Invasion Ability Of A549 Cells Research Articles

Investigating the efficacy and mechanisms of Jinfu'an decoction in treating non-small cell lung cancer using network pharmacology and in vitro and in vivo experiments

Ethnopharmacological relevanceJinfu'an Decoction (JFAD) is a traditional Chinese decoction used in lung cancer treatment to improve patient quality of life and survival. Previous research has established that JFAD has a significant therapeutic effect on non-small cell lung cancer (NSCLC), although the underlying molecular mechanisms have not been largely underexplored. Aim of the studyWe used network pharmacology to identify the putative active ingredients of JFAD and conducted experimental studies to determine the potential molecular mechanism of JFAD in NSCLC treatment. Materials and methodsThe herbal components in JFAD-containing serum were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and targets associated with the anti-lung cancer metastasis effects of JFAD were retrieved from various databases. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Next, the protein-protein interactions network and the "JFAD-Chemical Component-Target-KEGG Pathway" network were constructed. The network pharmacology findings were confirmed by in vitro and in vivo experiments. In vitro experiments were conducted to assess cell viability by CCK8 assay, cell cycle analysis by propidium iodide (PI) assay, and migration and invasion ability of cells by the transwell assay. In vivo experiments were performed to assess the efficacy of JFAD on the tumor by observing the growth of transplanted tumor models in nude mice and evaluated by in vivo bioluminescence imaging. Moreover, we assessed the effect of JFAD on the PI3K/Akt signaling pathway and proteins of Lumican, p120ctn, and specific RhoGTP enzyme family members (RhoA, Rac1, and RhoC) by Western Blot and immunohistochemistry. Results32 herbal components were identified in the JFAD-containing serum, which potentially acted on 229 targets related to lung cancer metastasis. Network pharmacology results suggested that JFAD may treat lung cancer metastasis by targeting the PI3K/Akt pathway via regulating multiple core targets. Our experiments showed that JFAD suppressed the proliferation of A549 cells in vitro, induced cell cycle arrest, and reduced the migration and invasion ability of A549 cells. Our in vivo study revealed that JFAD inhibited tumor growth in a nude mouse model. Additionally, we found that JFAD could downregulate the expression of the PI3K/Akt pathway and affect the expression of Lumican, p120ctn, and specific RhoGTPase family members. ConclusionsIn conclusion, through network pharmacology, we have unveiled the underlying mechanisms that link the various components, targets, and pathways influenced by JFAD in the context of lung cancer metastasis. Our experimental results suggest that the oncostatic effects of JFAD may be achieved by upregulating the expression of Lumican/p120ctn and downregulating the levels of specific RhoGTPase family members, which in turn block the PI3K/Akt signaling pathway.

MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34.

Lung adenocarcinoma (LUAD) represents the most prevalent subtype of lung cancer and typically has high incidence and fatality rates. Motor neuron and pancreas homeobox 1 (MNX1) and coiled-coil domain-containing 34 (CCDC34) serve as oncogenes in multiple types of cancer. However, their role in LUAD remains to be elucidated. In the present study, bioinformatics analysis and LUAD cell lines were adopted to examine the expression of MNX1 and CCDC34. The proliferation, migration and invasion abilities of A549 cells were determined using Cell Counting Kit-8, colony formation, wound-healing and Transwell assay, and flow cytometry was conducted to assess cell cycle distribution and apoptosis. The interaction between MNX1 and CCDC34 was verified by luciferase reporter and chromatin immunoprecipitation assays. In addition, an in vivo animal model of LUAD was established for validation. The results demonstrated that both MNX1 and CCDC34 were upregulated in LUAD cell lines. MNX1 knockdown significantly suppressed cell proliferation, migration and invasion, hindered cell cycle progression and promoted cell apoptosis in vitro and inhibited tumor growth in vivo. However, the antitumor effect of MNX1 knockdown was weakened by simultaneous CCDC34 overexpression in vitro. In terms of mechanism, MNX1 was demonstrated to directly bind to the CCDC34 promoter and transcriptionally activate CCDC34 expression. In conclusion, the present study highlighted a critical role of the MNX1/CCDC34 axis in regulating LUAD progression, providing novel therapeutic targets for LUAD treatment.

Streptococcus pneumoniae promotes migration and invasion of A549 cells in vitro by activating mTORC2/AKT through up-regulation of DDIT4 expression.

Dysbiosis of the lower airway flora is associated with lung cancer, of which the relationship between Streptococcus, especially pathogenic Streptococcus pneumoniae (S. pneumoniae), and the progression of lung cancer are unclear. Bronchoalveolar lavage fluid (BALF) samples were prospectively collected from patients with pulmonary nodules during diagnostic bronchoscopy, and finally included 70 patients diagnosed with primary lung cancer and 20 patients with benign pulmonary nodules as the disease control group. The differential flora was screened by 16S ribosomal RNA (rRNA) gene amplicon sequencing. An in vitro infection model of lung adenocarcinoma (LUAD) cells exposed to S.pneumoniae was established to observe its effects on cell migration and invasion ability. Exploring the molecular mechanisms downstream of DDIT4 through its loss- and gain-of-function experiments. 16S rRNA sequencing analysis showed that the abundance of Streptococcus in the lower airway flora of lung cancer patients was significantly increased. After exposure to S. pneumoniae, A549 and H1299 cells significantly enhanced their cell migration and invasion ability. The results of DDIT4 loss- and gain-of-function experiments in A549 cells suggest that up-regulation of DDIT4 activates the mTORC2/Akt signaling pathway, thereby enhancing the migration and invasion of A549 cells while not affecting mTORC1. Immunofluorescence (IF) and fluorescence in situ hybridization (FISH) showed that S. pneumoniae was enriched in LUAD tissues, and DDIT4 expression was significantly higher in cancer tissues than in non-cancerous tissues. The increased expression of DDIT4 was also related to the poor prognosis of patients with LUAD. The data provided by this study show that S. pneumoniae enriched in the lower airway of patients with lung cancer can up-regulate DDIT4 expression and subsequently activate the mTORC2/AKT signal pathway, thereby increasing the migration and invasion abilities of A549 cells. Our study provides a potential new mechanism for targeted therapy of LUAD.

MiR-367-3p Regulates Cells Proliferation and Invasion in NSCLC by Targeting ZEB2

microRNAs play an important role in the development and biological phenotype of lung cancer. The present study was to investigate miR-367-3p level in non-small cell lung cancer (NSCLC) patients and its biological function of NSCLC cells. Twenty-two patients with NSCLC (13 cases of adenocarcinoma and 9 cases of squamous carcinoma) admitted to our hospital and treated by surgery were included. During the operation, cancer tissue, paracancerous tissue and 5 mL peripheral blood were collected. Meanwhile, 22 healthy controls were selected and 5 mL peripheral blood was taken. Real-time PCR was applied to detected the expression of miR-367-3p in cancer tissues, peripheral blood of patients with NSCLC and healthy controls. miR-367-3p was detected in lung cancer cell lines (A549) and normal bronchial epithelial cells (BEAS-2B). The proliferation and invasion ability of A549 cells before and after infection were detected by MTT and Transwell assay after transfection with exogenous miR-367-3p. The downstream target gene of miR-367-3p was analyzed by bioinformatics. Zinc finger E-box binding homeobox 2 (ZEB2) was detected by Real-time PCR and Western blot. miR-367-3p in cancer tissues of 22 NSCLC patients was lower than corresponding normal tissues (P<0.05), and the serum miR-367-3p level in healthy subjects was higher than NSCLC subjects (P<0.05). The area under the receiver operating characteristic (ROC) curve of NSCLC was 0.95 (95%CI: 0.89-1.00) and 0.85 (95%CI: 0.74-0.97) respectively; The proliferation and migration ability of lung cancer cell line A549 transfected with exogenous miR-367-3p decreased significantly (P<0.05); Bioinformatics predicted that the downstream target of miR-367-3p was ZEB2 and up-regulating miR-367-3p expression, ZEB2 gene was decreased (P<0.05). The Cancer Genome Atlas (TCGA) data analysis showed that there was no significant difference in overall survival (OS) and disease free survival (DFS) between ZEB2 high expression group and low expression group (P>0.05). ZEB2 expression was positively correlated with infiltration of B lymphocytes (r=0.32, P<005), CD8⁺ T cells (r=0.44, P<005), CD4⁺ T cells (r=0.46, P<005), macrophages (r=0.65, P<005), neutrophils (r=0.73, P<005) and dendritic cells (r=0.71, P<005) in NSCLC. The expression of miR-367-3p is down regulated in NSCLC patients and participates in the biological process of proliferation and invasion of NSCLC by targeting ZEB2 gene.

Mechanism of lncRNA SNHG19 miR-299-5p MAPK6 signaling axis promoting metastasis of non-small cell lung cancer cells

Abstract Objective The aim of this study was to explore the mechanism behind lncRNA small nucleolar RNA host gene 19 (lncRNA SNHG19)/microrNA-299-5P (miR-299-5p)/mitogen-activated protein kinase 6 (MAPK6) signaling axis promoting metastasis of non-small cell lung cancer (NSCLC). Methods To analyze the abnormal expression of lncRNAs in NSCLC, 50 surgically resected NSCLC and adjacent tissue samples were collected from August 2021 to August 2022. The mRNA expression levels of lncRNA SNHG19, Mir-299-5p, and MAPK6 were detected by qRT-PCR. The functions of lncRNA SNHG19, Mir-299-5p and MAPK6 were investigated by CCK-8, clone formation, EdU, scratch, Transwell western blotting (WB)and in vivo xenograft assay. RNA fluorescence in-situ hybridization (FISH), RNA pull-down, dual luciferase reporter, and RNA co-immunoprecipitation assays were used to explore the mechanism of action between lncRNA SNHG19, miR-299-5p, and MAPK6. Results High expression of lncRNA SNHG19 was correlated with poor prognosis, tumor size, lymph node metastasis, and TNM stage in NSCLC patients (P &lt; 0.05). Cell function experiments showed that lncRNA SNHG19 could improve the proliferation, clone formation, migration, and invasion ability of A549 cells both in vitro and in vivo (all P &lt; 0.05) and increased the relative expression levels of vimentin and MAPK6 (P &lt; 0.05). The relative expression level of E-cadherin was decreased (P &lt; 0.05). lncRNA SNHG19 can interact with Mir-299-5p and regulate the expression level of MAPK6. Conclusion lncRNA SNHG19 is upregulated in NSCLC tissues and cells, and its high expression is associated with tumor progression and poor survival. Moreover, it can act as a molecular sponge for Mir-299-5p to regulate MAPK6 expression and promote the proliferation and metastasis of A549 cells.

Lobetyolin suppressed lung cancer in a mouse model by inhibiting epithelial-mesenchymal transition.

Traditional Chinese medicines are gaining more attention as promising adjuvant agents for conventional chemotherapy. Recent studies have shown that lobetyolin (LBT) is one of the main bioactive compounds of traditional Chinese medicines and it exhibits anticancer activity in several types of cancer. Therefore, this study aimed to investigate the mechanism by which LBT inhibits lung cancer. A549 human lung cancer cells were treated with LBT. In addition, A549 cells were injected into Balc/b nude mice to establish model of lung cancer. The mice were treated with cisplatin (DDP) or LBT alone or in combination, and tumor growth was monitored. Protein levels of E-cadherin, vimentin and matrix metalloproteinase 9 (MMP9) were detected. We found that the combination of LBT and DDP showed stronger effect to inhibit the proliferation of A549 cells compared to LBT or DDP treatment alone. Wound healing assay showed that the ratio of wound healing was significantly lower in LBT group and DDP group and was the lowest in LBT+DDP group. Transwell invasion assay showed that the invasion ability of A549 cells was the weakest in LBT+DDP group. Protein levels of E-cadherin were the highest while those of vimentin and MMP9 were the lowest in A549 cells treated with LBT+DDP. Nude mouse xenograft tumor model showed that the combination of LBT with DDP had the highest efficacy to inhibit the growth of lung cancer, and tumor tissues of mice treated with LBT+DDP had the lowest expression of vimentin and MMP9 and the highest expression of E-cadherin. In conclusion, LBT significantly enhances the efficacy of chemotherapy on lung cancer, and the mechanism may be related to the inhibition of epithelial-mesenchymal transition.

Regulation of Epithelial-Mesenchymal Transition of A549 Cells by Prostaglandin D2.

Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-β1 and TGFβ type I receptor (TGFβRI), and protein expressionwas assessed by immunoblotting and immunofluorescence. Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-β1 expression, and silencing of TGF-β1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFβRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

The mechanism of LncRNA01977 in lung adenocarcinoma through the SDF-1/CXCR4 pathway.

BackgroundA significant correlation has been identified between lncRNA and tumor cell resistance, diagnosis, and prognosis. Although mRNA studies have dominated the field of non-coding RNA biology in tumorigenesis in recent years, long-chain non-coding RNA (the biological function) has also attracted increasing attention. However, the lncRNA associated with lung adenocarcinoma (LUAD) remains unexplored. This study used bioinformatics analysis to screen and identify LncRNA01977 as a key oncogenic driver of LUAD.MethodsThe experiment was divided into blank serum group (normal serum medium) and lung compound low, medium and high dose groups (5%, 10%, 15% and 15% lung compound drug serum medium, respectively). Transwell invasion ability of A549 cells was detected, and Western blot tested A549 cells SDF-1 specific receptor CXCR4, and CXCR4 gene expression in A549 cells were determined by reverse transcription–polymerase chain reaction (RT-PCR). In addition, western blotting, MTT proliferation test, colony formation test and apoptosis detection techniques were used to explore the mechanism of LncRNA01977's effects on LUAD.ResultsIn vitro assays demonstrated that LncRNA01977 can significantly promote the progression of LUAD and that stromal cells in tumor microenvironment secrete chemokine CXCL12, also known as stromal derived factor-1 (SDF-1), and its receptor CXCR4 is low expressed in normal tissues and high expressed in LUAD tissues. Lung cancer patients with high expression of CXCR4 are more prone to metastasis.ConclusionsLncRNA01977 can be used as a new prognostic indicator of LUAD, and can help patients to find more effective target treatment options for LUAD.

The TCM Prescription Yi-Fei-Jie-Du-Tang Inhibit Invasive Migration and EMT of Lung Cancer Cells by Activating Autophagy.

Yi-Fei-Jie-Du-Tang (YFJDT) is a traditional Chinese medicine formulation. Our previous studies have demonstrated that YFJDT can be used to treat non-small-cell lung cancer (NSCLC), but its protective effect against NSCLC and its mechanisms remain unclear. In the present study, we evaluated the protective effects and potential mechanisms of YFJDT on a tumor-bearing mouse lung cancer model and A549 cell model. Tumor-bearing mice and A549 cells were treated with YFJDT, tumors were measured during the experiment, and tumor tissues and cell supernatants were collected at the end of the experiment to assess the levels of autophagy and epithelial-mesenchymal transition (EMT)-related proteins. The results showed that YFJDT treatment reduced tumor volume and mass, increased the expression of the autophagy marker LC3, and inhibited EMT-related proteins compared with the model group. Cell survival was reduced in the YFJDT-treated groups compared with the model group, and YFJDT also reduced the migration and invasion ability of A549 cells in a dose-dependent manner. Western blotting detected that YFJDT also upregulated FAT4 in the tumor tissue and A549 cells and downregulated the expression of vimentin. Meanwhile, apoptosis in both tissues and cells was greatly increased with treatment of YFJDT. We further interfered with FAT4 expression in cells and found that the inhibitory effect of YFJDT on EMT was reversed, indicating that YFJDT affects EMT by regulating FAT4 expression. Taken together, results of this study suggested that the inhibitory effect of YFJDT on EMT in lung cancer tumors is through upregulating FAT4, promoting autophagy, and thus inhibiting EMT in cancer cells.

Chemical composition, anticancer activities and related mechanisms of the essential oil from Alpinia coriandriodora rhizome

Alpinia coriandriodora is widely cultivated as a medicinal and edible Zingiberaceae plant in Guangxi, China. Since essential oils from Zingiberaceae plants can be used as a potential source of anticancer drugs, this study was designed to identify the chemical composition of A. coriandriodora rhizome essential oil (EO) and firstly elucidate its anticancer activities and related mechanisms. The GC-FID/MS results indicated that the most predominant component of EO was (E)-2-decenal (56.3%). Based on the MTT results, EO exhibited comparable cytotoxicity to A549 cells and lower cytotoxicity to non-cancerous cells compared with the positive control (elemene injection). For related mechanism studies, EO inhibited the A549 cells proliferation, resulting from inducing cell cycle arrest at G1 phase via downregulation of cyclin E2-CDK2 and cyclin D3-CDK4/CDK6 complexes and upregulation of p21. Meanwhile, it induced the apoptosis through mitochondrial pathway with the successive enhancement in the Bax/Bcl-2 ratio, Δψm decline, cyt c release, activation of caspase-9 and caspase-3, and PARP cleavage. Moreover, it inhibited the migration and invasion ability of A549 cells via downregulation of MMP-2 and N-cadherin, as well as upregulation of E-cadherin. Hence, EO has outstanding anticancer properties in vitro, and this fact encourages further studies using in vivo models.

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。

Downregulation of CENPK suppresses lung adenocarcinoma by regulating EMT

IntroductionCentromere protein K (CENPK) plays a key role in regulating the assembly and function of centromeres, which in turn can affect the occurrence and development of various tumors. However, little is known about the biological function of CENPK in lung adenocarcinoma (LUAD).Material and methodsWe evaluated the relationship between CENPK expression and the clinicopathological characteristics of LUAD patients via a bioinformatics method based on data taken from the TCGA database. Then, the role of CENPK in LUAD was investigated in vitro by using the human LUAD cell line A549. Cell Counting Kit-8 and colony formation assays were used detect the cell proliferation ability. Wound healing and transwell assays were used to detect the cell migration and invasion ability. Epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, Snail and vimentin) were measured by western blotting. In addition, a xenograft experiment was used to explore the function of CENPK in vivo.ResultsCENPK is highly expressed in LUAD tissues and cell lines. Moreover, high expression of CENPK in LUAD is significantly associated with stage, lymph node involvement and poor survival. CENPK knockdown significantly decreases the proliferation, migration and invasion ability of A549 cells by EMT both in vitro and in vivo.ConclusionsCENPK is highly expressed in LUAD and leads to a poor prognosis. CENPK knockdown can suppress the proliferation, migration, and invasion of lung cancer cells via EMT, which may be a valid target for treatment.

MiR-758-3p inhibits the proliferation and invasion of non-small cell lung cancer cells by targeting NUSAP1

Objective: To investigate the biological function of miR-758-3p and the underlying mechanism in non-small cell lung cancer (NSCLC). Methods: miR-758-3p mimics was transfected to A549 NSCLC cells, miRNA control was used as a negative control, cells transfected with nucleolar and spindle associated protein 1 (NUSAP1)-overexpression vector indicated as NUSAP1 group, cells co-transfected with miR-758-3p mimics and NUSAP1-overexpression vector indicated as miR-758-3p mimics+ NUSAP1 group. The effects of miR-758-3p on the proliferation, migration and invasion abilities of A549 cells were detected by cell counting kit-8 (CCK-8) and Transwell assays, respectively. Bioinformatics and dual luciferase reporter assay were used to predict and verify the target gene of miR-758-3p. Results: The expressions of miR-758-3p and NUSAP1 in A549 cells were significantly up-regulated at 24 hours after transfection with miR-758-3p mimics (P<0.05). Compared with the miRNA control group (1.15±0.06), the OD value of miR-758-3p mimic group (0.78±0.06) was significantly decreased at 72 hours after transfection (P<0.05). The number of migrated cells of miR-758-3p mimic group (119.04±11.49) was significantly lower than that of the control group (271.38±19.05) (P<0.05). The number of invaded cells of miR-758-3p mimic group (71.33±5.36) was significantly lower than the control group (164.30±8.11) (P<0.05). The result of dual-luciferase reporter assay showed that NUSAP1 was a direct target of miR-758-3p. Moreover, up-regulation of NUSAP1 abolished the inhibitory effects of miR-758-3p on the proliferation, migration and invasion abilities of A549 cells. Conclusion: miR-758-3p inhibits the proliferation, migration and invasion of NSCLC cells by targeting NUSAP1.

Correlation between Plasma Cellular Retinoic Acid-Binding Protein 2 and Proliferation, Migration, and Invasion of Non-Small-Cell Lung Cancer Cells.

Our primary aim of the current study was to explore the correlation between plasma CRABP2 and migration, proliferation and invasion of non-small cell lung cancer (NSCLC) cells. Human lung cancer cell line A549 was used in the present study, which was cultured in 6-well plates (1 × 106 cells/well) and then transfected with pcDNA-CRABP2 and pcDNA, siRNA with the use of Lipofectamine 2000 based on the manufacturer's protocol. The expression of CRABP2 mRNA was detected through real-time PCR. Proliferation was further detected using MTT assays, and apoptosis was monitored and recorded with the application of flow cytometry. The expression of E-cadherin, MMP9, vimentin and related pathway proteins was detected by Western blotting assays. Transwell assays and cell scratch assays were utilized for the detection of migration and invasion ability of A549 cells. RT-PCR results showed The CRABP2 mRNA transcript levels in the CRABP2 overexpression group were higher when comparing those of the empty vector group (P < 0.05). By MTT assays, CRABP2 overexpression promoted cellular proliferation, while CRABP2 downregulation inhibited cellular proliferation. CRABP2 overexpression inhibited cell apoptosis and promoted cellular proliferation. The number of TUNEL staining positive cells was the lowest in the CRABP2 overexpression group, and the siRNA transfection group had increased apoptosis. CRABP2 downregulation reduced EMT in cells and cell migration and invasion reflected from western blotting results and cell migration and invasion assay results, respectively. Inhibition of plasma CRABP2 expression offers the potential in terms of reducing the expression of MAPKs and proteins in the NF-κB pathway and inhibiting the proliferation and migration of NSCLC cells, which is ideally suited for further treatment for NSCLC.

RPB5-mediating protein promotes the progression of non-small cell lung cancer by regulating the proliferation and invasion.

BackgroundThis study aimed to investigate the relationship between RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) and clinicopathological characteristics of non-small cell lung cancer (NSCLC) patients by measuring the expression level of RMP in human NSCLC tissues and cell lines. At the same time, we studied the impact of RMP on the biological function of cancer, providing strong support for gene targeted therapy of NSCLC.MethodsReal-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to determine the expression levels of messenger (m)RNA and protein in NSCLC cell lines and tissues. Cell counting kit 8 (CCK8) assay and flow cytometry were selected to detect cell proliferation, cycle and apoptosis. The wound healing assay was chosen to detect the migration and invasion ability of cells. The xenograft model was performed to study the function of RMP in vivo. Immunohistochemical (IHC) staining showed the levels of RMP, Bcl-2, Bax and caspase-3.ResultsFirst, mRNA and protein levels of RMP were relatively overexpressed in NSCLC cells. Compared with the corresponding normal tissues, the mRNA and protein levels of RMP were significantly higher in human NSCLC tissues. Concurrently, we found that the expression of RMP was related to the status of lymph nodes (LNs) in cancer tissues and T stage. Then, RMP overexpression promoted the proliferation of A549. At the same time, RMP provided A549 cells the ability to resist chemotherapy and radiotherapy; when A549 cells were treated with gefitinib and radiation, RMP reduced apoptosis. We also found that RMP can protect A549 from G2 block caused by radiation. Over-irradiated RMP-overexpressed A549 cells had lower Bcl2-associated X protein (Bax) levels and higher B-cell lymphoma 2 (Bcl-2) levels. The migration and invasion ability of A549 cells was increased by RMP. Finally, RMP can promote tumor growth by increasing Bcl-2 levels and decreasing Bax and caspase-3 levels in the xenograft model.ConclusionsThere is potential for RMP to develop into a diagnostic and therapeutic target for NSCLC.

Liver Kinase B1 (LKB1) Regulates Proliferation and Apoptosis of Non-Small Cell Lung Cancer A549 Cells via Targeting ERK Signaling Pathway

ObjectiveTo study the effect and potential mechanism of LKB1 on non-small cell lung cancer (NSCLC) A549 cells.Material and MethodsA549 cells were divided into control group, LKB1 negative control (NC) group, LKB1 group, ERK inhibitor group and LKB1 + ERK activator group. Cell proliferation and apoptosis were detected by cell counting kit (CCK-8) assay and flow cytometry, respectively. Transwell assay was used to analyze the invasion ability of A549 cells. The expression of apoptosis and ERK signaling pathway-related proteins were studied by Western blot. Furthermore, a nude mouse xenograft model was constructed and treated with LKB1, ERK inhibitor and activator, respectively. The tumor volume and tumor weight were measured. Immunohistochemistry was used to test the expression of Ki-67 protein in tumor tissues, and TUNEL staining was used to test the apoptosis. Moreover, Western blot was used to detect ERK signaling pathway-related proteins in tumor tissues.ResultsCompared with control and NC groups, cell proliferation and invasion were inhibited in ERK inhibitor and LKB1 groups, while apoptosis and apoptosis-related proteins were increased (p < 0.05). Further study showed that ERK activator can reverse the effect of LKB1 in A549 cells. In nude mice, ERK inhibitor and LKB1 can reduce cell tumorigenicity and inhibit proliferation. Apoptosis was increased by ERK inhibitor and LKB1 treatment. Western blot showed that LKB1 and ERK inhibitor could reduce the protein expression of p-ERK1/2. However, the indicators above were the opposite in the ERK activator group.ConclusionLKB1 overexpression can inhibit proliferation and promote apoptosis of NSCLC A549 cells, and its mechanism may be related to inhibition of the ERK signaling pathway.

DEHP-elicited small extracellular vesicles miR-26a-5p promoted metastasis in nearby normal A549cells.

Small extracellular vesicles (sEV) are small lipid bilayer particles released by cells. sEV have been shown to play critical roles in intercellular communication. Di (2-ethylhexyl) phthalate (DEHP), widely used as plasticizers, has been detected in the environment and human beings. DEHP was found to exist in the air particles and showed pulmonary toxicity. However, there's little knowledge about the role of sEV in mediating the toxicity of DEHP-induced lung toxicity. We hypothesized that sEV mediated the toxicity of DEHP through their cargo. To validate this, lung epithelial cells (A549) were exposed to various concentrations (0, 0.2, 2 and 20μM) of DEHP for 48h. sEV extracted from DEHP-exposed A549cells were cultured with unexposed A549cells. Results showed that DEHP induced the epithelial-mesenchymal transition (EMT) and promoted the migration and invasion ability of A549cells. The number of released sEV significantly increased in the culture media in DEHP-exposed groups compared to unexposed groups. The sEV can enter the unexposed A549cells and enhance its EMT and the ability of migration and invasion. Treatment with GW4869 in DEHP-exposed A549cells almost blocked the effects of DEHP-elicited sEV in normal A549cells. Sequencing and functional analysis showed that the enrichment of significantly differentially expressed sEV miRNAs were related to tumor etiology. MiR-26a-5p was significantly enriched in DEHP-elicited sEV. Inhibition of miR-26a-5p in DEHP-exposed cells led to the downregulation of miR-26a-5p in sEV, and thus abolished the effects of DEHP-elicited sEV in normal A549cells, whereas overexpression of miR-26a-5p restored the effects. The transcription factors twist is one of the downstream targets in the effects of sEV-miR-26a-5p on EMT process. In all, our results showed that DEHP exposure promoted the secretion of miR-26a-5p in sEV, which subsequently enhanced the EMT, migration and invasion ability in neighboring normal cells via the twist.

Inhibition of formyl peptide receptor 1 activity suppresses tumorigenicity in vivo and attenuates the invasion and migration of lung adenocarcinoma cells under hypoxic conditions in vitro.

BackgroundTumor hypoxia has been widely reported to promote metastasis. However, the molecular mechanisms underlying metastasis-associated hypoxia remain unclear. Formyl peptide receptor 1 (FPR1) has been reported to be highly expressed under hypoxic conditions. This study aimed to explore the role of FPR1 in tumor cells under hypoxic conditions.MethodsThe expressions of FPR1 and hypoxia-inducible factor 1α (HIF-1α) in A549 cells under hypoxic conditions were detected using western blot. The expression of FPR1 in A549 cells under hypoxic conditions was suppressed using the FPR1 antagonist Boc2. Wound-healing and Transwell assays were performed to investigate the migration and invasion of cells. Furthermore, the tumorigenicity of A549 cells was evaluated by constructing a hypoxic mouse model of lung adenocarcinoma. The expression levels of HIF-1α and FPR1 in tumors were measured by real-time polymerase chain reaction (PCR) and western blot.ResultsThe expression levels of FPR1 and HIF-1α increased in a time-dependent manner after exposure to hypoxic conditions. Wound-healing and Transwell assays showed that hypoxia promoted the migration and invasion abilities of A549 cells, whereas downregulation of FPR1 blocked the effects of hypoxia on A549 cells. Our in vivo results demonstrated that the tumor volumes and weights of mice exposed to hypoxic conditions were significantly higher than those of untreated mice. Furthermore, the downregulation of FPR1 blocked the effects of hypoxia in the mice. Meanwhile, the expressions of HIF-1α and FPR1 at the protein and mRNA levels were increased after hypoxic exposure, whereas FPR1 antagonist Boc2 suppressed the effect of hypoxia on the expression of FPR1.ConclusionsOur results suggest that FPR1 could be a therapeutic target for suppressing the invasion and tumorigenicity of lung adenocarcinoma cells.

MiR-92b inhibits proliferation and invasion of lung cancer by targeting EZH2.

To verify that miR-92b inhibits proliferation and invasion of lung cancer by targeting EZH2. The expression levels of miR-92b and EZH2 in human bronchial epithelial cell line BEAS-2B and human lung cancer cell line (A549, NCI-H23, NCI-H358, NCI-H1975, PC-9) were detected, and miR-92b mimic, sh-EZH2 expression vector, and plasmid blank vector (blank group) were constructed. Blank group, miR-92b mimic, miR-92b mimic+sh-EZH2 group (combined group) were set up, MTT and transwell were used to detect the proliferation and invasion ability of A549 and NCI-H23 cells, and fluorescein report verified the regulatory relationship of miR-92b to EZH2. The expression level of miR-92b in A549, NCI-H23, NCI-H358, NCI-H1975, and PC-9 cells was lower than that in BEAS-2B cells (p<0.05). The expression level of EZH2 was higher than that of BEAS-2B cells (p<0.05). A549 and NCI-H23 cells were selected for transfection. After that, the expression level of miR-92 in miR-92b mimic, combined group A549 and NCI-H23 cells was higher than that in blank group (p<0.05), and miR-92b mimic had no difference with joint group (p>0.05). The expression level of EZH2 in cells of miR-92b mimic, blank group A549, and NCI-H23 was lower than that of combined group (p<0.05), and miR-92b mimic was lower than that of blank group (p<0.05). After the overexpression of miR-92b, pmirGLO-EZH2-3'UT Wt luciferase activity decreased significantly (p<0.05) but had no effect on pmirGLO-EZH2-3'UTR Mut Luciferase activity (p>0.05). Cell proliferation ability and invasion ability of A549 cells and NCI-H23 cells in miR-92b mimic group were lower than those in blank group (p<0.05), while those in combined group were higher than those in miR-92b mimic group (p<0.05). MiR-92b inhibits proliferation and invasion of lung cancer cells through targeted inhibition of EZH2, which is a potential target for future treatment of lung cancer.

The expression and the role of PIWI like RNA-mediated gene silencing 3 (PIWIL3) in lung cell line.

BackgroundTo determine the expression and role of PIWI-like RNA-mediated gene silencing 3 (PIWIL3) in human lung cancer.MethodsImmunohistochemistry was performed to measure the expression of PIWIL3 in 30 of pairs lung cancer and corresponding paracancer tissues. Quantitative PCR (qPCR) was conducted to analyze the expression of PIWIL3 in four breast cancer cell lines. siRNA was used to silence PIWIL3 in the lung cancer cells (A549). Methylthiazoletetrazolium (MTT) proliferation assay and colony formation assay were conducted to detect the growth of A549 cells. Fluorescence-activated cell sorting (FACS) was performed to measure the apoptosis of A549 cells. Transwell migration assay and wound scratch healing assay were used to analyze the migration and invasion ability of A549 cells. SPSS 20.0 was used to analyze the data.ResultsThe expression of PIWIL3 protein was higher in tumor tissues than that in paracancer tissues. In addition, PIWIL3 mRNA was highly expressed in all four lung cancer cell lines. Furthermore, RPS15A knockdown significantly suppressed cell proliferation (P<0.01), induced apoptosis (P<0.01) and inhibited metastasis (P<0.01) of A549 cells.ConclusionsPIWIL3 was highly expressed in lung cancer tissues and could promote the progression of lung cancer.