Biological pathways potentially affected by the differentially expressed microRNAs of significance from macrophages of active MTB disease, LTBI and healthy controls.

<p>Biological pathways potentially affected by the differentially expressed microRNAs of significance from macrophages of active MTB disease, LTBI and healthy controls.</p

miRNAs differentially expressed in human macrophages with active MTB and latent infections against healthy controls.

<p>^aIndicates miRNA expression in macrophages of latent group vs healthy controls.</p><p>^bIndicates miRNA expression in macrophages of active group vs healthy controls.</p><p>^cP-value was obtainedby an independent median test.</p><p>miRNAs differentially expressed in human macrophages with active MTB and latent infections against healthy controls.</p

Clustering analysis of the 11 miRNAs was performed using DataAssist 3.0v based on ΔCt-values of the TLDA results.

<p>Upregulated miRNAs are designated by various shades of red and down-regulated miRNAs by various shades of green. Clinical phenotypes are labelled in different colours: active MTB infection (red), latent infection (blue), and healthy controls (green).</p

miRNAs expression level in the THP-1 macrophages infected with Beijing/W and non-Beijing/W clinical TB strains.

<p>The relative quantity (RQ, 2-ΔΔCt) was used to normalize the relative gene expression data. Statistical analysis between two groups was performed using Mann-Whitney test. Individual values were denoted by black dots/squares from each group of Beijing/W versus non-Beijing strains. The mean RQ and S.D. of each group were represented by the ------- bar and short bars --- in each figure, respectively.</p

MicroRNAs differentially expressed in THP-1 macrophages infected with Beijing/W and non-Beijing/W clinical TB strains.

<p>^aFold difference in miRNA expression in THP-1 cells infected with Beijing/W clinical strains vs non-Beijing/W strains.</p><p>^bP-value was calculated by Mann-Whitney test.</p><p>MicroRNAs differentially expressed in THP-1 macrophages infected with Beijing/W and non-Beijing/W clinical TB strains.</p

Previously reported microRNAs with differential expression related to current study of MTB infections and their validated transcript targets.

<p>Previously reported microRNAs with differential expression related to current study of MTB infections and their validated transcript targets.</p

Potential biomarkers for latent and active TB infections based on miRNA or whole genome microarray studies.

<p>Potential biomarkers for latent and active TB infections based on miRNA or whole genome microarray studies.</p

Epigenetic Inactivation of <i>Inositol polyphosphate 4-phosphatase B</i> (<i>INPP4B</i>), a Regulator of PI3K/AKT Signaling Pathway in EBV-Associated Nasopharyngeal Carcinoma

<div><p>Nasopharyngeal carcinoma (NPC) is a common viral-associated neoplasm in which multiple signaling cascades are interfered with by Epstein-Bar virus (EBV) latent proteins and various genetic alterations. Aside from the previously reported <i>PIK3CA</i> amplification, we examined the role of INPP4B, a negative regulator of the PI3K/AKT signaling pathway in the development of NPC. By RT-PCR and Western blotting, we revealed that the expression of <i>INPP4B</i> was down-regulated in all five established EBV-positive tumor lines. While <i>INPP4B</i> was consistently expressed in normal nasopharyngeal epithelial cells, downregulation of <i>INPP4B</i> was found in 32/65 (49.2%) of primary tumors by immunohistochemistry. Furthermore, our study also demonstrated the hypermethylation of the 5′CpG island of <i>INPP4B</i> in the tumors in which <i>INPP4B</i> transcription was downregulated. Notably, the re-expression of <i>INPP4B</i> was detected in the NPC cells treated with the demethylation agent (5-aza-2′deoxycytidine). Our study showed that promoter hypermethylation was the major mechanism for transcriptional silencing of <i>INPP4B</i> in NPC. Furthermore, restoration of INPP4B expression significantly suppressed PI3K/AKT downstream signals in the NPC C666-1 cells. <i>In vivo g</i>rowth inhibition was clearly demonstrated in the tumor cells stably expressing INPP4B. The findings indicate that epigenetic inactivation of <i>INPP4B</i> is one of the key mechanisms in activating PI3K/AKT signaling cascade and playing a role in the tumorigenesis of NPC.</p></div

Activation of the PI3K/AKT pathway in EBV+ve NPC tumor cell line and xengrafts.

<p>(A) By Western blotting, p-AKT (Thr308) was detected in all EBV-positive NPC tumor lines and p-AKT (Ser473) was found in C666-1, C15 and xeno-99186. The weak expression of p-AKT was shown in the NP69 cells. No AKT activation was observed in the EBV-ve NPC cell line HK-1 and the breast cancer cell line MCF7. p-mTOR was found in all of the cell lines except C17 and xeno-1915 which also shown absence of mTOR expression. The phosphorylation of GSK-3β (Ser9) was detected in xeno-99186 and C17. A high level of PTEN expression was found in the EBV+ve NPC tumor lines. Relative protein expression was calculated using densitometry with C666-1 at 1. The ratios of the phosphorylation and total protein of AKT, mTOR and GSK-3β were also indicated. (B) Suppression of AKT and mTOR phosphorylation was detected in NPC C666-1 cells transient transfected with <i>INPP4B</i>. Relative protein expression was calculated using densitometry with vector control (24hr) at 1. The ratios of the phosphorylation and total protein of AKT and mTOR were indicated.</p

Expression of INPP4B in NPC. (A) RT-PCR analysis of <i>INPP4B</i> transcription in NPC, gastric and cervical cancer cell lines.

<p>The reduced expression of <i>INPP4B</i> was detected in the EBV+ve NPC tumor lines when compared with the EBV-negative NPC cell line HK1 and immortalized nasopharyngeal epithelial cells NP69. Loss of <i>INPP4B</i> transcription was also detected in a gastric cancer cell line NKM28 and a cervical cancer cell line HeLa. The RT-PCR experiments were performed in duplicate. (B) The reduced INPP4B protein expression of EBV-positive NPC tumor lines was detected by Western blotting. A high level of INPP4B expression was found in the EBV-negative NPC cell line HK1. The experiment were carried out in duplicate (C) By IHC staining, a reduction in, or loss of, INPP4B expression was detected in the NPC tumor T3, T5 and T10. In tumor T3, intensive staining of INPP4B was demonstrated in the normal nasopharyngeal epithelial cells (red arrow). Representative NPC cases with medium (T11 and T15) and high expression levels of INPP4B (T6) are shown. NPC tumor cells are indicated (*).</p