Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients

Apoptosis is genetically regulated and involves intrinsic and extrinsic pathways. We examined 133 genes within these pathways to identify whether they are expressed differently in colorectal carcinoma (CRC) and normal tissue (N?=?217) and if they are associated with similar differential miRNA expression. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0) were generated. We focused on dysregulated genes with a fold change (FC) of >?1.50 or <?0.67, that were significant after adjustment for multiple comparisons. miRNA:mRNA seed-region matches were determined. Twenty-three genes were significantly downregulated (FC?<?0.67) and 18 were significantly upregulated (FC?>?1.50). Of these 41 genes, 11 were significantly associated with miRNA differential expression. BIRC5 had the greatest number of miRNA associations (14) and the most miRNAs with a seed-region match (10). Four of these matches, miR-145-5p, miR-150-5p, miR-195-5p, and miR-650, had a negative beta coefficient. CSF2RB was associated with ten total miRNAs (five with a seed-region match, and one miRNA, miR-92a-3p, with a negative beta coefficient). Of the three miRNAs associated with CTSS, miR-20b-5p, and miR-501-3p, had a seed-region match and a negative beta coefficient between miRNA:mRNA pairs. Several miRNAs that were associated with dysregulated gene expression, seed-region matches, and negative beta coefficients also were associated with CRC-specific survival. Our data suggest that miRNAs could influence several apoptosis-related genes. BIRC5, CTSS, and CSF2R all had seed-region matches with miRNAs that would favor apoptosis. Our study identifies several miRNA associated with apoptosis-related genes, that if validated, could be important therapeutic targets.     

Over-expression of miR-146b and its regulatory role in intestinal epithelial cell viability,proliferation, and apoptosis in piglets

Background

Weaning stress affects the small intestine of piglets. MiR-146b is differentially expressed in suckling and weaned piglets. In this study, we evaluated the effects of miR-146b on cell viability, proliferation, and apoptosis in IPEC-J2 cells.

Results

Transfection with miR-146b mimics successfully increased miR-146b levels by 1000× (P?<?0.001). The over-expression of miR-146b significantly promoted the apoptosis (P?<?0.01) of IPEC-J2 cells, with no significant effects on cell viability or proliferation. MiR-146b suppressed the luciferase activity of the miR-TLR4-wt by 57% compared with the negative control, while mutation of the miR-146b binding site significantly blocked the suppressive effect (P?<?0.05). Western blot results showed that TLR4 levels decreased in IPEC-J2 cells transfected with miR-146b mimics (P?<?0.05).

Conclusions

The over-expression of miR-146b promotes IPEC-J2 cell apoptosis. TLR4 is a direct target of miR-146b in IPEC-J2 cells.

Reviewers

This article was reviewed by Eugene Berezikov and Jan B Hoek.

     

Plasma microRNAs are associated with acute exacerbation in idiopathic pulmonary fibrosis

Background

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) has high short-term mortality with unknown causes. To predict this malignant condition in clinics is challenging. In this study, we aim to demonstrate whether there are miRNAs that differ between AE-IPF and stable IPF, which may be served as reliable biomarker for AE-IPF prediction.

Methods

Human fibrotic-associated miRNAs arrays were designed to detect miRNAs expression in plasma of 3 AE-IPF patients, 3 Stable-IPF (S-IPF) patients and 3 normal controls (NC). Differentially expressed miRNAs between AE-IPF and S-IPF patients were selected for further analyses. The validation studies were carried out in plasma of 12 AE-IPF patients, 45 S-IPF patients and 51 healthy control subjects. Signaling pathways and cellular processes interacted with validated miRNAs were predicted by DIANA-miRPath.

Results

According to the array analysis, 6 miRNAs showed differentiated expression between AE-IPF and S-IPF patients (P?<?0.05). In the validation studies, let-7d-5p was decreased in S-IPF and further decreased in AE-IPF, when compared to NC (0.0003?±?0.0002 vs 0.003?±?0.002, P?<?0.01 and 0.0007?±?0.0005 vs 0.003?±?0.002, P?<?0.01). While miR-25-3p was obviously decreased in S-IPF (0.0002?±?0.0001 vs 0.0003?±?0.0003, P?<?0.01) but significantly increased in AE-IP (0.0023?±?0.002 vs 0.0003?±?0.0003, P?<?0.01). In receiver-operator characteristic (ROC) curve analysis, the areas under the curve (AUCs) of miR-25-3p and let-7d-5p were 0.83 and 0.75, respectively. The sensitivity at fixed specificity of 90% was improved from 50% to 66.7% when the two miRNAs were combined. The functional prediction of miRNAs suggested that the loss of anti-fibrotic capacity and the gain of uncontrolled cell growth may be required in AE-IPF pathogenesis.

Conclusions

In conclusion, miR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and S-IPF. A combination of these two miRNAs may be a potential biomarker for AE-IPF from IPF.

     

<Emphasis Type="Italic">miR-3075</Emphasis> Inhibited the Migration of Schwann Cells by Targeting Cntn2

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.     

MicroRNA expression patterns and target prediction in multiple myeloma development and malignancy

Epigenetic changes have emerged as key causes in the development and progression of multiple myeloma (MM). In this study, global microRNA (miRNA) expression profiling were performed for 27 MM (19 specimens and 8 cell lines) and 3 normal controls by microarray. miRNA-targets were identified by integrating the miRNA expression profiles with mRNA expression profiles of the matched samples (unpublished data). Two miRNAs were selected for verification by RT-qPCR (miR-150-5p and miR-4430). A total of 1791 and 8 miRNAs were over-expressed and under-expressed, respectively in MM compared to the controls (fold change ≥2.0; p?<?0.05). The miRNA-mRNA integrative analysis revealed inverse correlation between 5 putative target genes (RAD54L, CCNA2, CYSLTR2, RASGRF2 and HKDC1) and 15 miRNAs (p?<?0.05). Most of the differentially expressed miRNAs are involved in survival, proliferation, migration, invasion and drug resistance in MM. Some have never been described in association with MM (miR-33a, miR-9 and miR-211). Interestingly, our results revealed 2 miRNAs, which are closely related to B cell differentiation (miR-150 and miR-125b). For the first time, we suggest that miR-150 might be potential negative regulator for two critical cell cycle control genes, RAD54L and CCNA2, whereas miR-125b potentially target RAS and CysLT signaling proteins, namely RASGRF2 and CYSLTR2, respectively. This study has enhanced our understanding on the pathobiology of MM and opens up new avenues for future research in myelomagenesis.     

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p?<?0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p?<?0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p?<?0.05) and endothelial apoptosis (p?<?0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication—with implications in organ regeneration.     

Extracellular vesicles-mediated transfer of miR-208a/b exaggerate hypoxia/reoxygenation injury in cardiomyocytes by reducing QKI expression

Diabetes mellitus (DM) induces a variable degree of muscle sarcopenia, which may be related to protein degradation and to the expression of both E3 ubiquitin ligases and some specific microRNAs (miRNAs). The present study investigated the effect of diabetes and acute muscle contraction upon the TRIM63 and FBXO32 expression as well as the potential involvement of some miRNAs. Diabetes was induced by streptozotocin and studied after 30 days. Soleus muscles were harvested, stimulated to contract in vitro for twitch tension analysis (0.5 Hz), 30 min later for tetanic analysis (100 Hz), and 30 min later were frozen. TRIM63 and FBXO32 proteins were quantified by western blotting; Trim63 mRNA, Fbxo32 mRNA, miR-1-3p, miR-29a-3p, miR-29b-3p, miR-133a-3p, and miR-133b-3p were quantified by qPCR. Diabetes induced sarcopenia by decreasing (P < 0.05) muscle weight/tibia length index, maximum tetanic contraction and relaxation rates, and absolute twitch and tetanic forces (P < 0.05). Diabetes decreased (P < 0.05) the Trim63 and Fbxo32 mRNAs (30%) and respective proteins (60%), and increased (P < 0.01) the miR-29b-3p (2.5-fold). In muscle from diabetic rats, acute contractile stimulus increased TRIM63 protein, miR-1-3p, miR-29a-3p, and miR-133a/b-3p, but decreased miR-29b-3p (P < 0.05). Independent of the metabolic condition, after muscle contraction, both TRIM63 and FBXO32 proteins correlated significantly with miR-1-3p, miR-29a/b-3p, and miR-133a/b-3p. All diabetes-induced regulations were reversed by insulin treatment. Concluding, the results depict that muscle wasting in long-term insulinopenic condition may not be accompanied by increased proteolysis, pointing out the protein synthesis as an important modulator of muscle sarcopenia in DM.     

Integrating CNVs into meta-QTL identified <Emphasis Type="Italic">GBP4</Emphasis> as positional candidate for adult cattle stature

Copy number variation (CNV) of DNA sequences, functionally significant but yet fully ascertained, is believed to confer considerable increments in unexplained heritability of quantitative traits. Identification of phenotype-associated CNVs (paCNVs) therefore is a pressing need in CNV studies to speed up their exploitation in cattle breeding programs. Here, we provided a new avenue to achieve this goal that is to project the published CNV data onto meta-quantitative trait loci (meta-QTL) map which connects causal genes with phenotypes. Any CNVs overlapping meta-QTL therefore will be potential paCNVs. This study reported potential paCNVs in Bos taurus autosome 3 (BTA3). Notably, overview indexes and CNVs both highlighted a narrower region (BTA3 54,500,000–55,000,000 bp, named BTA3_INQTL_6) within one constructed meta-QTL. Then, we ascertained guanylate-binding protein 4 (GBP4) among the nine positional candidate genes was significantly associated with adult cattle stature, including body weight (BW, P?<?0.05) and withers height (WHT, P?<?0.05), fitting GBP4 CNV either with three levels or with six levels in the model. Although higher copy number downregulated the mRNA levels of GBP2 (P?<?0.05) and GBP4 (P?<?0.05) in 1-Mb window (54.0–55.0 Mb) in muscle and adipose, additional analyses will be needed to clarify the causality behind the ascertained association.     

The evaluation of protective and mitigating effects of vitamin C against side effects induced by radioiodine therapy

The goal of this study was to evaluate the protective and mitigative effect of vitamin C on oxidative stress in differentiated thyroid cancer (DTC) patients ablated with radioiodine. 58 DTC patients selected for radioactive iodine therapy (RAIT) with 5550 MBq ¹³¹Iodine were divided into four groups. Group 1 (control group) consisted of patients who underwent RAIT routinely. Other patients received 1500 mg vitamin C daily 2 days after (group 2), 2 days before to 2 days after (group 3) and 2 days before RAIT (group 4). Serum oxidative stress markers including malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) were measured immediately before and 2 days after RAIT. A significant increase in MDA after RAIT was observed in all groups (p?<?0.05). The concentrations of MDA were significantly higher in the control group compared to the intervention groups (p?<?0.05). A significant decrease in the control group (p?<?0.05) and increase in group 4 (p?<?0.05) were observed in GSH level after RAIT (p?<?0.05). Mean variation of GSH was significant between control group with groups 3 (p?<?0.01) and 4 (p?<?0.01). The results indicate that activity of SOD remained unchanged in all groups (p?>?0.05). A significant increase was observed in CAT activity after RAIT in all groups (p?<?0.05), which was higher in control group than intervention groups. In groups 3 (p?<?0.05) and 4 (p?<?0.05), this increase in CAT activity was significantly lower than the control group. RAIT causes serum oxidative stress, which can be ameliorated using vitamin C as an antioxidant. These results indicate that radioprotective effect of vitamin C is preferable to its mitigative effect.     

Targeting myomiRs by tocotrienol-rich fraction to promote myoblast differentiation

Background

Several muscle-specific microRNAs (myomiRs) are differentially expressed during cellular senescence. However, the role of dietary compounds on myomiRs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on myomiRs and myogenic genes during differentiation of human myoblasts. Young and senescent human skeletal muscle myoblasts (HSMM) were treated with 50 μg/mL TRF for 24 h before and after inducing differentiation.

Results

The fusion index and myotube surface area were higher (p?<?0.05) on days 3 and 5 than that on day 1 of differentiation. Ageing reduced the differentiation rate, as observed by a decrease in both fusion index and myotube surface area in senescent cells (p?<?0.05). Treatment with TRF significantly increased differentiation at days 1, 3 and 5 of young and senescent myoblasts. In senescent myoblasts, TRF increased the expression of miR-206 and miR-486 and decreased PTEN and PAX7 expression. However, the expression of IGF1R was upregulated during early differentiation and decreased at late differentiation when treated with TRF. In young myoblasts, TRF promoted differentiation by modulating the expression of miR-206, which resulted in the reduction of PAX7 expression and upregulation of IGF1R.

Conclusion

TRF can potentially promote myoblast differentiation by modulating the expression of myomiRs, which regulate the expression of myogenic genes.

     

Association between circulating levels of heat-shock protein 27 and aggressive periodontitis

Heat-shock protein (Hsp) 27 is a major intracellular molecular chaperone and controller of intracellular responses to inflammatory signals. In the extracellular space, recombinant Hsp27 has been described to exert anti-inflammatory activities. The aim of this study was to assess the association between circulating levels of Hsp27 and different types of periodontitis. Pro- and anti-inflammatory cytokines and the stress proteins Hsp27 and Hsp60 with proposed anti- and pro-inflammatory properties, respectively, were measured by two-site ELISA in the serum of patients with aggressive periodontitis (AgP, n?=?30), chronic periodontitis (CP, n?=?29) and periodontally healthy controls (H, n?=?28). Furthermore, Hsp27 and Hsp60 levels were also measured longitudinally in 12 AgP patients at 6 time points up to 3 months after treatment. AgP patients had lower levels of Hsp27 compared to CP patients and healthy subjects (adjusted one-way ANOVA, p?<?0.001, followed by post hoc Tukey HSD comparisons), while no differences in levels of Hsp60 or cytokines between the three groups were detected. In CP patients and H subjects, the systemic Hsp27 levels correlated with Hsp60 (r?=?0.43, p?<?0.001; r?=?0.59, p?<?0.001, respectively) and with pro-inflammatory cytokines TNF-α (r?=?0.48, p?<?0.001; r?=?0.55, p?<?0.001, respectively) and IL-6 (r?=?0.44, p?<?0.01). However, no such correlations were detected in AgP cases. No consistent temporal patterns of changes of Hsp27 concentration were detected across AgP patients following periodontal treatment. This study provides the first evidence that Hsp27 may be differentially expressed and regulated in AgP patients as compared with CP patients and healthy individuals.     

<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells

Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24 h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis.