Cause Of Aberrant Expression Research Articles

Patched 1 expression in Merkel cell carcinoma.

The relevance of Hedgehog signaling in Merkel cell carcinoma has only been addressed by a few studies with conflicting results. Thus, we aimed to establish the expression of Hedgehog signaling molecules in Merkel cell carcinoma to characterize causes of aberrant expression and to correlate these findings with the clinical course of the patients. Immunohistochemistry was performed for Sonic, Indian, Patched 1 (PTCH1) and Smoothened on patients' tumor tissue. Respective mRNA expression was analyzed in 10 Merkel cell carcinoma cell lines using quantitative real-time polymerase chain reaction. PTCH1 sequencing and DNA methylation microarray analyses were carried out on tumor tissues as well as cell lines. PTCH1 immunoreactivity in Merkel cell carcinoma was similar to that of basal cell carcinomas, which both significantly differed from PTCH1 immunoreactivity in healthy skin. Most PTCH1 mutations found were synonymous or without known functional impact. However, on average, the promoter regions of both PTCH1 were hypomethylated independently from PTCH1 gene expression or Merkel cell polyomavirus status. PTCH1 and GLI1/2/3 genes were differently expressed in different cell lines; notably, there was a significant correlation between GLI2 and PTCH1 mRNA expression. Similar to PTCH1 protein expression in patient tissues, PTCH1 gene expression in Merkel cell carcinoma cell lines is highly variable, but due to the similar methylation pattern across Merkel cell carcinoma cell lines, effects other than methylation seem to be the reason for the differential expression and PTCH1 appears to be upregulated by GLI as a classical Hedgehog target gene.

Inhibition of let-7b-5p contributes to an anti-tumorigenic macrophage phenotype through the SOCS1/STAT pathway in prostate cancer

BackgroundDysfunction of microRNAs (miRNAs) is a major cause of aberrant expression of inflammatory cytokines and contributes to macrophage polarization. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity, whereas M2 macrophages display regulatory functions in tissue repair and remodeling and promote Th2 immune responses. Previous studies have shown that miRNA let-7 is associated with cellular differentiation and that the expression of let-7b-5p is significantly augmented in M2 macrophages. However, the mechanism by which let-7b-5p regulates macrophage differentiation in prostate cancer (PCa) remains largely unknown.MethodsHuman macrophages were induced by blood monocytes from healthy male donors, and M1 macrophages were polarized by stimulating them overnight with 100 ng/ml of lipopolysaccharides and 100 ng/ml of IFN-γ. Conditioned medium from PC-3 cells was used to induce prostatic macrophages (M-CMs) in vitro, and we then transfected let-7b-5p mimics or inhibitors into M1 and M-CMs for 72 h. The expression of cluster of differentiation 206 (CD206) in each group was detected with the High-Throughput Connotation of Imaging System. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the expression of the inflammatory cytokines IL-10, IL-12, IL-13, TNF-alpha, and let-7b in macrophages. SOCS1 protein levels were evaluated by ELISA, and the phosphorylation difference in STAT family member proteins was analyzed using CST signal-pathway chip. Phagocytosis by macrophages and the effect of macrophages on the proliferation of prostate cancer PC-3 cells were evaluated with phagocytosis assay or the Cell Counting Kit-8 (CCK-8) and colony formation assay. The relationship between SOCS1 and let-7b-5p was confirmed with a dual-luciferase reporter.ResultsThe expression of cluster of differentiation 206 (CD206, a M2-like macrophage surface molecule) was significantly increased in M1 macrophages treated with let-7b-5p mimics, while CD206 expression was decreased in M-CMs treated with let-7b-5p inhibitors. Overexpression or knockdown of let-7b-5p significantly affected the expression of inflammatory factors in macrophages—including interleukin 10 (IL-10), IL-12, IL-13, and tumor necrosis factor alpha. Let-7b-5p downregulated the expression of suppressor of cytokine signaling 1 (SOCS1) and increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5a proteins in M-CMs and M1 macrophages with let-7b-5p mimics relative to the other groups. In addition, with the elevated expression of let-7b-5p, the phagocytosis by macrophages showed a commensurate and significant decrease. As a result, M-CMs treated with let-7b-5p inhibitors reduced the proliferation of PC-3 PCa cells.ConclusionsCollectively, these data indicated that let-7b-5p may regulate M2 polarization through the SOCS1/STAT pathway and that reversal of M2 differentiation by let-7b-5p inhibitors enhanced macrophage phagocytosis, ultimately inhibiting the proliferation of PCa cells.

MiR-149-3p reverses CD8+ T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells.

Blockade of inhibitory receptors (IRs) is one of the most effective immunotherapeutic approaches to treat cancer. Dysfunction of miRNAs is a major cause of aberrant expression of IRs and contributes to the immune escape of cancer cells. How miRNAs regulate immune checkpoint proteins in breast cancer remains largely unknown. In this study, downregulation of miRNAs was observed in PD-1-overexpressing CD8+ T cells using miRNA array analysis of mouse breast cancer homografts. The data reveal that miR-149-3p was predicted to bind the 3'UTRs of mRNAs encoding T-cell inhibitor receptors PD-1, TIM-3, BTLA and Foxp1. Treatment of CD8+ T cells with an miR-149-3p mimic reduced apoptosis, attenuated changes in mRNA markers of T-cell exhaustion and downregulated mRNAs encoding PD-1, TIM-3, BTLA and Foxp1. On the other hand, T-cell proliferation and secretion of effector cytokines indicative of increased T-cell activation (IL-2, TNF-α, IFN-γ) were upregulated after miR-149-3p mimic treatment. Moreover, the treatment with a miR-149-3p mimic promoted the capacity of CD8+ T cells to kill targeted 4T1 mouse breast tumour cells. Collectively, these data show that miR-149-3p can reverse CD8+ T-cell exhaustion and reveal it to be a potential antitumour immunotherapeutic agent in breast cancer.

Genomic Regions Supporting microRNA Expression and Their Biogenesis Are Globally Conserved in Diffuse Large B-Cell Lymphomas (DLBCL) and Display a Limited Number of Homozygous Deletions or Amplifications.

MicroRNAs are proposed to play a direct role in oncogenesis as they act as both oncogenes and/or tumour suppressor genes (TSG). MicroRNA deregulation can be explained in some instances by deletion, amplification, translocation or epigenetic mechanisms. However the cause of aberrant expression of specific microRNAs in cancer remains largely unknown. Chromosomal-comparative genomic hybridization (CGH) and higher resolution array-CGH experiments have shown that genomic gains and losses play a crucial role in the development of DLBCL. Whether these gene copy number aberrations may involve specifically microRNA coding regions and be therefore implicated in the dysfunctional expression of microRNAs is currently undetermined. To address this issue, we analyzed 65 de novo DLBCL by array-CGH, using a high resolution microarray that contains ∼ 43,000 probes, giving an average spatial resolution of 35 kB. For each of the 474 microRNAs identified in the human genome (microRNA.sanger, April 2007 registry), 286 probes located near microRNA coding genes or microRNA clusters were defined and used to detect DNA copy alterations involving microRNA coding genes. 21% of the microRNA associated probes displayed a deletion in more than 5% of cases, a rate lower than the rate observed for the entire genome (30%, p =.0008). By contrast, 58% of the microRNA coding regions displayed a DNA copy gain, similarly to the whole genome (61%). Notably, none of the genes coding for components of the biosynthetic pathway (including DGCR8, DICER1-2, Argonaute 1, EIF2C2-3-4, Exportin 5 and DROSHA) displayed recurrent deletions. These results indicate that genomic regions supporting microRNA expression and their biogenesis are globally conserved in DLBCL. However, homozygous microRNA deletions, occurring in more than 5% of cases were observed for some microRNA, namely mir-548a (6%), mir-588 (6%), mir-548b (6%), mir-587 (5%) (all located in the 6q21–23 region), and mir-31 (9p21, 5%), suggesting that these microRNA may act as TSG and/or are located in TSG containing regions. Mir-31 was constantly co-deleted with CDKN2A, a well-defined TSG located in the vicinity. The most frequent amplifications involved mir-92b (1q22, 9%), mir-210 (11p15, 6%), mir-196a-2 (12q13, 6%), mir-148b (12q13, 6%), mir-122a (18q21, 9%), mir-153-1 (2q35, 8%), mir-567 (3q13, 8%), mir-106b (7q22, 9%) and mir-126 (9q34, 8%). Similarly, these genes represent candidate oncogenes or are located in oncogene containing regions. For instance, mir122a was co-amplified with MALT1, located in a close vicinity in 6/6 cases. In addition, distinct patterns of microRNA coding gene copy abnormalities between GCB and ABC subtypes were identified. To conclude, we provide experimental genome wide documentation of DNA copy alterations involving microRNA coding genes in DLBCL and suggest that some still uncharacterized microRNAs may act as TSG or oncogenes. Concordance between microRNA gene copy changes and microRNA gene transcriptional expression is currently investigated.

Differential expression of the splicing regulatory factor genes during two-step chemical transformation in a BALB/3T3-derived cell line, MT-5

Although the alternative splicing of various genes is a common event in human tumors, the mechanisms behind it have not been characterized. We hypothesized that the expression of splicing regulatory factors would be changed during cellular transformation. Gene expression of three splicing regulatory factors, alternative splicing factor/splicing factor 2 (ASF/SF2), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 65 kDa subunit of U2 small nuclear ribonucleoprotein particles auxiliary factor (U2AF(65)), were examined by northern blotting in a two-step chemical transformation model. This in vitro model is composed of BALB/3T3 cells and a BALB/3T3-derived N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-initiated cell line (MT-5). MT-5 cells can be transformed on exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). ASF/SF2 mRNA levels were decreased 2-fold in both MNNG-initiated cells and TPA-induced transformed cells compared with the normal parental cells, whereas hnRNP A2 mRNA expression did not significantly change between these three types of cells. U2AF(65) mRNA levels were markedly increased ( approximately 4.7-fold) associated with progression of cellular transformation. Moreover, RT-PCR analysis showed that distinct forms of ASF/SF2 mRNA were present in the MNNG-initiated cells and TPA-induced transformed cells but not in the parental cells. These findings indicate that ASF/SF2 or U2AF(65) gene expression is altered during in vitro two-step chemical transformation. The data suggest that the differential expression of splicing regulatory factors is one cause of aberrant expression of alternatively spliced mRNAs encoded by various genes in tumor cells.