Comprehensive analysis of Epha10 as a predictor of clinical prognosis and immune checkpoint therapy efficacy in non-small cell lung cancer.

The EphA family belongs to a large group of membrane receptor tyrosine kinases. Emerging evidence indicates that the EphA family participates in tumour occurrence and progression. Nonetheless, the expression patterns and prognostic values of the nine EphAs in non-small cell lung cancer (NSCLC) have rarely been studied before. In the current study, we comprehensively analysed the expression and prognostic role of EphA family members by different means. The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis databases were used to investigate the expression of EphAs in NSCLC. The cBioPortal database was applied to analyse the prognostic values and genetic mutations of EphAs.We discovered that the expression of EphA10 was significantly higher in NSCLC tissues than in adjacent noncancerous tissues, and survival analyses showed that a higher level of EphA10 predicted poor prognosis. Further exploration into the role of EphA10 by ESTIMATE, CIBERSORT, and ssGSEA analysis found that it was also related to immune infiltration and higher expression of targets of ICI targets. In conclusion, this study revealed that among the EphA family members, EphA10 played an oncogenic role and was a promising biomarker for poor prognosis and better immunotherapy response in NSCLC.

LncRNA NEAT1 aggravates lipopolysaccharide-induced acute lung injury by regulating the miR-98-5p/TLR4 axis.

Although long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) was reported to be associated with acute lung injury (ALI), its specific mechanism has not been well studied. Mouse and cell ALI models were constructed by lipopolysaccharide (LPS). Cell viability was evaluated by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide assay. Cell death was evaluated by lactate dehydrogenase release (LDH) detection kit and flow cytometry. The levels of cytokines in lung tissues lysates were detected by quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The expression of apoptosis-related markers was detected by Western blot. The relationship between NEAT1, miR-98-5p, and toll-like receptor 4 (TLR4) was determined by bioinformatics prediction, luciferase reporter assay, and RNA immunoprecipitation (RIP) assay. Rescue experiments were performed to determine the role of NEAT1/miR-98-5p/TLR4 in ALI. NEAT1 was significantly upregulated during ALI both in vitro and in vivo. NEAT1 knockdown efficiently attenuated LPS-induced ALI and reduced LPS-induced elevation of cytokines both in vitro and in vivo. NEAT1 negatively regulated miR-98-5p by directly sponging it, and TLR4 was a target of miR-98-5p. MiR-98-5p inhibition or TLR4 overexpression could obviously attenuate the protective effects of NEAT1 knockdown in LPS-treated A549 cells. Our study demonstrated that NEAT1 knockdown alleviated LPS-induced ALI by targeting the miR-98-5p/TLR4 axis.

HIF-1α promoted vasculogenic mimicry formation in lung adenocarcinoma through NRP1 upregulation in the hypoxic tumor microenvironment.

Neovascularization is a key factor that contributes to tumor metastasis, and vasculogenic mimicry (VM) is an important form of neovascularization found in highly invasive tumors, including lung cancer. Despite the increasing number of studies focusing on VM, the mechanisms underlying VM formation remain unclear. Herein, our study explored the role of the HIF-1α/NRP1 axis in mediating lung adenocarcinoma metastasis and VM formation. HIF-1α, NRP1 expression, and VM in lung adenocarcinoma (LUAD) patient samples were examined by immunohistochemical staining. Quantitative real-time (qRT-PCR), western blot, transwell assay, wound healing assay, and tube formation assay were performed to verify the role of HIF-1α/NRP1 axis in LUAD metastasis and VM formation. ChIP and luciferase reporter assay were used to confirm whether NRP1 is a direct target of HIF-1α. In LUAD tissues, we confirmed a positive relationship between HIF-1α and NRP1 expression. Importantly, high HIF-1α and NRP1 expression and the presence of VM were correlated with poor prognosis. We also found that HIF-1α could induce LUAD cell migration, invasion, and VM formation by regulating NRP1. Moreover, we demonstrated that HIF-1α can directly bind to the NRP1 promoter located between -2009 and -2017 of the promoter. Mechanistically, MMP2, VE-cadherin, and Vimentin expression were affected. HIF-1α plays an important role in inducing lung adenocarcinoma cell metastasis and VM formation via upregulation of NRP1. This study highlights the potential therapeutic value of targeting NRP1 for suppressing lung adenocarcinoma metastasis and progression.

A Novel Long Non-coding RNA, MSTRG.51053.2 Regulates Cisplatin Resistance by Sponging the miR-432-5p in Non-small Cell Lung Cancer Cells.

Objective: The aim of this study was to investigate the molecular mechanisms underlying cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC) cells by constructing a competing endogenous RNA (ceRNA) network. Methods: The gene expression profiles of human lung adenocarcinoma DDP-resistant cell line (A549/DDP) and its progenitor (A549) were comparatively evaluated by whole-transcriptome sequencing. The differentially expressed genes (DEGs) were subjected to KEGG pathway analysis. The expression levels of mRNAs involved in several pathways associated with conferring DDP resistance to tumor cells were evaluated. The ceRNA network was constructed based on the mRNA expression levels and the sequencing data of miRNA and lncRNA. Several ceRNA regulatory relationships were validated. Results: We quantified the expression of 17 genes involved in the six pathways by quantitative real-time polymerase chain reaction (qRT-PCR). The differential protein expression levels of eight genes were quantified by western blotting. Western blot analysis revealed six differentially expressed proteins (MGST1, MGST3, ABCG2, FXYD2, ALDH3A1, and GST-ω1). Among the genes encoding these six proteins, ABCG2, ALDH3A1, MGST3, and FXYD2 exhibited interaction with 8 lncRNAs and 4 miRNAs in the ceRNA regulatory network. The expression levels of these lncRNAs and miRNAs were quantified in cells; among these, 6 lncRNAs and 4 miRNAs exhibited differential expression between A549/DDP and A549 groups, which were used to construct a ceRNA network. The ceRNA regulatory network of MSTRG51053.2-miR-432-5p-MGST3 was validated by luciferase reporter assay. Conclusion: The MSTRG51053.2 lncRNA may function as a ceRNA for miR-432-5p to regulate the DDP resistance in NSCLC. The MGST1, MGST3, GST-ω1, and ABCG2 mRNAs, miR-432-5p and miR-665 miRNAs, and MSTRG51053.2 and PPAN lncRNAs can serve as potential DDP drug targets to reverse DDP resistance in NSCLC.

Neuropilin 1 modulates TGF­ß1­induced epithelial­mesenchymal transition in non­small cell lung cancer.

Previously, the authors reported that neuropilin­1 (NRP1) was significantly increased and acted as a vital promoter in the metastasis of non­small cell lung cancer (NSCLC). However, the regulatory mechanism of NRP1 in NSCLC cell migration and invasion remained unclear. The present study aimed to explore the regulatory mechanism of NRP1 in the transforming growth factor­ß (TGF­ß) 1­induced migration and invasion of NSCLC cells. The expression level of NRP1 was determined by RT­qPCR analysis in human tissue samples with or without lymph node metastasis. Transwell assay and wound healing assay were conducted to determine the cell migration. Lentivirus­mediated stable knockdown and overexpression of NRP1 cell lines were constructed. Exogenous TGF­ß1 stimulation, SIS3 treatment, western blot analysis and in vivo metastatic model were utilized to clarify the underlying regulatory mechanisms. The results demonstrated that the expression of NRP1 was increased in metastatic NSCLC tissues. NRP1 promoted NSCLC metastasis in vitro and in vivo. The Transwell assays, wound healing assays and western blot analysis revealed that the knockdown of NRP1 significantly inhibited TGF­ß1­mediated EMT and migratory and invasive capabilities of NSCLC. Furthermore, the overexpression of NRP1 weakened the inhibitory effect of SIS3 on the NSCLC migration and invasion. Co­IP assay revealed that NRP1 interacted with TGFßRII to induce EMT. On the whole, the findings of this study demonstrated that NRP1 was overexpressed in metastatic NSCLC tissues. NRP1 could contributes to TGF­ß1­induced EMT and metastasis in NSCLC by binding with TGFßRII.

Integrative analysis of mRNA and miRNA expression profiles reveals seven potential diagnostic biomarkers for non­small cell lung cancer.

The present study aimed to explore specific molecular targets for the diagnosis and treatment of non­small cell lung cancer (NSCLC). The expression profiles of microRNAs (miRNAs) and mRNAs were downloaded from the GEO (GSE102286 and GSE101929) and TCGA databases. After data preprocessing, differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) in cancer and normal tissues were selected and used to construct a DEM­DEG regulatory network and a protein­protein interaction (PPI) network. The genes and miRNAs in these networks were subjected to functional enrichment and survival analyses. Several key DEMs and DEGs were verified using RT­qPCR, and the results were statistically interpreted using a multivariate logistic regression analysis. In this study, 25 DEMs and 789 DEGs common to all datasets were identified, which were then used for the construction of a DEM­DEG regulatory network and a PPI network module. Survival analyses of 19 DEMs in the DEM­DEG regulatory network and 36 DEGs in the PPI network module revealed that 34 DEGs (including TOP2A, CCNB1, BIRC5, and TTK) and two miRNAs (miR­21­5p and miR­31­5p) were significantly associated with NSCLC prognosis. Moreover, RT­qPCR analysis identified three DEGs and five DEMs that had changes in expression consistent with those observed in the bioinformatic analysis. Finally, a multivariate logistic regression analysis of the data showed that TOP2A, CCNB1, BIRC5, miR­21­5p, miR­193b­3p, miR­210­3p and miR­31­5p could be combined for the diagnosis of NSCLC. In conclusion, TOP2A, CCNB1, BIRC5, miR­21­5p, miR­193b­3p, miR­210­3p and miR­31­5p may therefore serve as important biomarkers and diagnostic targets for NSCLC.

Cytological effects of honokiol treatment and its potential mechanism of action in non-small cell lung cancer.

PURPOSE: In this study, we aimed to explore key micro(mi)RNAs and their potential regulatory mechanisms induced by honokiol treatment in non-small cell lung cancer (NSCLC) cells. METHODS: NSCLC A549 cells were treated with 0 (control) or 45 µM honokiol. Cell proliferation and migration were determined using CCK-8 and transwell assay, respectively, and apoptosis was determined using flow cytometry. RNA-sequencing was performed to detect the transcript expression levels. The differentially expressed miRNAs (DE-miRNAs) between the honokiol group and the control group were screened and analyzed for their functions and pathways. Then, protein-protein interaction (PPI) networks and miRNA-mRNA regulatory networks were constructed. In addition, survival analysis based on the key miRNAs was performed. Finally, the expression of the key miRNAs and their target genes were determined, and their effects on drug sensitivity were validated using their inhibitors. RESULTS: Cell proliferation and migration were inhibited (P < 0.01), and the apoptosis rate was increased (P < 0.01) after honokiol treatment compared to that in the control group. A total of 26 upregulated and 20 downregulated DE-miRNAs were screened. DE-miRNAs were enriched in 10 pathways and 48 biological processes, such as the PI3K/AKT signaling pathway (involving miR-148a-3p). The miRNA-mRNA regulatory networks involved eight upregulated (including miR-148a-3p and let-7c-5p) and seven downregulated miRNAs (including miR-7-5p) and 190 target mRNAs. Survival analysis revealed that let-7c-5p, miR-148a-3p, and miR-148a-5p levels correlated with NSCLC prognosis. The expression of let-7c-5p, miR-148a-3p, and miR-148a-5p was significantly increased and negatively correlated with the expression of their target genes. The cytological effects of honokiol on A549 cells was partly reversed by treatment with the inhibitors of Let-7c-5p and miR-148a-3p. CONCLUSION: Let-7c-5p, miR-148a-3p, miR-148a-5p, and miR-7-5p are favorable indicators of NSCLC patients treated with honokiol.

The regulation of Neuropilin 1 expression by miR-338-3p promotes non-small cell lung cancer via changes in EGFR signaling.

Neuropilin 1 (NRP1) is a transmembrane glycoprotein that acts as a co-receptor for multiple extracellular ligands and typically performs growth-promoting functions in cancer cells. Accumulating evidence indicates that NRP1 is upregulated, and may be an independent predictor of cancer relapse and poor survival, in many cancer types, including non-small cell lung cancer (NSCLC). Recent evidence suggests that NRP1 affects tumour cell viability via the epidermal growth factor receptor (EGFR) and Erb-B2 receptor tyrosine kinase 2 (ErbB2) signalling pathways in venous endothelial cells and in multiple cancer cells. In the present study, we aimed to evaluate the role of NRP1 in NSCLC tumourigenesis and to explore a new post-transcriptional mechanism of NRP1 regulation via a microRNA that mediates EGFR signalling regulation in lung carcinogenesis. The results showed that miR-338-3p is poorly expressed and NRP1 is overexpressed in NSCLC tissues relative to their levels in adjacent noncancerous tissues. Luciferase reporter assays, quantitative real-time reverse transcription PCR, and Western blot analyses showed that NRP1 is a direct target of miR-338-3p. Overexpression of miR-338-3p in NSCLC cell lines inhibited cell proliferation in vitro and in vivo. Moreover, cell migration and invasion were inhibited by miR-338-3p overexpression. These effects occurred via the EGF signalling pathway. Our data revealed a new post-transcriptional mechanism by which miR-338-3p directly targets NRP1; this mechanism plays a role in enhancing drug sensitivity in EGFR wild-type patients with NSCLC.

High expression of Copine 1 promotes cell growth and metastasis in human lung adenocarcinoma.

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients is poor. Further understanding of the disease mechanism and treatment strategies is required. Copines are a family of calcium-dependent phospholipid-binding proteins that are evolutionally conserved in various eukaryotic organisms and protists. Copine 1, encoded by CPNE1, is a soluble membrane-binding protein, which includes two tandem C2 domains at the N-terminus and an A domain at the C­terminus. A previous study reported that Copine 1 binds with various intracellular proteins via its A domain and C  omain. However, the role of CPNE1 in lung cancer remains unclear. In the presented study, CPNE1 expression level was demonstrated to be positively associated with the stage (P=0.002) and significantly associated with lymph node status (P=0.011) and distant metastasis (P=0.042). Furthermore, the function of CPNE1 in regulation of cell growth, migration and invasion was investigated, and it was demonstrated that knockdown of CPNE1 inhibits the cell cycle in NSCLC cells. Collectively, these data suggest that CPNE1 is an oncogene in NSCLC and serves an important role in tumorigenesis of NSCLC progression.

CPNE1 is a target of miR-335-5p and plays an important role in the pathogenesis of non-small cell lung cancer.

BACKGROUND: Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor. There is therefore a strong need to identify potential molecular targets for the treatment of NSCLC. In the present study, we investigated the function of CPNE1 in the regulation of cell growth, migration and invasion. METHODS: Quantitative real-time PCR (qRT-PCR) was used to detect the expression of CPNE1 and miR-335-5p. Western blot and immunohistochemical assays were used to investigate the levels of CPNE1 and other proteins. Flow cytometry was used to determine cell cycle stage and apoptosis. CCK-8 and clonogenic assays were used to investigate cell proliferation. Wound healing, migration and invasion assays were used to investigate the motility of cells. A lung carcinoma xenograft mouse model was used to investigate the in vivo effects of CPNE1 overexpression. RESULTS: We observed that knockdown of CPNE1 and increased expression of miR-335-5p inhibits cell proliferation and motility in NSCLC cells, and found that CPNE1 was a target of miR-335-5p. In addition, our data indicated that CPNE1 inhibition could improve the clinical effects of EGFR-tyrosine kinase inhibitors. CONCLUSIONS: The present results indicate that CPNE1 may be a promising molecular target in the treatment of NSCLC.

MiR-484 promotes non-small-cell lung cancer (NSCLC) progression through inhibiting Apaf-1 associated with the suppression of apoptosis.

Increasing studies have indicated that the dysregulated microRNAs (miRNAs) are associated with tumorigenesis, development and even the poor prognosis of a variety of tumors, including the non-small-cell lung cancer (NSCLC). Here in our study, we found that miRNA-484 was expressed highly in NSCLC clinical tumor samples in comparison to the matched adjacent tissues. In addition, high and low expression of miRNA-484 was observed in NSCLC cell lines and lung normal cells, respectively. Furthermore, the capability of migration and proliferation changed accompanied with the altered expression of miR-484 in NSCLC. Apoptotic protease activating factor-1 (APAF-1), frequently down-regulated in a number of types of cancer, was found to be reduced in NSCLC tissue samples or NSCLC cell lines along with high expression of miR-484, which were inversely expressed in Apaf-1 over-expressed tissues or cells. Moreover, miR-484 triggered the migration and proliferation, and simultaneously reduced the cleavage of poly (ADP-ribose) polymerase-2 (PARP-2) and Caspase-3 of A549 cells, which could be suppressed by the improvement of Apaf-1. And the inhibition of Apaf-1 could reverse the function caused by miR-484 in A549 cells, suggesting that Apaf-1 was targeted by miR-484 directly and it could be acted as a potential therapeutic target against NSCLC. In conclusion, the reductive Apaf-1 regulated by miR-484 accelerated the NSCLC cell progression associated with the inhibition of apoptosis via down-regulating Caspase-3 and PARP cleavage.

Methylated +322-327 CpG site decreases hOGG1 mRNA expression in non-small cell lung cancer.

hOGG1 plays a role in several disease pathways, including various cancers. Despite such functional importance, how hOGG1 is regulated at the transcriptional level in human non-small cell lung cancer (NSCLC) remains unknown, particularly via DNA methylation changes. We obtained NSCLC tissues and adjacent non-cancerous tissues and examined hOGG1 mRNA expression levels. NSCLC cells were treated with 5-Aza to test whether DNA methylation can influence the expression of hOGG1. The MassARRAY EpiTYPER and luciferase reporter gene assays were used to define the functional region of the hOGG1 gene (including CpG sites). Finally, ChIP assay was utilized to verify transcription factor binding to the hOGG1 5'-UTR region. Our previous studies supported the idea that the methylation of the hOGG1 gene promoter region occurs frequently in NSCLC. Treatment with 5-Aza, a demethylating agent, led to a significant restoration of hOGG1 expression in NSCLC cell lines. Quantitative PCR and MassARRAY EpiTYPER assays demonstrated that methylation of the +322-327 CpG site in the 5'-UTR region of hOGG1 was higher in NSCLC tissues compared with adjacent non-cancerous tissues. Notably, the methylation level of +322-327 site (T/N) was inversely correlated with that of hOGG1 mRNA level (T/N) in 25 NSCLC tissues. ChIP assay and in silico prediction showed an association between the +322-327 CpG site and Sp1, which has been reported to be an activator of transcription. Importantly, luciferase reporter gene and ChIP assays showed that +322-327 CpG site methylation particularly reduced the recruitment of Sp1 to the 5'-UTR sequence in hOGG1 and reduced transcriptional activity ~50%. In summary, we have demonstrated that hOGG1 mRNA is downregulated in NSCLC tissues. Moreover, we identified that the methylated +322-327 CpG site in the hOGG1 5'-UTR is associated with reduced expression of hOGG1 by decreasing the recruitment of Sp1 to the 5'-UTR of hOGG1.

Valproic acid (VPA) enhances cisplatin sensitivity of non-small cell lung cancer cells via HDAC2 mediated down regulation of ABCA1.

Valproic acid (VPA) has been suggested to be a histone deacetylase inhibitor (HDACI). Our present study revealed that VPA at 1 mm, which had no effect on cell proliferation, can significantly increase the sensitivity of non-small cell lung cancer (NSCLC) cells to cisplatin (DDP). VPA treatment markedly decreased the mRNA and protein levels of ABCA1, while had no significant effect on ABCA3, ABCA7 or ABCB10. Luciferase reporter assays showed that VPA can decrease the ABCA1 promoter activity in both A549 and H358 cells. VPA treatment also decreased the phosphorylation of SP1, which can bind to -100 and -166 bp in the promoter of ABCA1. While the phosphorylation of c-Fos and c-Jun were not changed in VPA treated NSCLC cells. Over expression of HDAC2 attenuated VPA induced down regulation of ABCA1 mRNA expression and promoter activities. Over expression of HDAC2 also attenuated VPA induced DDP sensitivity of NSCLC cells. These data revealed that VPA can increase the DDP sensitivity of NSCLC cells via down regulation of ABCA1 through HDAC2/SP1 signals. It suggested that combination of VPA and anticancer drugs such as DDP might be great helpful for treatment of NSCLC patients.

A 2-DE-based proteomic study on the toxicological effects of cisplatin in L02 cells.

Cisplatin is a chemotherapeutic agent for the treatment of various cancers. In this study, cisplatin-induced effects were characterized in vitro model of human liver cells (L02) using 2-DE-based proteomics. Results indicated that different cisplatin treatments primarily induced disturbances in protein synthesis and oxidative stress via differential mechanisms. Since the experimental concentrations of cisplatin described a hormesis effect in cell proliferation of L02 cells, it was expected to reveal the hormesis effects using proteomic markers. However, only confilin-1 was commonly up-regulated in three concentrations of cisplatin treatments showing a hormesis effects with a U-shape regulation. These results were highly consistent with many other toxico-proteomic studies, indicating that the toxico-proteomic responses based on dose-dependent protein responses were incongruent with the theoretically linear or hormetic concentration-effect relationship. Our findings suggested that a macroscopic hormesis phenomenon on the cell proliferation could not be reflected by proteomic responses induced by cisplatin treatments.

Dose responsive effects of cisplatin in L02 cells using NMR-based metabolomics.

Cisplatin is an effective chemotherapeutic agent for the treatment of various cancers, such as bladder cancer, epithelial ovarian cancer, cervical cancer, and so on. However, cisplatin can cause various side effects. In this study, the dose-responsive effects of cisplatin were investigated in an in vitro model of human liver cells (L02) using NMR-based metabolomics. The inverted U-shaped curve of cell proliferation confirmed the hormetic effects of cisplatin (from 1 nM to 1 mM) in L02 cells. However, the metabolite changes revealed both U-shaped (ethanol, lactate, aspartate, choline, etc.) and inverted U-shaped (glutamate, glutamine, 4-aminobutyrate, myo-inositol, etc.) curves induced by three typical concentrations of cisplatin which covered the inverted U-shaped curve as indicated by the cell proliferation assay. These findings suggested that a macroscopic hormesis phenomenon on the cell proliferation could be reflected by both stimulated and inhibited metabolites and corresponding metabolic pathways to cisplatin treatments. Therefore, a global analysis using metabolomics may give a broader view into the dose-response relationship than using a single endpoint at molecular levels.

[Effect of Akt on the function of esophageal squamous cancer cell Eca109 and the expression of vasculogenic mimicry-related genes].

OBJECTIVE: To investigate the inhibitory effect of siRNA targeting Akt on the biological behavior of esophagus squamous cell carcinoma cell line in vitro and to explore the relationship between Akt and vasculogenic mimicry (VM)-related genes in esophageal squamous cell carcinoma. METHODS: The plasmid-harboring small interfering RNA targeting Akt was introduced into Eca109 cells by liposome-mediated transfection. The proliferation of Eca109 cell was determined by colony formation assay. The cellular migration was evaluated by Transwell migration assay. And three dimensional cell culture was employed to observe and count the number of capillary structure for each cell group. Flow cytometry (FCM) was used to detect the apoptotic rate of Eca109, Eca109/Neo and Eca109/siRNA Akt cells under normoxia exposure. The apoptotic rate was assessed by Annexin V/7-AAD double labeling. And the expressions of Akt and matrix metalloproteinase-2 (MMP-2) protein were detected by Western blotting. RESULTS: The results of Western blotting showed that the expression of Akt in stably transfected group were significantly lower than empty carrier and untransfected groups (0.03 ± 0.01 vs 1.49 ± 0.39 and 1.47 ± 0.41, both P < 0.05). Transwell migration assay showed that fewer Eca109/8 cells could move through the artificial basement membrane as compared with untransfected and empty carrier groups (48 ± 9 vs 128 ± 10 and 122 ± 11, both P < 0.05). Clone formation number of stably transfected group was significantly lower than empty carrier and untransfected groups (63 ± 7 vs 148 ± 11 and 163 ± 15, both P < 0.05). Annexin V/7-AAD double standard method demonstrated that the apoptotic rate of stably transfected group was much more than those of untransfected and empty carrier groups (12.2% ± 1.6% vs 4.8% ± 0.8% and 4.2% ± 0.8%, both P < 0.05). Eca109 and Eca109/Neo cells were capable of forming the in vitro structures of VM. And the number of tube-shaped structure in stably transfected group was markedly less than those of untransfected and empty carrier groups (14.0 ± 1.2 vs 30.0 ± 1.2 and 27.7 ± 1.5, both P < 0.05). MMP-2 protein expression in stably transfected group was less than those of untransfected and empty carrier groups (both P < 0.05). CONCLUSIONS: The PI(3)K/Akt pathway is involved in the regulation of VM formation in esophageal squamous cell carcinoma through the action of MMP-2. Blockade of this pathway may provide a new therapeutic approach to human esophagus squamous cell carcinoma.