Invasion Of A549 Cells Research Articles

SP13786 Inhibits the Migration and Invasion of Lung Adenocarcinoma Cell A549 by Supressing Stat3-EMT via CAFs Exosomes

背景与目的成纤维细胞活化蛋白（fibroblast activation protein, FAP）是肿瘤相关成纤维细胞（cancer-associated fibroblasts, CAFs）的表面标志物之一，与CAFs的恶性表征关系密切，SP13786是FAP的特异性小分子抑制剂。本研究探讨SP13786作用于CAFs后，CAFs外泌体（exosomes, exo）对A549细胞迁移、侵袭的影响与机制。方法原代提取CAFs和癌旁成纤维细胞（peri-tumer fibroblasts, PTFs）；MTT实验检测不同浓度SP13786对CAFs增殖的影响；聚合物沉淀法提取PTFs-exo、CAFs-exo以及CAFs+SP13786-exo。将A549细胞设对照组、PTFs组、CAFs组及CAFs+SP13786组并分别以等体积的DMEM、PTFs-exo、CAFs-exo及CAFs+SP13786-exo孵育细胞。激光共聚焦实验检测A549细胞摄取外泌体的情况；免疫荧光、免疫组化和Western blot方法检测α平滑肌肌动蛋白（alpha-smooth muscle actin, α-SMA）、FAP在PTFs和CAFs中的表达及E-cadherin、N-cadherin、Slug、Stat3、P-Stat3在各组A549细胞中的表达；划痕实验和Transwell实验检测各组细胞的迁移和侵袭能力。结果免疫荧光、免疫组化和Western blot结果均显示α-SMA、FAP在CAFs中高表达，在PTFs中低表达（P < 0.05），表明从肺腺癌组织和癌旁组织中分别成功获得了CAFs和PTFs。MTT实验测得SP13786对于CAFs细胞的半数抑制浓度（50% inhibitory concentration, IC50）约为3.3 nmol/L。免疫组化和Western blot结果显示与CAFs组相比，CAFs+SP13786组的α-SMA与FAP的表达显著降低（P < 0.05），说明抑制FAP可以显著降低CAFs的恶性表征。激光共聚焦结果显示外泌体能够被A549细胞所摄取。划痕实验与Transwell实验显示SP13786可抑制CAFs-exo对A549细胞迁移和侵袭的促进作用（P < 0.05）。与CAFs组比较，SP13786组A549细胞E-cadherin表达增多，N-cadherin与Slug表达降低（P < 0.05）；免疫荧光与Western blot显示SP13786组A549细胞的P-Stat3较CAFs组明显降低（P < 0.05），而总Stat3无显著差异。Stat3的特异性抑制剂WP1066明显抑制CAFs组A549细胞上皮间质转化（epithelial-mesenchymal transition, EMT），P-Stat3显著降低（P < 0.05），而加入WP1066后再加入SP13786-exo，P-Stat3未见进一步减低，EMT的抑制亦未见显著变化（P > 0.05）。结论FAP的小分子特异性抑制剂SP13786通过影响CAFs外泌体间接抑制A549细胞的迁移、侵袭，其可能机制是抑制Stat3的磷酸化从而影响A549细胞的EMT。

MicroRNA-34c-5p Inhibition of NUF2 Suppresses Lung Adenocarcinoma Cell Viability and Invasion

Background. Lung cancer continues to be a burden worldwide with an estimated 2.09 million new cases of lung cancer and 1.76 million deaths in 2018. MicroRNAs (miRs) are key regulators of gene expression and show their oncogenic or antioncogenic role in human cancers including lung cancer. In this study, we test the hypothesis that miR-34c-5p functions as a candidate antioncomiR in lung adenocarcinoma by targeting NUF2. Methods. The expression pattern of miR-34c-5p and NUF2 was evaluated in 202 biopsy specimens from patients with lung adenocarcinoma and 176 biopsy specimens from patients with benign lung diseases. Interaction between miR-34c-5p and NUF2 was verified by the luciferase-based assay. Cell viability and invasion assays were carried out in cultured A549 cells treated with miR-34c-5p mimic, inhibitor, and siRNA against NUF2. Results. NUF2 was highly expressed in lung adenocarcinoma samples and related to the differentiation degree, TNM stage, and presence of lymph node metastasis (LNM). Patients with NUF2 overexpression had reduced overall survival (OS) and disease-free survival (DFS) compared to patients with underexpression. Cox multivariate analysis revealed that high expression of NUF2, advanced TNM stage, well/moderate differentiation, and existence of LNM were unfavorable prognostic factors. siRNA-mediated knockdown of NUF2 inhibits A549 cell viability and invasion. miR-34c-5p was expressed at a poor level in lung adenocarcinoma samples and related to the differentiation degree, TNM stage, and presence of LNM. miR-34c-5p underexpression contributes to reduced OS and DFS, which was demonstrated as an unfavorable prognostic factor by Cox multivariate analysis. siRNA-mediated knockdown of NUF2 could ablate miR-34c-5p inhibition-mediated effects on A549 cells. Conclusion. Our results prove the hypothesis that miR-34c-5p could suppress lung adenocarcinoma progression by binding to the NUF2 gene. The study is a significant step towards extending our understanding of the mode of miRNA regulation in lung adenocarcinoma.

Upregulated osteoprotegerin expression promotes lung cancer cell invasion by increasing miR-20a expression.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor superfamily and a major regulatory factor in osteoclast development. OPG has been previously associated with the malignant behavior of various types of cancer, particularly that of cancer metastasis. However, information on the link between the expression profile of OPG and lung cancer metastasis remained elusive. In the present study, the expression levels of OPG in the serum samples of patients with non-small cell lung cancer (NSCLC) was measured using ELISA. The expression of miRNAs was assessed using reverse transcription-quantitative PCR. A549 or H3122 cell invasion was assessed using Transwell invasion assays. The effect of OPG on the invasiveness of lung cancer cells was evaluated using an experimental mouse lung metastasis model. OPG expression was found to be upregulated in the serum of patients with NSCLC compared with that in healthy individuals. The serum levels of OPG in patients with distant metastasis were observably higher compared with those in patients without metastasis. Functionally, overexpression of OPG in NSCLC cells markedly promoted cell invasion. Mechanistically, increased expression of OPG resulted in upregulation of microRNA (miR)-20a in NSCLC cells. Furthermore, miR-20a promoted NSCLC cell invasion, whilst miR-20a inhibition partially abrogated the effect of OPG on NSCLC cell invasion. Taken together, the present results demonstrated that the OPG/miR-20a axis serve an important role in lung cancer metastasis, which potentially provide an additional novel target for lung cancer treatment.

Shikonin inhibits the Warburg effect, cell proliferation, invasion and migration by downregulating PFKFB2 expression in lung cancer.

Lung cancer is one of the most lethal diseases and therefore poses a significant threat to human health. The Warburg effect, which is the observation that cancer cells predominately produce energy through glycolysis, even under aerobic conditions, is a hallmark of cancer. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB) is an important regulator of glycolysis. Shikonin is a Traditional Chinese herbal medicine, which has been reported to exert antitumor effects. The present study aimed to investigate the anticancer activity of shikonin in lung cancer. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to analyze proliferation in A549 and H446 cells. Wound healing and Transwell assays were used to measure migration and invasion in A549 and H446 cells. Cell apoptosis was analyzed using flow cytometry. Lactate levels, glucose uptake and cellular ATP levels were measured using their corresponding commercial kits. Western blotting was performed to analyze the protein expression levels of key enzymes involved in aerobic glucose metabolism. Reverse transcription-quantitative PCR was used to analyze the mRNA expression levels of PFKFB2. The results of the present study revealed that PFKFB2 expression levels were significantly upregulated in NSCLC tissues. Shikonin treatment decreased the proliferation, migration, invasion, glucose uptake, lactate levels, ATP levels and PFKFB2 expression levels and increased apoptosis in lung cancer cells in a dose-dependent manner. The overexpression of PFKFB2 increased the proliferation, migration, glucose uptake, lactate levels and ATP levels in lung cancer cells, while the knockdown of PFKFB2 expression exerted the opposite effects. Moreover, there were no significant differences in lung cancer cell migration, apoptosis, glucose uptake, lactate levels and ATP levels between cells with knocked down PFKFB2 expression or treated with shikonin and the knockdown of PFKFB2 in cells treated with shikonin. In conclusion, the results of the present study revealed that shikonin inhibited the Warburg effect and exerted antitumor activity in lung cancer cells, which was associated with the downregulation of PFKFB2 expression.

MiR-144-3p Inhibits the Invasion and Metastasis of Lung Adenocarcinoma Cells by Targeting IRS1

背景与目的MicroRNAs（miRNAs）是短的非编码RNA，是能够调控基因表达、影响细胞过程、促进疾病发展的重要分子。在许多疾病中可以观察到miRNA表达的变化，包括肝炎、心血管疾病和癌症。本研究旨在探讨miR-144-3p靶向调控胰岛素受体底物1（insulin receptor substrate 1, IRS1）对肺腺癌细胞侵袭和转移的影响。方法通过生物信息学数据库查询miR-144-3p在肺腺癌患者中的表达情况；利用mirTarPathway对miRNA进行KEGG通路富集分析；定量逆转录聚合酶链式反应（quantitative reverse transcription polymerase chain reaction, qRT-PCR）检测miR-144-3p在肺腺癌细胞系中的表达情况与质粒转染效率；Transwell实验检测不同组细胞侵袭迁移能力的变化。生物信息学确定miR-144-3p的关键基因（Hub基因）；双荧光素酶靶标实验检测miR-144和IRS1的相互结合情况；Western blot实验检测IRS1在不同细胞系的表达及过表达miR-144后IRS1的表达情况。结果miR-144-3p在肺腺癌组织中表达降低，qRT-PCR结果提示miR-144-3p在肺腺癌细胞A549中的表达显著降低（P < 0.05）且过表达质粒转染成功（P < 0.05）；过表达miR-144后，细胞的迁移、侵袭和增殖能力下降（P < 0.05）；IRS1在肺腺癌组织中表达上调；生存分析表明高表达IRS1的肺腺癌患者预后不良（P < 0.05）；双荧光素酶实验结果显示，miR-144可特异识别IRS1的3’-UTR抑制报告酶的表达（P < 0.05）；Western blot结果提示IRS1在A549细胞中表达升高（P < 0.05），过表达miR-144后，IRS1蛋白表达水平下降（P < 0.05）；Transwell实验证明，miR-144-3p可通过靶向调控IRS1抑制肺腺癌细胞的侵袭和转移（P < 0.05）。结论miR-144-3p通过靶向调控IRS1抑制A549细胞的侵袭和迁移能力，进而在肿瘤中发挥抑癌作用。

Deciphering the Molecular Mechanism Responsible for Efficiently Inhibiting Metastasis of Human Non-Small Cell Lung and Colorectal Cancer Cells Targeting the Matrix Metalloproteinases by Selaginella repanda.

Selaginella species are known to have antimicrobial, antioxidant, anti-inflammatory, anti-diabetic as well as anticancer effects. However, no study has examined the cytotoxic and anti-metastatic efficacy of Selaginella repanda (S. repanda) to date. Therefore, this study aimed to evaluate the potential anti-metastatic properties of ethanol crude extract of S. repanda in human non-small-cell lung (A-549) and colorectal cancer (HCT-116) cells with possible mechanisms. Effect of S. repanda crude extract on the growth, adhesion, migration and invasion of the A-549 and HCT-116 were investigated. We demonstrated that S. repanda crude extract inhibited cell growth of metastatic cells in a dose and time dependent manner. Incubation of A-549 and HCT-116 cells with 100–500 µg/mL of S. repanda crude extract significantly inhibited cell adhesion to gelatin coated surface. In the migration and invasion assay, S. repanda crude extract also significantly inhibited cellular migration and invasion in both A-549 and HCT-116 cells. Moreover, reverse transcription-polymerase chain reaction, and real-time PCR (RT-PCR) analysis revealed that the activity and mRNA level of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-2 (MMP-2) and membrane type 1-matrix metalloproteinase (MT1-MMP) were inhibited. While the activity of tissue inhibitor matrix metalloproteinase 1 (TIMP-1); an inhibitor of MMPs was stimulated by S. repanda crude extract in a concentration-dependent manner. Therefore, the present study not only indicated the inhibition of motility and invasion of malignant cells by S. repanda, but also revealed that such effects were likely associated with the decrease in MMP-2/-9 expression of both A-549 and HCT-116 cells. This further suggests that S. repanda could be used as a potential source of anti-metastasis agent in pharmaceutical development for cancer therapy.

Procyanidins mediates antineoplastic effects against non-small cell lung cancer via the JAK2/STAT3 pathway.

BackgroundLung cancer is a malignant tumor with one of the highest rates of cancer-related morbidity and mortality worldwide. Non-small cell lung cancer (NSCLC) account for 85% of all lung cancers and have a poor prognosis. Proanthocyanidins (PCs) are polyphenolic compounds that are found widely in natural plants. The present study aimed to determine the effects of PC on lung cancer and identify its possible mechanism.MethodsA cell growth assay was used to detect the cell growth ability of A549 cancer cells, and a clonal formation assay was used to detect the cloning ability of A549 cancer cells. Flow cytometry was used to detect the effect of PCs on apoptosis and the cell cycle. The wound healing test, Transwell migration, and invasion test were used to detect the migration and invasion of human NSCLC A549 cells. Western blotting was utilized to detect the expression levels of N-cadherin, E-cadherin, vimentin, Janus kinase 2 (JAK2), p-signal transducer and activator of transcription 3 (p-STAT3), STAT3, matrix metalloproteinase 2 (MMP-2), MMP-9, and the apoptosis-related proteins, B-cell lymphoma-2 (Bcl-2) and BCL2-associated X (Bax). Cell immunofluorescence was used to detect the expression levels of the p-STAT3 primary antibody.ResultsPCs reduced the proliferation and cloning ability of A549 cells and significantly inhibited the migration and invasion of A549 cells in a dose-dependent manner. At the same time, PCs induced apoptosis in A549 cells and G2/M cell cycle arrest. PCs increased the pro-apoptotic protein expression, Bax, and down-regulated the anti-apoptotic protein expression, Bcl-2. PCs also inhibited the epithelial-mesothermal transition (EMT) process of A549 cells. We also found that the JAK2/STAT3 signaling pathway inhibitor, AG490, cooperated with PCs to inhibit A549 cell invasion and migration. Our results demonstrated that PCs could mediate the antitumor effect of NSCLC via the JAK2/STAT3 pathway.ConclusionsPCs can inhibit NSCLC A549 cell proliferation, invasion, metastasis, clone formation, EMT, and induced apoptosis and G2/M cell cycle arrest. They work by inhibiting the JAK2/STAT3 signaling pathway. As a novel antitumor drug, PCs have broad application prospects for the treatment of NSCLC.

GTSE1 Facilitates the Malignant Phenotype of Lung Cancer Cells via Activating AKT/mTOR Signaling.

The expression of G2 and S phase-expressed-1 (GTSE1) was upregulated in human cancer. However, its expression and roles in lung cancer have not been identified yet. In our study, we reported that GTSE1 expression was statistically higher in lung tissues than in the adjacent noncancerous tissues which might be a consequence of hypomethylation of the GTSE1 promoter. The upregulated expression of GTSE1 mRNA predicted the poorer survival of the lung patients. Ectopic expression of GTSE1 in lung cancer cells significantly increased while knockdown of GTSE1 decreased cell proliferation, cell migration, and cell invasion in H460 and A549 cells. Furthermore, knockdown of GTSE1 regulated the cell cycle and promoted cell apoptosis in H460 and A549 cells. Finally, we presented that GTSE1 was able to activate AKT/mTOR signaling in H460 and A549 cells. In conclusion, these results indicated that the overexpressed GTSE1 was involved in the progress of lung cancer by promoting proliferation migration and invasion and inhibiting apoptosis of lung cancer cells via activating AKT/mTOR signaling.

A Combined Phytochemistry and Network Pharmacology Approach to Reveal Potential Anti-NSCLC Effective Substances and Mechanisms in Marsdenia tenacissima (Roxb.) Moon (Stem).

Marsdeniae tenacissimae Caulis is a traditional Chinese medicine, named Tongguanteng (TGT), that is often used for the adjuvant treatment of cancer. In our previous study, we reported that an ethyl acetate extract of TGT had inhibitory effects against adenocarcinoma A549 cells growth. To identify the components of TGT with anti-tumor activity and to elucidate their underlying mechanisms of action, we developed a technique for isolating compounds, which was then followed by cytotoxicity screening, network pharmacology analysis, and cellular and molecular experiments. We isolated a total of 19 compounds from a TGT ethyl acetate extract. Two novel steroidal saponins were assessed using an ultra-performance liquid chromatography-photodiode array coupled with quadrupole time-of-flight mass (UPLC-ESI-Q/TOF-MS). Then, we screened these constituents for anti-cancer activity against non-small cell lung cancer (NSCLC) in vitro and obtained six target compounds. Furthermore, a compound-target-pathway network of these six bioactive ingredients was constructed to elucidate the potential pathways that controlled anticancer effects. Approximately 205 putative targets that were associated with TGT, as well as 270 putative targets that were related to NSCLC, were obtained from online databases and target prediction software. Protein–protein interaction networks for drugs as well as disease putative targets were generated, and 18 candidate targets were detected based on topological features. In addition, pathway enrichment analysis was performed to identify related pathways, including PI3K/AKT, VEGF, and EGFR tyrosine kinase inhibitor resistance, which are all related to metabolic processes and intrinsic apoptotic pathways involving reactive oxygen species (ROS). Then, various cellular experiments were conducted to validate drug-target mechanisms that had been predicted using network pharmacology analysis. The experimental results showed the four C21 steroidal saponins could upregulate Bax and downregulate Bcl-2 expression, thereby changing the mitochondrial membrane potential, producing ROS, and releasing cytochrome C, which finally activated caspase-3, caspase-9, and caspase-8, all of which induced apoptosis in A549 cells. In addition, these components also downregulated the expression of MMP-2 and MMP-9 proteins, further weakening their degradation of extracellular matrix components and type IV collagen, and inhibiting the migration and invasion of A549 cells. Our study elucidated the chemical composition and underlying anti-tumor mechanism of TGT, which may be utilized in the treatment of lung cancer.

Long Noncoding RNA MLK7-AS1 Promotes Non-Small-Cell Lung Cancer Migration and Invasion via the miR-375-3p/YWHAZ Axis.

Long noncoding RNAs act essential regulators in lung cancer tumorigenesis. Our research aimed to investigate the potential function and molecular mechanisms of MLK7-AS1 in NSCLC (non-small-cell lung cancer). QRT-PCR results indicated that the MLK7-AS1 expression level was upregulated in NSCLC cells and tissues. MLK7-AS1 strengthened cell migration and invasion in H1299 and A549 cells. Luciferase reporter assay found that MLK7-AS1 functioned as an endogenous sponge for miR-375-3p. Transwell assay results showed that miR-375-3p suppressed cell migration and invasion in H1299 and A549 cells. YWHAZ was confirmed as a target gene of miR-375-3p by Targetscan. YWHAZ overexpression promoted the invasion of H1299 and A549 cells. MLK7-AS1 upregulated YWHAZ expression and enhanced H1299 and A549 cell invasion by sponging miR-375-3p. MLK7-AS1 improved the metastasis ability of A549 in vivo. In conclusion, MLK7-AS1 was identified as a novel oncogenic RNA in NSCLC and can function as a potential therapeutic target for NSCLC treatment.

125I Seed Promotes Apoptosis in Non-small Lung Cancer Cells via the p38 MAPK-MDM2-p53 Signaling Pathway.

Purpose: 125I seeds were effective in the treatment of non-small cell lung cancer in previous research. However, the exact signaling pathway-mediated apoptosis mechanism is still unclear. The present study analyzed the effects and potential mechanisms of 125I seed on the growth and migration of A549 cells.Methods: Lung cancer A549 cells were irradiated with 125I seed for various times. MTT, invasion assay, and flow cytometry were used to detect the proliferation, invasion, and apoptosis of treated cells, respectively. A Nimblegen genome-wide expression profile chip was used to evaluate gene expression changes in 125I seed-treated A549 cells. Validation studies were performed using phosphorylated protein chip technology, Western blot, nude mouse tumor xenograft assay, and immunohistochemical experiments. All statistical analyses were performed using unpaired Student's t tests and Kruskal-Wallis test.Results: Irradiation with 125I seed inhibited A549 cell proliferation and invasion and induced apoptosis (primarily early apoptosis). Irradiation with 125I seed also caused the downregulation of p38MAPK, degradation of mouse double-minute 2 homolog (MDM2), and higher expression of p53, which eventually resulted in non-small cell lung cancer cell apoptosis.Conclusion: 125I seed irradiation activated the p38MAPK/MDM2/p53 signaling pathway and promoted non-small cell lung cancer cell apoptosis. Future clinical studies targeting this signal may provide a new potential therapeutic approach for non-small cell lung cancer.

MiR-598 Represses Cell Migration and Invasion of Non-Small-Cell Lung Cancer by Inhibiting MSI2

Non-small-cell lung cancer (NSCLC) is one of the most frequent solid tumors and regarded as a significant threat to individual health around the world. MicroRNAs (miRs) are recognized as critical governors of gene expression during carcinogenesis, while their clinical significance and mechanism in NSCLC occurrence and development are required for further investigation. In this report, we characterized the functional role of miR-598 and its regulation mechanism in NSCLC. The expression level of miR-598 in NSCLC tissues and cell lines was detected by qRT-PCR. A549 cells were transiently transfected with miR-598 mimics or miR-598 inhibitors. Scratch assay and Transwell assay were used to detect cell transfection, migration, and invasion. Possible binding sites of miR-598 in MSI2 mRNA were predicted by bioinformatics and validated by dual-luciferase reporter gene system. The ability of migration and invasion was examined on cells transfected with MSI2 alone or cotransfected A549 cells with miR-598. The expression of miR-598 in NSCLC tissues was significantly lower than that in adjacent tissues, and the expression of miR-598 in NSCLC cell lines (A549, H1650, and H1299) was also significantly lower than that of normal lung epithelial cell line BEAS-2B. A549 cells were significantly inhibited in migration and invasion after transfection with miR-598 mimics, while miR-598 inhibitors were significantly enhanced in migration and invasion. MSI2 was a direct target gene of miR-598. MSI2 can promote the migration and invasion of A549 cells, but the ability to promote cell migration and invasion was reversed when miR-598 was introduced. In conclusion, miR-598 inhibits the migration and invasion of NSCLC by downregulating the target gene MSI2.

Inhibitory effects of terrein on lung cancer cell metastasis and angiogenesis.

Cancer metastasis is the leading cause of mortality in cancer patients. Over 70% of lung cancer patients are diagnosed at advanced or metastatic stages, and this results in an increased incidence of mortality. Terrein is a secondary bioactive fungal metabolite isolated from Aspergillus terreus. Numerous studies have demonstrated that terrein has anticancer properties, but in the present study, the cellular mechanisms underlying the inhibition of lung cancer cell metastasis by terrein was investigated for the first time. Using MTT assays, the cytotoxic effects of terrein were first examined in human lung cancer cells (A549 cells) and then compared with its cytotoxic effects in three noncancer control cell lines (Vero kidney, L6 skeletal muscle and H9C2 cardiomyoblast cells). The results indicated that terrein significantly reduced the viability of all these cells but exhibited a different level of toxicity in each cell type; these results revealed a specific concentration range in which the effect of terrein was specific to A549 cells. This significant cytotoxic effect of terrein in A549 cells was verified using LDH assays. It was then demonstrated that terrein attenuated the proliferation of A549 cells using IncuCyte image analysis. Regarding its antimetastatic effects, terrein significantly inhibited A549 cell adhesion, migration and invasion. In addition, terrein suppressed the angiogenic processes of A549 cells, including vascular endothelial growth factor (VEGF) secretion, capillary-like tube formation and VEGF/VEGFR2 interaction. These phenomena were accompanied by reduced protein levels of integrins, FAK, and their downstream mediators (e.g., PI3K, AKT, mTORC1 and P70S6K). All these data indicated that terrein was able to inhibit all the major metastatic processes in human lung cancer cells, which is crucial for cancer treatment.

Overexpression of PELP1 in Lung Adenocarcinoma Promoted E2 Induced Proliferation, Migration and Invasion of the Tumor Cells and Predicted a Worse Outcome of the Patients.

The expression of Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) has been reported to be dysregulated in non-small cell lung carcinoma, especially in lung adenocarcinoma (LUAD). Therefore, we aimed to investigate the functional and prognostic roles of PELP1 in LUAD in this study. We first immunolocalized PELP1 in 76 cases of LUAD and 17 non-pathological or tumorous lung (NTL) tissue specimens and correlated the findings with the clinicopathological parameters of the patients. We then performed in vitro analysis including MTT, flow cytometry, wound healing, and transwell assays in order to further explore the biological roles of PELP1 in 17-β-estradiol (E2) induced cell proliferation, migration, and invasion of LUAD cells. We subsequently evaluated the prognostic significance of PELP1 in LUAD patients using the online survival analysis tool Kaplan-Meier Plotter. The status of PELP1 immunoreactivity in LUAD was significantly higher than that in the NTL tissues and significantly positively correlated with less differentiated features of carcinoma cells, positive lymph node metastasis, higher clinical stage as well as the status of ERα, ERβ, and PCNA. In vitro study did reveal that E2 promoted cell proliferation and migration and elevated PELP1 protein level in PELP1-high A549 and H1975 cells but not in PELP1-low H-1299 cells. Knock down of PELP1 significantly attenuated E2 induced cell proliferation, colony formation, cell cycle progress as well as migration and invasion of A549 and H1975 cells. Kaplan-Meier Plotter revealed that LUAD cases harboring higher PELP1 expression had significantly shorter overall survival. In summary, PELP1 played a pivotal role in the estrogen-induced aggressive transformation of LUAD and could represent adverse clinical outcome of the LUAD patients.

LncRNA BLACAT1 Accelerates Non-small Cell Lung Cancer Through Up-Regulating the Activation of Sonic Hedgehog Pathway.

Recently, increasing evidence has displayed that lncRNAs can exhibit crucial function in cancer progression, including lung cancer. LncRNA bladder cancer-associated transcript 1 (BLACAT1) is reported to participate in various cancers. The aim of our current study was to investigate the function of BLACAT1 in non-small cell lung cancer progression and study the functional pathway. Here, we reported BLACAT1 was significantly up-regulated in lung cancer tissues in comparison to the adjacent normal tissues, which suggested BLACAT1 might act as an oncogene in lung cancer. Then, A549 and PC9 cells were infected with BLACAT1 overexpression plasmid and shRNA. As shown, we proved up-regulation of BLACAT1 greatly induced the growth of non-small cell lung cancer cells. Reversely, knockdown of BLACAT1 reduced A549 and PC9 cell proliferation, migration and invasion. Sonic hedgehog (shh) signaling is able to exert a significant role in carcinogenesis, including lung cancer. Currently, we proved that up-regulation of BLACAT1 activated shh signaling pathway, via inducing shh, Gli-1 and Smo expression. shh pathway inhibitor GANT-61 reversed the effect of overexpression of BLACAT1 on non-small cell lung cancer. Moreover, we manifested that loss of BLACAT1 remarkably reduced the in vivo growth and metastasis of A549 cells via enhancing infiltrating CD3+ T cells. In conclusion, our research revealed a critical role of BLACAT1 in the modulation of non-small cell lung cancer via modulating shh pathway.

Cyclic RGD Pentapeptide Cilengitide Enhances Efficacy of Gefitinib on TGF-β1-Induced Epithelial-to-Mesenchymal Transition and Invasion in Human Non-Small Cell Lung Cancer Cells.

During non-small cell lung cancer (NSCLC) progression, transforming growth factor (TGF)-β mediated epithelial-to-mesenchymal transition (EMT) is an important process leading to high mortality and poor prognosis. The EMT is a fundamental process for morphogenesis characterized by the transformation of cancer cells into invasive forms that can be transferred to other organs during human lung cancer progression. Gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, has shown anti-proliferative effects in EGFR-mutated NSCLC cells and an inhibitory effect on migration and invasion of NSCLC cells to other organs. In this study, we evaluated the combinatorial treatment effect of cilengitide, a cyclic RGD pentapeptide, on TGF-β1-induced EMT phenotype and invasion. Gefitinib suppressed the expression of TGF-β1-induced mesenchymal markers by inhibiting Smad and non-Smad signaling pathways. Cilengitide enhanced the inhibitory effect of gefitinib on TGF-β1-induced expression of mesenchymal markers, phosphorylation of Smad2/3, and invasion of NSCLC A549 cells. We suggested that the use of cilengitide can improve the efficacy of anti-cancer drugs in combination drug-based chemotherapy. These results provide an improved therapeutic strategy for treating and preventing EMT-related disorders, such as NSCLC, lung fibrosis, cancer metastasis, and relapse.

MicroRNA-217 inhibits the proliferation and invasion, and promotes apoptosis of non-small cell lung cancer cells by targeting sirtuin 1.

Non-small cell lung cancer (NSCLC) is a common malignancy worldwide. MicroRNA (miR)-217 and sirtuin 1 (SIRT1) have been reported to play significant roles in different types of cancer, such as osteosarcoma and prostate cancer; however, the association between miR-217 and SIRT1 in the cell proliferation, apoptosis and invasion of NSCLC remain unknown. Thus, the present study aimed to investigate the roles of miR-217 and SIRT1 in NSCLC. The expression levels of miR-217 and SIRT1 were detected via reverse transcription-quantitative (RT-q)PCR and western blot analyses. The effect of miR-217 on A549 and H1299 cell proliferation, apoptosis and invasion was assessed via the Cell Counting Kit-8, flow cytometry and Transwell assays, respectively. In addition, the association between SIRT1 and miR-217 was predicted using the TargetScan database, and verified via the dual-luciferase reporter assay, and RT-qPCR and western blot analyses. The results demonstrated that miR-217 expression was significantly downregulated, while SIRT1 expression was significantly upregulated in A549 and H1299 cells compared with the human bronchial epithelial cells. Furthermore, transfection with miR-217 mimic significantly inhibited A549 and H1299 cell proliferation and invasion, and induced A549 and H1299 cell apoptosis. The results of the dual-luciferase reporter assay and western blot analysis confirmed that SIRT1 is a target gene of miR-217. In addition, miR-217 inhibited the activation of AMP-activated protein kinase (AMPK) and mTOR signaling. Taken together, the results of the present study suggest that miR-217 inhibits A549 and H1299 cell proliferation and invasion, and induces A549 and H1299 cell apoptosis by targeting SIRT1 and inactivating the SIRT1-mediated AMPK/mTOR signaling pathway. Thus, miR-217 may be used as a potential therapeutic target for the treatment of patients with NSCLC.

Ectodermal‑neural cortex 1 affects the biological function of lung cancer through the MAPK pathway.

Ectodermal-neural cortex 1 (ENC1), a highly expressed protein in lung cancer tissues, was identified from the Cancer Genome Atlas (TCGA) database. The objective of the present study was to examine the effects of ENC1 on the biological functions of lung cancer cells. For this purpose, the expression of ENC1 was examined by RT-qPCR to compare mRNA expression levels between 28 lung cancer tissue samples and para-cancerous tissue samples. The association between ENC1 expression and clinicopathological features was evaluated between the 2 tissue types. Using RT-qPCR and western blot analysis, the expression of ENC1 was investigated in a normal lung cell line (16HBE) and 2 lung cancer cell lines (A549 and H1299). The effect of siRNA targeting ENC1 (si-ENC1) on the proliferation of A549 and H1299 cells was detected by CCK-8 assay at the indicated time points. Transwell assay was used to measure the migration and invasion of A549 and H1299 cells following transfection with siRNA targeting ENC1 (si-ENC1). The expression levels of several proteins related to migration and invasion were examined by western blot analysis. A mouse model of subcutaneous tumor xenotransplantation was established in nude mice to examine the effects of ENC1 downregulation on cancer cells. The results revealed that the expression of ENC1 in lung cancer tissues and lung cancer cells was significantly higher than that in para-cancerous tissues and non-cancer lung cells, respectively. The knockdown of ENC1 in the A549 and H1299 cells using si-ENC1 significantly decreased cell proliferation, migration and invasion compared with the untransfected cells. The knockdown of ENC1 significantly downregulated the levels of matrix metalloproteinase (MMP)2, MMP9, N-cadherin, p-c-Jun N-terminal kinase (JNK), p-extracellular signal-regulated kinase (ERK) and p-p38. The levels of E-cadherin were upregulated. In the mouse lung tumor model, reduced levels of ENC1 inhibited the growth of lung tumors. On the whole, the present study demonstrates that ENC1 is involved in the proliferation, migration and invasion of lung cancer cells, and may thus be an effective diagnostic target for certain cancers. The inhibition or reduction of ENC1 activity may represent a breakthrough in the treatment of lung cancer.

Effect of Carbon Ion Radiation Induces Bystander Effect on Metastasis of A549 Cells and Metabonomic Correlation Analysis.

ObjectiveTo analyze the effect of carbon ion (12C6+) radiation may induce bystander effect on A549 cell metastasis and metabonomics.MethodsA549 cell was irradiated with carbon ion to establish the clone survival model and the transwell matrix assay was applied to measure the effect of carbon ion on cell viability, migration, and invasion, respectively. Normal human embryonic lung fibroblasts (WI-38) were irradiated with carbon ions of 0 and 2 Gy and then transferred to A549 cell co-culture medium for 24 h. The migration and invasion of A549 cells were detected by the Transwell chamber. The analysis of metabonomic information in transfer medium by liquid phase mass spectrometry (LC-MS), The differential molecules were obtained by principal pomponent analysis (PCA) and the target proteins of significant differences (p = 1.7 × 10−3) obtained by combining with the STICH database. KEGG pathway was used to analyze the enrichment of the target protein pathway.ResultsCompared with 0 Gy, the colony formation, migration, and invasion of A549 cells were significantly inhibited by carbon ion 2 and 4 Gy irradiation, while the inhibitory effect was not significant after 1 Gy irradiation. Compared with 0 Gy, the culture medium 24 h after carbon ion 2 Gy irradiation significantly inhibited the metastasis of tumor cells (p = 0.03). LC-MS analysis showed that 23 differential metabolites were obtained in the cell culture medium 24 h after carbon ion 0 and 2 Gy irradiation (9 up-regulated and 14 down-regulated). Among them, two were up-regulated and two down-regulated (p = 2.9 × 10−3). 41 target proteins were corresponding to these four differential molecules. Through the analysis of the KEGG signal pathway, it was found that these target molecules were mainly enriched in purine metabolism, tyrosine metabolism, cysteine and methionine metabolism, peroxisome, and carbon metabolism. Neuroactive ligand-receptor interaction, calcium signaling pathway, arachidonic acid metabolism, and Fc epsilon RI signaling pathway.ConclusionThe bystander effect induced by 2 Gy carbon ion radiation inhibits the metastasis of tumor cells, which indicates that carbon ions may change the metabolites of irradiated cells, so that it may indirectly affect the metabolism of tumor cell growth microenvironment, thus inhibiting the metastasis of malignant tumor cells.

Red Raspberry Extracts Inhibit A549 Lung Cancer Cell Migration, Invasion, and Epithelial-Mesenchymal Transition Through the Epidermal Growth Factor Receptor/Signal Transducer and Activator of Transcription-3 Signaling Pathway

The incidence and mortality of lung cancer ranks first among all malignant tumors in the world. Because it is relatively asymptomatic at early stages, most patients do not become aware of the disease until it has progressed to an advanced stage. Advanced lung cancer metastasis results in systemic cachexia and effective treatment becomes challenging, leading to poor response and outcome. Therefore, the development of new drugs for the treatment of lung cancer is paramount. In this study, A549 cells were treated with different concentrations of red raspberry extract and the proliferation, migration, and invasion of cells were evaluated. The results indicated that red raspberry extract reduced the proliferation, migration, and invasion of A549 cells. Western blot analysis was used to detect the expression of the cyclin D1, N-cadherin, vimentin, E-cadherin, EGFR, and STAT3 proteins. Treatment with red raspberry extract reduced the expression of cyclin D1, N-cadherin, vimentin, EGFR, and STAT3, whereas the expression of E-cadherin increased. Following transfection of an EGFR overexpression vector into A549 cells, we observed a reduced inhibitory effect of the red raspberry extract on the proliferation, migration, and invasion of A549 cells. In addition, EGFR overexpression abrogated the increased expression of cyclin D1, N-cadherin, vimentin, EGFR, and STAT3 protein expression in A549 cells following extract treatment. In contrast, E-cadherin protein expression was decreased under these treatment conditions. Overall, this study suggests that red raspberry extract may reduce the proliferation, migration, invasion, and epithelialmesenchymal transition of A549 lung cancer cells by inhibiting the activation of the EGFR/STAT3 signaling pathway. These findings may lead to the development of new strategies to treat advanced lung cancer.