exosomal miR‐3473b was responsible for that by hindering the NFKB inhibitor delta's (NFKBID) function. Blocking miR‐3473b could reverse the exosome‐mediated NF‐κB activation of fibroblasts and decrease intrapulmonary colonization of lung tumour cells. Together, this study demonstrated that the miR‐3473b in exosomes could mediate the interaction of lung tumour cells and local fibroblasts in metastatic sites and, therefore, enhance the metastasis of lung tumour cells.     

KITLG is a novel target of miR‐34c that is associated with the inhibition of growth and invasion in colorectal cancer cells

MiR‐34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR‐34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR‐34c and KITLG mRNA expression levels in CRC cell lines, including HT‐29, HCT‐116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR‐34c in the 3′ untranslated region (3′UTR) of KITLG mRNA. A dual‐luciferase reporter assay further confirmed that KITLG is a direct target of miR‐34c. Then, the cell lines were infected with lentiviruses expressing miR‐34c or a miR‐34c specific inhibitor. Restoration of miR‐34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR‐34c increased the expression of KITLG protein. The miR‐34c‐mediated down‐regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down‐regulation for the tumour‐suppressive effects of miR‐34c in CRC cells. In conclusion, our results demonstrated that miR‐34c might interfere with KITLG‐related CRC and could be a novel molecular target for CRC patients.     

Identification of HLA‐A*11:01‐restricted Mycobacterium tuberculosis CD8+ T cell epitopes

New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette‐Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug‐resistant tuberculosis (MDR‐TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8⁺ T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8⁺ T cell epitopes specific for MTB is essential for the design of peptide‐based vaccines. To identify CD8⁺ T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01‐binding motifs. HLA‐A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon‐γ release and CD8⁺ T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8⁺ T cells from active pulmonary TB patients. In addition, a significant level of epitope‐specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA‐A*11:01 dextramer carrying the peptide Rv3130c_194‐204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8⁺ T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB.     

The p53‐inducible long noncoding RNA TRINGS protects cancer cells from necrosis under glucose starvation

The tumor suppressor p53 is activated in response to cellular stress to prevent malignant transformation. However, several recent studies have shown that p53 can play protective roles in tumor cell survival under adversity. Whether p53‐regulated long noncoding RNAs are involved in this process remains to be fully understood. Here, we show that under glucose starvation condition, p53 directly upregulates a novel lncRNA named TRINGS (Tp53‐regulated inhibitor of necrosis under glucose starvation) in human tumor cells. TRINGS binds to STRAP and inhibits STRAP–GSK3β–NF‐κB necrotic signaling to protect tumor cells from cell death. Interestingly, TRINGS appears to respond to glucose starvation specifically, as it is not activated by serum, serine, or glutamine deprivation. Collectively, our findings reveal that p53‐induced lncRNA TRINGS controls the necrotic pathway and contributes to the survival of cancer cells harboring wild‐type p53 under glucose stress.     

MicroRNA‐144‐3p suppressed TGF‐β1‐induced lung cancer cell invasion and adhesion by regulating the Src–Akt–Erk pathway

Lung cancer remains a leading cause to cancer‐related death worldwide. The anti‐cancer ability of microRNA‐144‐3p has been reported in many cancer types. This study focused on the mechanisms underlying miR‐144‐3p in inhibiting lung cancer. The expression levels of miR‐144‐3p and steroid receptor coactivator (Src) in different lung cancer cell lines and those in bronchial epithelial cells (16HBE) were compared. miR‐144‐3p mimic and siSrc were transfected into A549 cells. Under the conditions of transforming growth factor‐β1 (TGF‐β1). Small interfering transfection or TGF‐β1 treatment, cell invasive and adhesive abilities were analyzed by Transwell and cell adhesion assays. miR‐144‐3p inhibitor and siSrc were co‐transfected into A549 cells and the changes in cell invasion and adhesion were detected. The activation of Src–protein kinase B–extracellular‐regulated protein kinases (Src–Akt–Erk) pathway was determined using Western blot. The downregulated miR‐144‐3p and upregulated Src were generally detected in lung cancer cell lines and were the most significant genes in A549 cells. Both miR‐144‐3p overexpression and Src inhibition could obviously inhibit the invasion and adhesion abilities of A549 cells in the presence or absence of the effects of TGF‐β1. The inhibition of Src could block the promotive effects of miR‐144‐3p inhibitor and TGF‐β1 on cell invasion and adhesion. Furthermore, we found that miR‐144‐3p could negatively regulate the phosphorylation levels of Akt and Erk. Our data indicated the essential role of Src in the mechanisms underlying TGF‐β1‐induced cell invasion and adhesion of lung cancer, and that miR‐144‐3p could effectively suppress TGF‐β1‐induced aggressive lung cancer cells by regulating Src expression.