MicroRNA-148a Suppresses Invasion and Metastasis of Human Non-Small-Cell Lung Cancer.

BACKGROUND/AIMS: microRNAs (miRNAs) are noncoding RNAs that regulate multiple targets through either the degradation of mRNAs or the inhibition of protein translation, thereby altering several functions simultaneously. Growing evidence indicates that miRNAs are involved in carcinogenesis and tumor progression in non-small-cell lung cancer (NSCLC). METHODS: In this study, the mRNA expression levels of miR-148a were examined in NSCLC cell lines and patient specimens using quantitative reverse transcription-PCR. The functions of miR-148a in migration/invasion and lung metastasis formation were determined by using transwell and tail vein injection assays, respectively. RESULTS: We demonstrated that miR-148a was down-regulated in NSCLC metastatic samples, and its expression was suppressed in NSCLC compared with the corresponding nonmalignant lung tissues. Clinical analysis indicated that miR-148a expression was lower in NSCLC patients compared with nonmalignant lung tissues . Decreased miR-148a was significantly associated with tumor node metastasis stage and lymph node metastasis. Furthermore, functional assays showed that miR-148a expression suppressed NSCLC cell invasive and migratory abilities in vitro and suppressed cancer metastasis in vivo, while inhibition of miR-148a enhanced NSCLC cell invasion and lung metastasis formation in a mouse model. CONCLUSIONS: Evidence from this study demonstrated that miR-148a exerts tumor-suppressive effects in NSCLC and suggests a new therapeutic option for NSCLC.

Quantitative proteomic analysis identifies CPNE3 as a novel metastasis-promoting gene in NSCLC.

To discover metastasis-associated proteins within cancer cells, we used the isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis to identify proteins that were differentially expressed between lung adenocarcinoma cancer cell lines SPC-A-1sci cells with high metastatic potential and parent SPC-A-1 cells with low metastatic potential. By employing biological and technical replicates, we identified 5818 nonredundant proteins and quantified 5443 proteins, 256 of which were differentially expressed in the two cell lines. Through si-RNA-mediated functional screens, Myosin heavy chain 9 (MYH9) and Copine III (CPNE3) were indicated as positively correlating with the migration and invasion properties of SPC-A1sci cells, and the same function of CPNE3 was confirmed in another lung cancer cell line, H1299. Furthermore, overexpressing CPNE3 promoted nonsmall-cell lung cancer (NSCLC) cell line (SPC-A-1 and XL-2) migration and invasion in vitro. Moreover, the targeted knock-down of CPNE3 inhibited the in vivo metastatic abilities of H1299 cells in mouse models. Lastly, immunohistochemistry revealed that the CPNE3 expression level was positively correlated with the clinical stage and TNM classification in NSCLC patients. Taken together, our results indicate that CPNE3 could play a critical role in NSCLC metastasis.

Systematic analysis of mRNA expression profiles in NSCLC cell lines to screen metastasis-related genes.

Lung cancer is the most prevalent cancer in humans and has the lowest survival outcomes due to its high metastatic potential. The aim of the present study was to screen for metastasis­related genes (MRGs) by investigating the differential expression genes (DEGs) identified by the mRNA expression profiles in SPC­A­1sci (highly metastatic) and SPC-A-1 (parental) cells. DEGs were screened using Genespring software. Gene Ontology and pathway enrichment analyses of these DEGs were performed. Interaction networks between the proteins encoded by the DEGs were identified using the database BioGRID and were visualized by Cytoscape. Modular analysis of the protein­protein interaction network was performed in CFinder. Among these DEGs, the expression levels of 18 genes were examined in SPC­A­1sci and SPC­A­1 cell lines with reverse transcription­quantitative polymerase chain reaction, and 10 of the 18 genes were assessed by western blotting to validate the results of the microarray. Furthermore, the role of metallothionein 1X (MT1X) in non­small cell lung cancer was explored in functional assays and 72 pairs of clinical samples in vitro. Finally, 4,838 DEGs were screened, including 798 upregulated and 4,040 downregulated genes. The significantly enriched functions included gene expression, cytosol and poly­(A) RNA binding, and the most enriched pathway was biosynthesis of antibiotics. Furthermore, MT1X was revealed to promote the migration and invasion ability in SPC­A­1sci and PC­9 lung cancer cell lines. Therefore, MT1X was identified as a candidate MRG through systematic analysis in the present microarray, which was demonstrated to offer potential reference value in screening MRGs.

Establishment of a highly metastatic model with a newly isolated lung adenocarcinoma cell line.

Lung cancer is the leading cause of malignancy-related death worldwide, and metastasis always results in a poor prognosis. However, therapeutic progress is hampered by a deficiency of appropriate pre-clinical metastatic models. To bridge this experimental gap, we developed an in vivo metastatic model via subcutaneous (s.c.) injection. The original cell line (XL-2) adopted in this model was newly isolated from the ascites of a patient with extensive metastases of lung adenocarcinoma, thereby avoiding any alteration of its initial molecular biology features from artificial serial cultivation. After comprehensive phenotypical and histological analysis, it was identified as a lung adenocarcinoma cell line. Additionally, the drug test showed that XL-2 cell line was sensitive to docetaxel, and resistant to doxorubicin, indicating it might serve as a cell line model of drug resistance for identifying mechanisms of tumors resistant to doxorubicin. Through this s.c. model, we further obtained a highly metastatic cell line (designated XL-2sci). The metastatic rate of mice in XL-2 group was 3/10, in contrast to the rate of 9/10 in XL-2sci group. Optical imaging, micro-computed tomography (micro-CT) scanning and Transwell assays were further applied to identify the enhanced metastatic capacity of Xl-2sci cells both in vivo and in vitro. Compared with XL-2 cells, ITRAQ labeled proteomics profiling study showed that some tumor metastasis-associated proteins were upregulated in XL-2sci cells, which also indicated the reliability of our model. Proliferation ability of XL-2 and XL-2sci were also evaluated. Results showed that highly metastatic XL-2sci possessed a decreased proliferation capacity versus XL-2, which demonstrated that its increased metastatic activity was not facilitated by a faster growth rate. In conclusion, we successfully developed an in vivo metastatic model using a newly established lung adenocarcinoma cell line, which will be beneficial to further investigations of lung cancer metastasis and to the development of anti-metastasis drugs.

Integrative analyses identify osteopontin, LAMB3 and ITGB1 as critical pro-metastatic genes for lung cancer.

OBJECTIVE: To explore the key regulatory genes associated with lung cancer in order to reduce its occurrence and progress through silencing these key genes. METHODS: To identify the key regulatory genes involved in lung cancer, we performed a combination of gene array and bioinformatics analyses to compare gene transcription profiles in 3 monoclonal cell strains with high, medium or low metastatic abilities, which were separated from the SPC-A-1sci and SPC-A-1 cell lines by limiting dilution monoclone assay. We then analyzed those genes' biological activities by knocking down their expression in SPC-A-1sci cells using siRNA and lenti-viral shRNA vectors, followed by determinations of the invasion and migration capabilities of the resulting cell lines in vitro as well as their potential for inducing occurrence and metastasis of lung cancer in vivo. To examine the clinical relevance of these findings, we analyzed the expression levels of the identified genes in human lung cancer tissues (n = 135) and matched adjacent normal tissues by immunohistochemical (IHC) staining. RESULTS: Three monoclonal cell strains characterized with high, medium or low metastatic abilities were successfully selected. Gene array and bioinformatics analyses implied that osteopontin, LAMB3 and ITGB1 were key genes involved in lung cancer. Knockdown of these genes suppressed human lung cancer cell invasion and metastasis in vitro and in vivo. Clinical sample analyses indicated that osteopontin, LAMB3 and ITGB1 protein expression levels were higher in lung cancer patients, compared to non-cancerous adjacent tissues, and correlated with lymphatic metastasis. CONCLUSIONS: We confirmed that osteopontin, LAMB3 and ITGB1 played important roles in the occurrence and metastasis of lung cancer, thus provided important clues to understanding the molecular mechanism of metastasis and contributing to the therapeutic treatment of lung cancer.

[Antitumor effect of endothelial progenitor cells with TRAIL gene transfection on ovarian carcinoma xenografts in nude mice].

BACKGROUND & OBJECTIVE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can induce apoptosis in various cancer cell lines with little toxicity toward normal cells. It offers a promising therapeutic method against ovarian cancer. Endothelial progenitor cells (EPCs) could home to tumor lesion, and take part in angiogenesis. This study was to observe antitumor effect of EPCs with TRAIL gene transfection on human ovarian epithelial cancer xenografts in nude mice. METHODS: EPCs were isolated from human cord blood by magnetic bead selection, and cultured and amplified in vitro. Human ovarian epithelial cancer cell line 3AO cells were transplanted subcutaneously in immunodeficiency nude mice. EPCs were transfected with hTRAIL plasmid (TRAIL-EPCs). TRAIL-EPCs, TRAIL, or culture media IMDM (control) was injected into the mice model of 3AO xenograft by caudal vein. The changes of tumor volume were observed, and the tumor growth inhibition rate was calculated. RESULTS: EPCs with TRAIL gene transfection obviously inhibited the growth of 3AO xenograft in nude mice; the tumor weight of control, TRAIL, and TRAIL-EPCs groups were (0.226 +/- 0.209) g, (0.118 +/- 0.164) g, and (0.075 +/- 0.084) g, respectively, the maximum tumor growth inhibition rate was significantly higher in TRAIL-EPCs group than in TRAIL group (66.9% vs. 48.1%, P < 0.05). HE staining showed that there were more hemorrhage and necrosis regions in TRAIL and TRAIL-EPCs groups than in control group. No severe toxic reaction was observed in the 2 groups. CONCLUSIONS: Both TRAIL and TRAIL-EPCs can inhibit the growth of ovarian epithelial carcinoma xenograft in nude mice. EPCs have certain guidance effect in tumor xenografts in nude mice.