MicroRNA-26 regulates the expression of CTGF after exposure to ionizing radiation.

Radiation-induced fibrosis (RIF) is a serious complication that occurs after irradiation and which is caused by the deposition of extracellular matrix (ECM) proteins such as collagen. However, the underlying mechanisms, including the expression of the cytokines, that promote the RIF process, are not yet fully understood. MicroRNAs (miRNAs) have recently been suggested to act as post-transcriptional repressors for many genes; however, their role in the process of RIF remains to be elucidated. Our previous study showed that ionizing radiation increased the type I collagen expression through the activation of transforming growth factor (TGF)-ß, while miR-29 repressed this increase. This study aimed to investigate the mechanisms by which the expression of connective tissue growth factor (CTGF), a downstream mediator of TGF-ß, is controlled by miRNAs post-transcriptionally after exposure to ionizing radiation. The expression of CTGF in NIH-3T3 cells and mouse embryonic fibroblasts was increased by ionizing radiation. However, this increase was suppressed with a specific inhibitor of TGF-ß receptor. Among the predictable miRNAs that target the CTGF gene, the expression of miR-26a was downregulated after exposure to ionizing radiation and this regulation was negatively mediated by TGF-ß signaling. miR-26a negatively regulated the CTGF expression at the post-transcriptional level; however, ionizing radiation suppressed this negative regulation. In addition, the overexpression of miR-26a inhibited the expression of CTGF and type I collagen after irradiation. In conclusion, miR-26a modulates the expression of CTGF via TGF-ß signaling in irradiated fibroblasts. The results suggest the potential application of miR-26a in the treatment of RIF.

Regulation of type I collagen expression by microRNA-29 following ionizing radiation.

Radiation-induced fibrosis (RIF) is thought to involve the excessive accumulation of collagen and other extracellular matrix components; previously, we reported that ionizing radiation increased the type I collagen expression and that transforming growth factor (TGF)-ß was involved in this increase through activating its downstream mediator, Smad3. A recent study found that microRNAs (miRNAs)-small, noncoding sequences approximately 20 nucleotides long-negatively regulate the gene expression posttranscriptionally, and it has been suggested that miRNAs play essential roles in cellular processes, including fibrosis. However, their role in the development of RIF remains unexplored. In the present study, we examined the effects of miRNA on the expression of type I collagen induced by ionizing radiation and the mechanisms underlying the miRNA expression observed following ionizing radiation. We analyzed the regulation of miRNA following ionizing radiation by an miRNA real-time PCR, and found that miR-29 family members were downregulated in irradiated mouse fibroblasts and directly targeted type I collagen genes by specifically binding to the 3' untranslated region. We also found that the overexpression of miR-29 inhibited the ionizing radiation-induced expression of type I collagen, whereas the knockdown of miR-29 enhanced it. In addition, TGF-ß/Smad-signaling significantly decreased the transcription of miR-29, whereas the inhibition of this signaling pathway cancelled this decrease. In conclusion, miR-29 was involved in the regulation of type I collagen expression through the TGF-ß/Smad-signaling pathway in irradiated cells, suggesting that miR-29 may be an important regulator of RIF.

Overexpression of cannabinoid receptor 1 in esophageal squamous cell carcinoma is correlated with metastasis to lymph nodes and distant organs, and poor prognosis.

In patients with esophageal squamous cell carcinoma (ESCC), the status of metastasis to lymph nodes is strongly associated with prognosis. Consequently, development of a biomarker to detect the presence of metastasis would be clinically valuable. In this study, we found that overexpression of cannabinoid receptor 1 (CB1R) was applicable as a marker for prediction of metastasis in ESCC. CB1R overexpression was detected immunohistochemically in 54 of 88 cases (61.4%). The intensity of CB1R expression was uniform in both intraepithelial and invasive regions in each case, and was significantly correlated with the status of metastasis to lymph nodes (P = 0.046) and distant organs (P = 0.047). Furthermore, multivariate analysis revealed that CB1R overexpression was independently associated with poor prognosis (P = 0.019). Biological analysis of CB1R overexpression using ESCC cell lines revealed that CB1R activation appeared to promote cell proliferation and invasion. On the basis of these findings, we propose that evaluation of CB1R expression status in biopsy specimens of ESCC using immunohistochemistry might be clinically useful for prediction of metastasis to lymph nodes and distant organs.

Expression of DDX27 contributes to colony-forming ability of gastric cancer cells and correlates with poor prognosis in gastric cancer.

Previously, we have reported that gain at chromosome 20q13 is the most common genomic copy number aberration in gastric cancer (GC) (29/30 cases), and that among the genes located in this region, we have identified DDX27, whose expression level shows the highest correlation with genomic copy number, as a candidate therapeutic target for GC. Here, we analyzed the clinicopathological significance of DDX27 using immunohistochemistry and studied its functions using knockdown assays. We found that DDX27 was frequently upregulated in GC tissues (98 of 140 cases, 70%), and significantly associated with venous invasion and liver metastasis. Furthermore, multivariate analysis of GC patients showed that high expression of DDX27 was independently associated with poorer prognosis. In functional assays, knockdown of DDX27 reduced the ability of GC cells to form colonies both on conventional plates and soft agar, but had little effect on their invasiveness. We also found that knockdown of DDX27 reduced the viability of GC cells through inhibition of cell cycle progression independently of apoptosis. Interestingly, DDX27 depletion induced accumulation of TP53 in a TP53 wild-type cell line, AGS, but not in a TP53-deleted cell line, 44As3, although DDX27 knockdown commonly reduced the viability of both, indicating the TP53-dependent and independent cell cycle control of DDX27. Thus, our results suggest that expression of DDX27 contributes to colony formation by GC cells through cell cycle control and may be a potential therapeutic target for GC patients with chromosome gain at 20q13.

A new murine osteoblastic cell line immortalized with the SV40 large T antigen.

The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis.

Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1 and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-ß1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-ß receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.

Increased globotriaosylceramide levels in a transgenic mouse expressing human alpha1,4-galactosyltransferase and a mouse model for treating Fabry disease.

Fabry disease is a lysosomal storage disorder caused by an α-galactosidase A (α-Gal A) deficiency and resulting in the accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3). A transgenic mouse expressing the human α-Gal A R301Q mutant in an α-Gal A-knockout background (TgM/KO) should be useful for studying active-site-specific chaperone (ASSC) therapy for Fabry disease. However, the Gb3 content in the heart tissue of this mouse was too low to detect an ASSC-induced effect. To increase the Gb3 levels in mouse organs, we created transgenic mice (TgG3S) expressing human α1,4-galactosyltransferase (Gb3 synthase). High levels of Gb3 were observed in all major organs of the TgG3S mouse. A TgG3S (+/-)M(+/-)/KO mouse was prepared by cross-breeding the TgG3S and TgM/KO mice and the Gb3 content in the heart of the TgG3S(+/-)M(+/-)/KO mouse was 1.4 µg/mg protein, higher than in the TgM(+/-)/KO (<0.1 µg/mg protein). Treatment with an ASSC, 1-deoxygalactonojirimycin, caused a marked induction of α-Gal A activity and a concomitant reduction of the Gb3 content in the TgG3S(+/-) M(+/-)/KO mouse organs. These data indicated that the TgG3S(+/-) M(+/-)/KO mouse was suitable for studying ASSC therapy for Fabry disease, and that the TgG3S mouse would be useful for studying the effect of high Gb3 levels in mouse organs.

Polo-like kinases (Plks) and cancer.

Deregulated centrosome duplication or maturation often results in increased centrosome size and/or centrosome number, both of which show a positive and significant correlation with aneuploidy and chromosomal instability, thus contributing to cancer formation. Given the role of Polo-like kinases (Plks) in the centrosome cycle, it is not unexpected that deregulated expression of Plks is detected in many types of cancer and is associated with oncogenesis. Extensive studies have shown that Plk1 expression is elevated in non-small-cell lung cancer, head and neck cancer, esophageal cancer, gastric cancer, melanomas, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, gliomas, and thyroid cancer. Plk1 gene and protein expression has been proposed as a new prognostic marker for many types of malignancies, and Plk1 is a potential target for cancer therapy. In contrast to Plk1, several studies have observed that Plk3 expression is negatively correlated with the development of certain cancers.