UPR-Induced miR-616 Inhibits Human Breast Cancer Cell Growth and Migration by Targeting c-MYC.

C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible protein 153 (GADD153), belongs to the CCAAT/enhancer-binding protein (C/EBP) family. CHOP expression is induced by unfolded protein response (UPR), and sustained CHOP activation acts as a pivotal trigger for ER stress-induced apoptosis. MicroRNA-616 is located within an intron of the CHOP gene. However, the regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. Here we show that the expression of miR-616 and CHOP (host gene of miR-616) is downregulated in human breast cancer. Both miR-5p/-3p arms of miR-616 are expressed with levels of the 5p arm higher than the 3p arm. During conditions of ER stress, the expression of miR-616-5p and miR-616-3p arms was concordantly increased primarily through the PERK pathway. Our results show that ectopic expression of miR-616 significantly suppressed cell proliferation and colony formation, whereas knockout of miR-616 increased it. We found that miR-616 represses c-MYC expression via binding sites located in its protein coding region. Furthermore, we show that miR-616 exerted growth inhibitory effects on cells by suppressing c-MYC expression. Our results establish a new role for the CHOP locus by providing evidence that miR-616 can inhibit cell proliferation by targeting c-MYC. In summary, our results suggest a dual function for the CHOP locus, where CHOP protein and miR-616 can cooperate to inhibit cancer progression.

miRNA-378 Is Downregulated by XBP1 and Inhibits Growth and Migration of Luminal Breast Cancer Cells.

X-box binding protein 1 (XBP1) is a transcription factor that plays a crucial role in the unfolded protein response (UPR), a cellular stress response pathway involved in maintaining protein homeostasis in the endoplasmic reticulum (EnR). While the role of XBP1 in UPR is well-characterised, emerging evidence suggests its involvement in endocrine resistance in breast cancer. The transcriptional activity of spliced XBP1 (XBP1s) is a major component of its biological effects, but the targets of XBP1s in estrogen receptor (ER)-positive breast cancer are not well understood. Here, we show that the expression of miR-378 and PPARGC1B (host gene of miR-378) is downregulated during UPR. Using chemical and genetic methods, we show that XBP1s is necessary and sufficient for the downregulation of miR-378 and PPARGC1B. Our results show that overexpression of miR-378 significantly suppressed cell growth, colony formation, and migration of ER-positive breast cancer cells. Further, we found that expression of miR-378 sensitised the cells to UPR-induced cell death and anti-estrogens. The expression of miR-378 and PPARGC1B was downregulated in breast cancer, and higher expression of miR-378 is associated with better outcomes in ER-positive breast cancer. We found that miR-378 upregulates the expression of several genes that regulate type I interferon signalling. Analysis of separate cohorts of breast cancer patients showed that a gene signature derived from miR-378 upregulated genes showed a strong association with improved overall and recurrence-free survival in breast cancer. Our results suggest a growth-suppressive role for miR-378 in ER-positive breast cancer where downregulation of miR-378 by XBP1 contributes to endocrine resistance in ER-positive breast cancer.

The Serum/Glucocorticoid-Regulated Kinase 1 Is Targeted by miR-19a in CD4+ T Cells.

The polarization of CD4+ T cells into different T helper subsets is an important process in many diseases, including asthma. Part of the adaptive immune system, T cells are responsible for propagating signals to alert and prime the immune system. MicroRNAs (miRNAs) are small non-coding RNAs that act on numerous targets in the cell to regulate a variety of cellular processes, including roles in T cell polarization. In this study, we aimed to identify genes dysregulated in peripheral blood mononuclear cells from individuals with asthma. Moreover, we sought to examine miRNAs that may regulate the candidate genes and explore their functional relationship. Utilizing a focused gene array, we identified the serum/glucocorticoid-regulated kinase 1 (SGK1) gene to be upregulated in circulating peripheral blood mononuclear cells, which included T cells, from individuals with asthma. Several miRNAs were bioinformatically identified to target SGK1, but miR-19a was the only screened candidate that negatively correlated to SGK1 expression. Further analysis of the miR-19a-SGK1 relationship showed a negative correlation in CD4+ T cells in situ and direct binding in vitro during T cell activation. Moreover, we observed a negative correlation of miR-19a and SGK1 during early type 2 polarization of CD4+ naïve human T cells. Thus, we suggest that miR-19a has a role in binding and regulating SGK1 transcript levels during T cell development.

Relative and Absolute Expression Analysis of MicroRNAs Associated with Luminal A Breast Cancer- A Comparison.

MicroRNAs, as small non-coding regulatory RNAs, play crucial roles in various aspects of breast cancer biology. They have prognostic and diagnostic value, which makes them very interesting molecules to investigate. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is the gold standard method to analyse miRNA expression in breast cancer patients. This study investigated two RT-qPCR methods (absolute and relative) to determine the expression of ten miRNAs in whole blood samples obtained from luminal A breast cancer patients compared to healthy controls. Whole blood samples were collected from 38 luminal A breast cancer patients and 20 healthy controls in Paxgene blood RNA tubes. Total RNA was extracted and analysed by relative and absolute RT-qPCR. For relative RT-qPCR, miR-16 was used as an endogenous control. For absolute RT-qPCR, standard curves were generated using synthetic miRNA oligonucleotides to determine the absolute copy number of each miRNA. Of the ten miRNAs that were analysed, the absolute RT-qPCR method identified six miRNAs (miR-16, miR-145, miR-155, miR-451a, miR-21 and miR-486) that were upregulated and one miRNA (miR-195) that was downregulated. ROC curve and AUC analysis of the data found that the combination of three miRNAs (miR-145, miR-195 and miR-486) had the best diagnostic value for luminal A breast cancer with an AUC of 0.875, with 76% sensitivity and 81% specificity. On the other hand, the relative RT-qPCR method identified two miRNAs (miR-155 and miR-486) that were upregulated and miR-195, which was downregulated. Using this approach, the combination of three miRNAs (miR-155, miR-195 and miR-486) was showed to have an AUC of 0.657 with 65% sensitivity and 69% specificity. We conclude that miR-16 is not a suitable normalizer for the relative expression profiling of miRNAs in luminal A breast cancer patients. Compared to relative quantification, absolute quantification assay is a better method to determine the expression level of circulating miRNAs in Luminal A breast cancer.

Epigenetic changes in gastric cancer induction by Helicobacter pylori.

Epigenetic disorder mechanisms are one of the causes of cancer. The most important of these changes is the DNA methylation, which leads to the spread of Helicobacter pylori and inflammatory processes followed by induction of DNA methylation disorder. Mutations and epigenetic changes are the two main agents of neoplasia. Epithelial cells infection by H. pylori associated with activating several intracellular pathways including: MAPK, NF-κB, Wnt/ß-catenin, and PI3K are affects a variety of cells and caused to an increase in the production of inflammatory cytokines, changes in apoptosis, proliferation, differentiation, and ultimately leads to the transformation of epithelial cells into oncogenic. The arose of free radicals impose the DNA cytosine methylation, and NO can increase the activity of DNA methyltransferase. H. pylori infection causes an environment that mediates inflammation and signaling pathways that probably caused to stomach tumorigenicity. The main processes that change by decreasing or increasing the expression of various microRNAs expressions include immune responses, apoptosis, cell cycle, and autophagy. In this review will be describe a probably H. pylori roles in infection and mechanisms that have contribution in epigenetic changes in the promoter of genes.

Are probiotics useful for therapy of Helicobacter pylori diseases?

Chronic infection with Helicobacter pylori (H. pylori) is a known risk factor for gastric cancer. Eradication rate of H. pylori infection by the classic triple treatment of PPIs and antibiotics is low. Therefore, probiotics are a useful tool for improving the rate of eradication and reduction of side effects. Several studies in animal models showed that Lactobacillus spp. alone and in combination with other probiotic strains have inhibitory effects on growth and suppression of inflammatory responses in H. pylori infections. However, some studies showed significant effects of Pediococcus strains on suppression, survival, and eradication of H. pylori infections. Therefore, it is suggested that in the treatment of H. pylori infections along with the usual probiotic strains, different strains of Pediococcus could be used. Recent studies showed that Lactobacillus reuteri and Lactobacillus gasseri alone with PPIs in human have a high eradication effect on H. pylori infections and it is suggested as the probiotic treatment of patient's in future therapeutic protocols. In relation to the probiotic treatment process, it should not be recommended that probiotics could be used as a single treatment for H. pylori eradication. However, use of probiotics as a supplement will increase eradication and reduce side effects associated with treatment. It is widely believed that probiotics could improve the eradication of H. pylori and reduce side effects during standard treatment, but some probiotic bacterial species could be useful with drug therapy. Generally, probiotic supplements could increase the eradication rate of H. pylori infections and reduced the side effects of antibiotics.

microRNA expression profiles in two- and three-dimensional culture conditions of human-umbilical-cord blood-derived CD34+ cells.

Human umbilical cord blood (HUCB) is a suitable source of hematopoietic stem cells (HSCs) for therapeutic transplantation. Different approaches have been used to expand the number of HSCs to increase the rate of HSC transplantation success in patients, such as using different cocktails of cytokines, feeder cell layers, and biocompatible scaffolds. microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. They play crucial roles in hematopoiesis including stem cell proliferation, differentiation, stemness, and self-renewal properties. Here, we studied the UCB-derived CD34+ cell expansion and the miRNA signatures of CD34+ cells on two- and three-dimensional (2D and 3D) culture conditions. We successfully expanded the UCB-derived CD34+ cells in both liquid culture (2D) and on aminated polyethersulfone nanofiber scaffolds (3D). Next, we identified the miRNA signature of CD34+ cells and their target genes. We found 58 dysregulated miRNAs in 3D culture condition and 34 dysregulated miRNAs in 2D culture condition when compared to the freshly isolated CD34+ cells. Various types of target genes were also predicted in both conditions using two online databases.

Importance of probiotics in the prevention and treatment of colorectal cancer.

Colorectal cancer (CRC) remains one of the most common and deadly cancers. Intestinal gut microflora is important to maintain and contributes to several intestinal functions, including the development of the mucosal immune system, absorption of complex macromolecules, synthesis of amino acids/vitamins and the protection against pathogenic microorganisms. It is well known that the gut microbiota changes or dysbiosis may have an essential impact in the initiation and promotion of chronic inflammatory pathways and also have a profound different genetic and epigenetic alterations leading to dysplasia, clonal expansion, and malignant transformation. Probiotic bacteria has antitumor activity with various mechanisms such as nonspecific physiological and immunological mechanisms. This review evaluates the effects of microbiota and probiotics in clinical trials, in vitro and animal model studies that have explored how probiotic against cancer development and also discusses the possible immunomodulatory mechanisms. Several mechanisms alteration of the intestinal microflora; inactivation of cancerogenic compounds; competition with putrefactive and pathogenic microbiota; improvement of the host's immune response; antiproliferative effects via regulation of apoptosis and cell differentiation; fermentation of undigested food; inhibition of tyrosine kinase; reduces the enteropathogenic complications before and after colon cancer surgery and improve diarrhea and it's have been able to create the integrity of gut mucosal and have stimulatory effects on the systemic immune system and prevent the CRC metastasis. Research in clinical trials encouraging findings that support a role of probiotics in CRC prevention and improve the safety and effectiveness of cancer therapy even though additional clinical research is still necessary.

Current information on the association of Helicobacter pylori with autophagy and gastric cancer.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium and causative agent of gastric cancer. H. pylori induce defective autophagy or inhibit it by means of CagA and vacuolating cytotoxin A (VacA) toxins leading to the gastric cancer induction. Impaired or defective autophagy leads to the accumulation of cytotoxic materials, such as ROS and P62 that lead to increased mutations in the DNA, genome instability, and risk of cancer formation. H. pylori CagA may inhibit autophagy through the c-Met-PI3k/Akt-mTOR signaling pathway. However, VacA induces autophagy by some signaling pathways. In the gastric epithelial cells, VacA is a necessary and sufficient factor for the creation of autophagy. While CagA is a negative regulator of this phenomenon, the elimination of this gene from H. pylori has increased autophagy and the production of inflammatory cytokines is reduced. In gastrointestinal cancers, some of the microRNAs (miRNAs) act as tumor suppressors and some other are oncogenes by regulating various genes expression. H. pylori can also modify autophagy through a mechanism that includes the function of miRNAs. In autophagy, oncogenic miRNAs inhibit activation of some tumor suppressor signaling pathways (e.g., ULK1 complex, Beclin-1 function, and Atg4 messaging), whereas tumor suppressor miRNAs can block the activation of oncogenic signaling pathways. For instance, Beclin-1 is negatively regulated by miRNA-376b (oncogenic miRNA) and miRNA-30a (tumor suppressor miRNA). Similarly, Atg4 by miRNA-376b (oncogenic miRNA) and miRNA-101 (tumor suppressor miRNA). So, this apparent paradox can be explained as that both Beclin-1 and Atg4 play different roles in a particular cell or tissue.

Hyperactivation of nuclear receptor coactivators induces PERK-dependent cell death.

Nuclear receptor coactivators (NCOAs) function as coactivators for nuclear receptors as well as several other transcription factors and potentiate their transcriptional activity. NCOAs play an important role in biology of hormone-dependent and -independent cancers. MCB-613 is a recently described, small molecule stimulator of NCOAs and anti-neoplastic compound that leads to the death of tumour cells due to increased cellular stress. In the present study we investigated the molecular mechanism of MCB-613-induced cell death. We report that absence of NCOA3 leads to compromised activation of PERK signalling pathway during unfolded protein response (UPR). We found that chemical and genetic inhibition of NCOA3 attenuated the expression of PERK at mRNA and protein level. We show that loss of NCOA3 renders cells hypersensitive to UPR induced cell death. Our results show that MCB-613 induced cell death is attenuated in NCOA3 knockout HeLa cells and MCB-613 leads to enhanced PERK signalling in wild-type HeLa cells. The knockdown of PERK provides resistance to MCB-613 mediated cell death while knockdown of XBP1 and ATF6 have no such effect. Our results suggest that hyperstimulation of NCOA3 by MCB-613 induces cell death by evoking constitutive PERK signalling. Taken together our results point to NCOA3 as an important determinant in regulating cell fate during ER stress, with too little and too much NCOA3 both producing deleterious effects.

Amplification-free detection of microRNAs via a rapid microarray-based sandwich assay.

The detection and profiling of microRNAs are of great interest in disease diagnosis and prognosis. In this paper, we present a method for the rapid amplification-free detection of microRNAs from total RNA samples. In a two-step sandwich assay approach, fluorescently labeled reporter probes were first hybridized with their corresponding target microRNAs. The reaction mix was then added to a microarray to enable their specific capture and detection. Reporter probes were Tm equalized, enabling specificity by adjusting the length of the capture probe while maintaining the stabilizing effect brought about by coaxial base stacking. The optimized assay can specifically detect microRNAs in spiked samples at concentrations as low as 1 pM and from as little as 100 ng of total RNA in 2 h. The detection signal was linear between 1 and 100 pM (R2 = 0.99). Our assay data correlated well with results generated by qPCR when we profiled a select number of breast cancer related microRNAs in a total RNA sample.

Dormant phase and multinuclear cells: two key phenomena in early culture of murine bone marrow mesenchymal stem cells.

Special features of mesenchymal stem cells (MSCs) have made them a popular tool in cell therapy and tissue engineering. Although mouse animal models and murine MSCs are common tools in this field, our understanding of the effect of in vitro expansion on the behavior of these cells is poor and controversial. In addition, in comparison to human, isolation of MSCs from mouse has been reported to be more difficult and some unexplained features such as heterogeneity and slow growth rate in the culture of these cells have been observed. Here we followed mouse bone marrow MSCs for >1 year after isolation and examined the effect of expansion on changes in morphology, growth kinetics, plasticity, and chromosomal structure during in vitro culture. Shortly after isolation, the growth rate of the cells decreased until they stopped dividing and entered a dormant state. In this state the size of the cells increased and they became multinuclear. These large multinuclear cells then gave origin to small mononuclear cells, which after a while resumed proliferation and could be expanded immortally. The immortal cells had diminished plasticity and were aneuploid but could not form tumors in nude mice. These results suggest that mouse bone marrow MSCs bear several modifications when expanded in vitro, and therefore, the interpretation of the data obtained with these cells should be done more cautiously.