TGF-β regulation of microRNA miR-497-5p and ocular lens epithelial cell mesenchymal transition

Science China Life Sciences - The purpose of this study was to investigate the role of a human lens microRNA (miR-497-5p) in regulating epithelialmesenchymal transition (EMT) under the control of...     

Ceramide and palmitic acid inhibit macrophage-mediated epithelial–mesenchymal transition in colorectal cancer

Molecular and Cellular Biochemistry - Accumulating evidence indicates that ceramide (Cer) and palmitic acid (PA) possess the ability to modulate switching of macrophage phenotypes and possess...     

Neuropilin-2 expression promotes TGF-β1-mediated epithelial to mesenchymal transition in colorectal cancer cells

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.     

miR-29b suppresses tumor growth and metastasis in colorectal cancer via downregulating Tiam1 expression and inhibiting epithelial–mesenchymal transition

Recently, the role of miR-29b in colorectal carcinoma (CRC) development appears to be controversial. Until now, the expression and function of miR-29b in CRC have not been clarified clearly. We showed that decreased expression of miR-29b usually occurred in CRC cell lines and tissue samples. Loss- and gain-of-function assays in vitro revealed suppressive effects of miR-29b on cell proliferation and migration. Endogenous overexpression of miR-29b was sufficient to suppress aggressive behavioral phenotypes in mice. Proteomic analysis showed that miR-29b involved in integrate several key biological processes. In addition, miR-29b mediated the inhibition of epithelial–mesenchymal transition (EMT) and the inactivation of mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signal transduction pathway. Further studies found that T lymphoma invasion and metastasis 1 (Tiam1) was identified as a direct target of miR-29b. In contrast to the phenotypes induced by miR-29b restoration, Tiam1-induced cell proliferation and migration partly rescued miR-29b-mediated biological behaviors. Our results illustrated that miR-29b as a suppressor has a critical role in CRC progression, which suggests its potential role in the molecular therapy of patients with advanced CRC.Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer death. In China, the incidence of CRC is continually increasing despite advances in treatment and subsequent improvement in prognosis. Metastasis leads to most of the mortalities and has a critical role in the poor prognosis.^{1, 2} The underlying molecular mechanisms in CRC metastasis are still unclear. Hence, it is urgent to identify important molecules in cancer progression, which may be used to develop new diagnostic strategies and drugs targeting these markers.MicroRNAs (miRNAs) are a class of diverse, small, noncoding RNAs that are processed from precursors with a characteristic hairpin secondary structure.³ They usually function as critical gene regulators. In recent years, a large number of studies have confirmed that miRNAs have important roles in tumorigenesis and metastasis by targeting different mRNAs.⁴ To date, abnormal expression of several miRNAs, such as miR-21,⁵ miR-124,⁶ miR-625,⁷ miR-339-5p⁸ and miR-27b,⁹ has been identified in CRC and may contribute to the development and progression of CRC. In our recent study, miR-133a was identified as a tumor-suppressive miRNA in human CRC that acts by repressing LIM and SH3 protein 1, which has been previously identified as tumor metastasis-associated protein,¹⁰ provides additional evidence of a pivotal role for miRNAs in CRC tumorigenesis and progression.¹¹miR-29b belongs to the miR-29b family that comprises three members: miR-29a, -29b and -29c. Recently, several studies have showed that miR-29b was dysregulated and represses tumor progression in hepatocellular,¹² ovarian,¹³ prostate,¹⁴ breast¹⁵ and gastric¹⁶ cancer. In colorectal cancer, increased miR-29b was found in colon cancer cells following exposure to a Hexane extract of American Ginseng (HAG) and suppressed the migration of colon cancer cells.¹⁷ In the other study, increased miR-29b was observed in ulcerative colitis-related CRC compared with ulcerative colitis, suggesting its function as oncogene.¹⁸ Thus, the role of miR-29b in CRC development appears to be controversial. Until now, the expression and function of miR-29b in CRC have not been clarified clearly.In this study, we detected miR-29b expression in CRC cells and tissue samples. Gain- or loss-of-function assays were used to analyze the effect of miR-29b on cell behaviors. We performed xenograft mice models to investigate its therapeutic role in tumor genesis and metastasis in vivo. Finally, we also explored the molecular mechanisms underlying the function of miR-29b and its potential targets.     

MiR-876-5p suppresses epithelial–mesenchymal transition of lung cancer by directly down-regulating bone morphogenetic protein 4

Lung cancer is the leading cause of cancer-related death throughout the world. We aimed to investigate the role of a novel microRNA-876-5p and its potential molecular target bone morphogenetic protein 4 (BMP-4), in the epithelial–mesenchymal transition (EMT) of lung cancer. Expressions of microRNA-876-5p and its potential target BMP-4 were analysed in lung cancer cells and patient tissues. Luciferase activity assay was conducted to verify direct targeting of microRNA-876-5p to the 3′-UTR of BMP-4 mRNA. Migration, invasion capacities of lung cancer cells expressing microRNA-876-5p were analysed, and characteristics of lung cancer EMT protein markers were also evaluated. A xenograft tumour mouse model was established to address the roles of microRNA-876-5p and BMP-4 in lung cancer EMT in vivo. MicroRNA-876-5p was decreased while BMP-4 was increased in lung cancer cells and tissues. MicroRNA-876-5p directly targeted 3′-UTR of BMP-4 mRNA to inhibit its expression. MicroRNA-876-5p expression significantly inhibited the migration, invasion and EMT of lung cancer cells in vitro, as well as metastasis in vivo, which required BMP-4 expression. MicroRNA-876-5p suppresses EMT of lung cancer by directly down-regulating BMP-4, both of which could serve as potential therapeutic targets in the treatment of lung cancer.     

Long noncoding RNA AC073284.4 suppresses epithelial–mesenchymal transition by sponging miR-18b-5p in paclitaxel-resistant breast cancer cells

Breast cancer (BC) is the most prevalent malignant cancer in the world, is the leading cause of cancer-related death female. Recently, there is accumulating evidence that long noncoding RNAs (lncRNAs) might as an important role in the progression of BC. (epithelial-mesenchymal transition (EMT) is considered to play a vital role in tumor cells migration and invasion. Nevertheless, the entire biological mechanisms and functions of lncRNAs in tumor migration, invasion, and EMT remain uncertain. In the present research, we observed that the expression of lncRNA AC073284.4 was downregulated in BC paclitaxel-resistant (PR) cells (MCF-7/PR) and tissues. Bioinformatics analysis predicted that miR-18b-5p was a direct target of AC073284.4, which has been validated by dual-luciferase reporter gene assay. We further proved that AC073284.4 could directly bind to miR-18b-5p and relieve the suppression for dedicator of cytokinesis protein 4 (DOCK4). Furthermore, the underlying functional experiments demonstrated that AC073284.4 might sponge miR-18b-5p to attenuate the invasion, metastasis, and EMT of BC cell through upregulating DOCK4 expression. In summary, AC073284.4 might serve as a competing endogenous RNA (ceRNA) in BC progression via modulating miR-18b-5p/DOCK4 axis, which weakens EMT and migration of BC. These results suggesting that AC073284.4 might function as a potential novel diagnostic biomarker in the progression of BC.     

Signaling roadmap to epithelial–mesenchymal transition in pterygium,TWIST1 centralized

Pterygium as a complex disease shares common features with other malignant cells in its onset recurrence and especially epithelial–mesenchymal transition (EMT) transition. Although using different approaches including conjunctival autografts, amniotic membrane, radiotherapy, mitomycin C (MMC) has shown promising insights in the inhibition of pterygium recurrence, it needs to be investigated in more details in molecular pathways to present adjuvant target therapy. In this study, we aimed to evaluate the expression of and then illustrate the role of signaling pathways on EMT in pterygium. Using real-time polymerase chain reaction, the twist-related protein 1 (TWIST1) expression was compared in primary pterygium and normal conjunctiva. This study assessed the mRNA expression, as well as the association between the clinicopathological indices and the gene expression level. The expression level of TWIST1 was overexpressed in 36% of our cohort ( n = 76). There was a significant positive correlation between recurrence with grade T, grade V and a significant negative correlation with growth activity. Our vast literature review on different signaling pathways in pterygium showed that EMT has centralization role in recurrence of this disease. Our data confirmed that EMT is important in the recurrence of pterygium samples and different signaling pathways end up activating the EMT markers. It is suggested to evaluate the environmental factors and their correlation with molecular markers to select favorable treatment for this kind of diseases.     

The lncRNA ZEB1-AS1 sponges miR-181a-5p to promote colorectal cancer cell proliferation by regulating Wnt/β-catenin signaling

Long noncoding RNAs (lncRNAs) are important regulators of the biological functions and underlying molecular mechanisms of colorectal cancer (CRC). However, the role of the lncRNA ZEB1-AS1 in CRC is not thoroughly understood. In this study, we found that ZEB1-AS1 was markedly upregulated in CRC. ZEB1-AS1 knockdown significantly suppressed CRC cell proliferation and induced apoptosis, whereas enhanced expression of ZEB1-AS1 had the opposite effect. Bioinformatics analysis identified miR-181a-5p as a candidate target of ZEB1-AS1. Moreover, we found an inverse correlation between ZEB1-AS1 and miR-181a-5p expression in CRC tissue. Inhibition of miR-181a-5p significantly upregulated ZEB1-AS1, whereas overexpression of miR-181a-5p had the opposite effect, suggesting that ZEB1-AS1 is negatively regulated by miR-181a-5p. Using luciferase reporter and RIP assays, we found that miR-181a-5p directly targets ZEB1-AS1. Importantly, ZEB1-AS1 may act as an endogenous ‘sponge’ to regulate miRNA targets by competing for miR-181a-5p binding. In summary, our findings provide the evidence supporting the role of ZEB1-AS1 as an oncogene in CRC. Our study also demonstrates that miR-181a-5p targets not only protein-coding genes but also the lncRNA ZEB1-AS1.     

lncRNA HNF1A-AS1 modulates non–small cell lung cancer progression by targeting miR-149-5p/Cdk6

Growing evidence have shown the important regulation of lncRNAs (long noncoding RNAs) in non–small cell lung cancer (NSCLC). lncRNA hepatocyte nuclear factor 1 homeobox A (HNF1A)-antisense RNA 1 (AS1), an “oncogene”, was reported to regulate human tumors progression. However, the molecular mechanism of HNF1A-AS1 involved in the development of NSCLC is still under investigation. In the current study, we found that HNF1A-AS1 was relatively upregulated in both NSCLC patient tissues and cell lines. Functional studies established that overexpression of HNF1A-AS1 promoted cell proliferation, cell cycle, invasion, and migration of NSCLC cells in vitro. The promotion abilities of HNF1A-AS1 on NSCLC cell progression were suppressed via knockdown of HNF1A-AS1. miR-149-5p was then proved to be a novel target of HNF1A-AS1, whose expression was negatively correlated with HNF1A-AS1 in NSCLC patient tissues and cell lines. HNF1A-AS1 increased the expression of cyclin-dependent kinase 6 (Cdk6) via sponging with miR-149-5p. Gain- and loss-of-functional studies indicated that HNF1A-AS1 promoted NSCLC progression partially through inhibition of miR-363-3p and induction of Cdk6. Subcutaneous xenotransplanted tumor model confirmed that interference of HNF1A-AS1 suppressed the tumorigenic ability of NSCLC via upregulation of miR-149-5p and downregulation of Cdk6 in vivo. In conclusion, our findings clarified the biologic significance of the HNF1A-AS1/miR-149-5p/Cdk6 axis in NSCLC progression and provided novel evidence that HNF1A-AS1 may be a new potential therapeutic target for the treatment of NSCLC.     

miR-363-3p inhibits migration,invasion, and epithelial–mesenchymal transition by targeting NEDD9 and SOX4 in non-small-cell lung cancer

miR-363-3p is downregulated in lung adenocarcinoma and can inhibit tumor growth. Here, we aimed to investigate the effect of miR-363-3p on non-small-cell lung cancer (NSCLC) metastasis. In our study, miR-363-3p overexpression inhibited cell migration and invasion via epithelial–mesenchymal transition inhibition, while miR-363-3p knockdown exhibited the opposite effects. Further studies demonstrated that miR-363-3p bound to 3′-untranslated regions of NEDD9 and SOX4, and negatively regulated their levels. Interestingly, NEDD9 or SOX4 knockdown rescued the metastasis-promoting effects of antagomiR-363-3p. The inhibitory effects of agomiR-363-3p were also blocked by NEDD9 or SOX4 overexpression. Moreover, lentivirus particles carrying pre-miR-363 (LV-pre-miR-363) significantly decreased, while LV-miR-363-3p inhibitor increased metastatic nodule numbers and the levels of NEDD9 and SOX4 in lungs. In conclusion, tumor suppressor miR-363-3p may be a potential target in NSCLC therapy.     

S1PR3 deficiency alleviates radiation-induced pulmonary fibrosis through the regulation of epithelial–mesenchymal transition by targeting miR-495-3p

Radiation-induced pulmonary fibrosis (RIPF) is a life-threatening complication of thoracic radiotherapy, which contributes to continued deterioration in pulmonary function. Sphingosine-1 phosphate receptor 3 (S1PR3) has been identified as a crucial molecule in fibrosis. Accumulating evidence indicated that the inhibition of the S1PRs ameliorates fibrogenesis. Thus, this study aims to explore whether S1PR3 participates in RIPF and elucidates the molecular mechanisms underlying S1PR3-modulated epithelial–mesenchymal transition (EMT) in transforming growth factor-β1-induced pulmonary epithelia. A recombinant adeno-associated viral-mediated S1PR3 (AAV-S1PR3) gene therapy analyzed the effect of S1PR3 gene deficiency on the altered histology structure and molecular mechanisms in the lung of mice with whole-lung irradiation. Compared with the AAV-negative control mice, AAV-mediated S1PR3 knockdown in the lung of mice attenuated pulmonary fibrosis induced by the radiation, as indicated by the alleviation of collagen accumulation, lessened histopathological alterations, and the suppression of inflammatory cells infiltration. S1PR3 deficiency reversed the RIPF concomitantly with abrogated EMT-related protein (α-smooth muscle actin). Consistently, S1PR3-deficient pulmonary epithelia inhibited the EMT process changes and fibrosis formation. Furthermore, S1PR3 was designated as one of the target genes for microRNA-495-3p (miR-495-3p). The inhibition of miR-495-3p promoted the expression of S1PR3 in pulmonary epithelia, whereas the overexpression of miR-495-3p inhibited the S1PR3/SMAD2/3 pathway and suppressed the EMT process. Collectively, miR-495-3p might be a negative regulator of the EMT process in fibrosis formation by inhibiting the targeted S1PR3 gene. These results established a link between the S1PR3 gene, the EMT process, and the fibrosis, suggesting the pharmacological blockage of S1PR3 as a potential therapeutic strategy for RIPF.     

The SDF-1/CXCR4 axis induces epithelial–mesenchymal transition in hepatocellular carcinoma

Targeted immunotherapy has become a popular research topic in cancer. The development and metastasis of cervical carcinoma are closely related to epidermal growth factor (EGF) and EGF-1 receptor (EGFR). We successfully constructed a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein (scFV/tP) expression vector using overlap extension PCR. Enzyme-linked immunosorbent assay and gel shift assay showed that the fusion protein retained the DNA and antigen-binding activity of the original antibody. Using the non-viral scFv/tP vector as a delivery tool, small interfering RNA (siRNA) of the human wings apart-like gene (hWAPL) was effectively transfected into cervical cancer HeLa cells. The hWAPL mRNA expression levels were reduced by 97.23 % in contrast with control cells, and the proliferation capability declined by 66.71 %, indicating significant inhibition. The present results provide a novel strategy for targeted gene therapy and siRNA therapy of EGFR-positive cervical cancers.