A regulatory loop involving miR-29c and Sp1 elevates the TGF-ß1 mediated epithelial-to-mesenchymal transition in lung cancer.

Specificity protein1 (Sp1) is required for TGF-ß-induced epithelial-to-mesenchymal transition (EMT) which has been demonstrated to aggravate the progression of cancer including lung cancer. microRNA-29c (miR-29c) is identified to inhibit EMT, but the correlation between miR-29c and Sp1 in human lung cancer remain incompletely clarified. Here, we confirmed decreased expression of miR-29c and enhanced expression of Sp1 in lung cancer tissues (n = 20) and found that Sp1 could be targeted and inhibited by miR-29c. Besides, the expression of miR-29c was down-regulated in high-metastatic lung cancer cell lines and TGF-ß1-treated cells. The inhibition of miR-29c or overexpression of Sp1 in 95C and A549 cells dramatically enhanced the cell migration and invasion, and also induced the decrease in the expression of epithelial markers, e.g. thyroid transcription factor 1 (TTF-1) and E-cadherin, together with an increase in mesenchymal markers including vimentin, α-smooth muscle actin (α-SMA), which could be restored by overexpression of miR-29c mimics during the TGF-ß-induced EMT. Moreover, dual-luciferase reporter assay was performed and the results indicated that miR-29c/Sp1 could form an auto-regulatory loop with TGF-ß1, which impaired TGFB1 transcription. Furthermore, miR-29c overexpression could abrogate the tumor progression and inhibit the Sp1/TGF-ß expressions in vivo, indicating that miR-29c could be a tumor suppressor and repress the Sp1/TGF-ß axis-induced EMT in lung cancer.

Downregulation of inhibitor of apoptosis­stimulating protein of p53 inhibits proliferation and promotes apoptosis of gastric cancer cells.

Gastric cancer (GC) remains one of the leading causes of cancer-associated mortality. Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is a member of the inhibitory apoptosis-stimulating protein p53 family. The overexpression of iASPP has been detected in several types of tumor in humans. However, the role of iASPP in GC remains to be elucidated. The objectives of the present study were to detect the expression of iASPP in GC and examine the potential role of iASPP in GC cell lines. Using reverse transcription-quantitative polymerase chain reaction and western blot analyses, it was identified that the expression of iASPP in GC tissues and GC cell lines was higher compared with that in adjacent normal tissues and in a normal gastric mucosa cell line (GES-1). To examine the role of iASPP in GC cells, the expression of iASPP was inhibited using a small interfering (si)RNA against iASPP and it was observed that iASPP expression was significantly downregulated. Using MTT assays, colony-formation assays and flow cytometry, it was identified that the inhibition of iASPP was able to significantly inhibit the proliferation and colony forming ability and promote apoptosis in GC cells. To examine the role of iASPP in GC cells in vivo, GC cells, which were infected with iASPP-siRNA or control-siRNA were subcutaneously injected into nude mice. It was identified that downregulation of iASPP significantly inhibited tumor growth in vivo. Thus, iASPP may be a potential molecular target in GC therapy.

Gastric tumor-initiating CD44+ cells and epithelial-mesenchymal transition are inhibited by γ-secretase inhibitor DAPT.

It has been proposed that the Notch signaling pathway may serve a pivotal role in cellular differentiation, proliferation and apoptosis. However, the function of Notch signaling in gastric cancer stem cells (GCSCs) is largely unknown. The present study aimed to delineate the role of the Notch1 pathway in GCSCs and during epithelial-mesenchymal transition (EMT). Flow cytometry was used to isolate CD44+ cells from the human gastric cancer cell line, MKN45. CD44+ cells displayed the characteristics of CSCs and exhibited higher Notch1 expression compared with CD44- cells. To investigate the role of the Notch1 pathway in GCSCs, CD44+ cells were treated with the γ-secretase inhibitor DAPT. DAPT treatment inhibited the expression of the Notch1 downstream target Hes1 and EMT markers, suppressed the properties of CSCs and impaired the invasion and proliferation capabilities of CD44+ cells. In addition, intraperitoneal treatment with DAPT effectively inhibited the growth of CD44+ cell xenograft tumors. The present study indicated that CD44+ GCSCs possess the characteristics of CSCs and that the Notch1 pathway serves a critical role in the maintenance of CSCs and EMT.

microRNA-503 inhibits gastric cancer cell growth and epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) is believed to be associated with cancer cell malignancy, and also to cause cancer invasion and metastasis. Recent evidence indicates that small non-protein coding RNA [microRNAs (miRNAs/miRs)] may act as powerful regulators of EMT. The present study aimed to systematically delineate miR-503 expression in gastric cancer and analyse the function of miR-503 in gastric cancer EMT. In the present study, miR-503 expression was detected in gastric cancer cell lines and gastric cancer tissues by quantitative polymerase chain reaction. Gastric cancer cell migration, invasion and proliferation capabilities were analysed by Transwell, MTT and clonability assays. The expression of mesenchymal markers, including fibronectin, vimentin, N-cadherin, SNAIL and the epithelial marker, E-cadherin, was examined by immunoblot analysis following miR-503 transfection. miR-503 expression was found to be reduced in gastric cancer cell lines compared with normal gastric mucosa cell lines, and the expression of miR-503 was upregulated in non-metastatic-derived gastric cancer cell lines compared with metastatic-derived lines. miR-503 expression levels were significantly reduced in tumour tissues in comparison with adjacent normal mucosa tissues, and the miR-503 expression levels in patients with metastases were significantly lower than those in patients without. miR-503 inhibited gastric cancer cell migration, invasion and proliferation. Fibronectin, vimentin, N-cadherin and SNAIL protein levels were decreased, but E-cadherin expression was increased in an AGS cell line transfected with miR-503. Taken together, the present findings indicate that miR-503 acts as a novel tumour suppressor gene in gastric cancer and can inhibit EMT in gastric cancer cells.

Gastric cancer cell growth and epithelial-mesenchymal transition are inhibited by γ-secretase inhibitor DAPT.

The Notch signaling pathway may be important in the development and progression of several malignancies. However, the functions of Notch signaling in epithelial-mesenchymal transition (EMT) remain largely unknown. The aim of the present study was to delineate Notch1 expression in gastric cancer (GC) and its function in GC EMT. Using quantitative polymerase chain reaction and western blot analysis, the expression of Notch1 was found to increase in GC cell lines compared with the normal gastric mucosa cell line. In addition, Notch1 expression was found to be downregulated in the non-metastatic-derived GC cell line compared with the metastatic-derived cell line. Furthermore, Notch1 expression was significantly increased in the tumor tissues compared with the adjacent normal mucosa tissues, as well as in patients with metastases than in patients without metastases. To explore the role of the Notch1 signaling pathway in EMT, the GC cell lines, AGS and MKN45, were treated with γ-secretase inhibitor DAPT. Using MTT, Transwell and clonality assays, DAPT was found to inhibit the expression of the Notch1 downstream target, Hes1, and impair the ability of the GC cell lines to migrate, invade and proliferate. The protein levels of the mesenchymal markers, vimentin, neural cadherin and Snail, were decreased; however, the expression of the epithelial marker, epithelial cadherin, was increased in the GC cell lines treated with DAPT. These results indicated that the Notch1 signaling pathway may be important in the development and progression of GC. In conclusion, DAPT inhibits the Notch1 signaling pathway, as well as the growth, invasion, metastasis and EMT of GC cells.

MicroRNA-338 inhibits growth, invasion and metastasis of gastric cancer by targeting NRP1 expression.

NRP1 as multifunctional non-tyrosine-kinase receptors play critical roles in tumor progression. MicroRNAs (miRNAs) are an important class of pervasive genes that are involved in a variety of biological functions, particularly cancer. It remains unclear whether miRNAs can regulate the expression of NRP1. The goal of this study was to identify miRNAs that could inhibit the growth, invasion and metastasis of gastric cancer by targeting NRP1 expression. We found that miR-338 expression was reduced in gastric cancer cell lines and in gastric cancer tissues. Moreover, we found that miR-338 inhibited gastric cancer cell migration, invasion, proliferation and promoted apoptosis by targeting NRP1 expression. As an upstream regulator of NRP1, miR-338 directly targets NRP1. The forced expression of miR-338 inhibited the phosphorylation of Erk1/2, P38 MAPK and Akt; however, the expression of phosphorylated Erk1/2, P38 MAPK and Akt was restored by the overexpression of NRP1. In AGS cells infected with miR-338 or transfected with SiNRP1, the protein levels of fibronectin, vimentin, N-cadherin and SNAIL were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-338-expressing cells was restored to normal levels by the restoration of NRP1 expression. In vivo, miR-338 also decreased tumor growth and suppressed D-MVA by targeting NRP1. Therefore, we conclude that miR-338 acts as a novel tumor suppressor gene in gastric cancer. miR-338 can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as gastric cancer EMT, by attenuating the expression of NRP1.