Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.     

Inheritable changes in miRNAs expression in HeLa cells after X-ray and mitomycin C treatment

We identified 40 miRNAs with inherited aberrant expression by multiple parallel sequencing of human HeLa cells irradiated with X rays and mitomycin C. Twenty-two miRNAs were repressed and 15 miRNAs were induced after radiation and mytomycin C treatment. The expression of three miRNAs (miR-10b-5p, miR-148a-3p, and miR-340-5p) decreased after X-ray exposure and increased after mitomycin C treatment. The spectrum of aberrantly expressed miRNAs after X-ray and mitomycin C treatment is different, except for three miRNAs (mir-100-5p, miR-99b-5p, miR-501-3p), which showed the inherited decreased expression after both mutagens. It has been ascertained that for five miRNAs (miR-21-3p, miR-182-5p, miR-19b-3p, miR-30a-3p, and miR-30e-3p) with increased inherited expression, the targets are well-described tumor suppressor genes. For 9 miRNAs (miR-99b-5p, miR-148a-3p, miR-365a-3p, miR-193a-3p, miR-100-5p, miR-99a-5p, miR-29b-3p, miR-340-5p, and miR-23b-3p) with reduced inherited expression, the targets are oncogenes. The obtained results provide further support of the idea that induced epigenetic changes in the genome should be considered when assessing the long-term genetic effects of ionizing radiation and chemical compounds.     

Cellular differences in ring glands of flesh fly pupae as a consequence of diapause programming

The ultrastructure of the ring gland (corpus cardiacum (CC), prothoracic gland (PG) and corpus allatum (CA)) was examined in diapausing and nondiapausing flesh fly pupae. The diapause developmental state, which is environmentally regulated and coordinated by the brain-ring gland complex, is associated with differences in the ultrastructure of PG and CA cells but not in the CC. During diapause the PG and CA cells have extensive arrays of rough endoplasmic reticulum and spherical mitochondria. The PG cells also contain lipid droplets surrounded by an electron dense amorphous coat not seen in PG cells from nondiapausing pupae. In nondiapausing pupae, the PG and CA cells contain large amounts of ribosomes throughout the cytoplasm but very little rough endoplasmic reticulum and elongated mitochondria. The fact that ring glands from diapausing pupae readily incorporate (35)S-methioninc indicates that the gland is actively synthesizing proteins, thus the contrasts in ring gland ultrastructure are not due to cellular quiescence during diapause but reflect fundamental cellular and physiological differences between the diapause and nondiapause developmental program.     

Profiles of serum microRNAs; miR-125b-5p and miR223-3p serve as novel biomarkers for HBV-positive hepatocellular carcinoma

Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions including cancer. Certain microRNAs (miRNAs) have been shown early diagnostic potential for many types of cancer. The objective of this study was to investigate the potential of certain serum/plasma miRNAs as novel non-invasive biomarkers for early diagnosis of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). For this reason, the expression levels of 24 miRNA (let-7c, miR-92a-3p, 423-5p, 150-5p, 223-3p, 125b-5p, 342-3p, miR-206, 122-5p, 375, 223-5p, 10a-5p, 23b-5p, 99a-5p, 23a-5p, 10a-3p, 122-3p, 125b-1-3p, 23b-3p, 125b-2-3p, 23a-3p, 92a-1-5p, 92a-2-5p, 99a-3p) were analyzed in plasma of patients with chronic hepatitis B, HBV-positive cirrhosis and HBV-positive HCC and compared with control group samples. Totally 94 plasma samples; 28 control and 66 patient plasma (24 CHB, 22 HBV-positive cirrhosis, 20 HBV-positive HCC) and were included in this study. The expression levels of 24 miRNAs were detected for all control and patient group plasma samples by qRT-PCR using BioMark? 96.96 Dynamic Array (Fluidigm Corporation) system. The expression levels of miR-125b-5p were detected 2.85 fold, 2.46 fold and 1.89 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) up regulated in CHB, HBV-positive cirrhosis and HBV-positive HCC, respectively when compared versus control group individually by Mann–Whitney U test. The expression levels of miR-223-3p were detected 5.55 fold, 13.88 fold and 12.65 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) down regulated in same comparisons. When all groups were compared versus control group by one-way ANOVA test, the expression levels of miR-223-3p were also found statistically significant (p < 0.05). Although not statistically significant, miR-125b-5p tended to be upregulated. (p = 0.07192). These results significantly imply that miR-125b-5p and miR223-3p could be used as novel non-invasive biomarkers of HBV-positive HCC in very early, even at CHB stage of liver disease.     

Upregulated microRNAs in membranous glomerulonephropathy are associated with significant downregulation of IL6 and MYC mRNAs

Membranous glomerulonephropathy (MGN) is a glomerulopathy characterized by subepithelial deposits of immune complexes on the extracapillary side of the glomerular basement membrane. Insertion of C5b-9 (complement membrane-attack complex) into the membrane leads to functional impairment of the glomerular capillary wall. Knowledge of the molecular pathogenesis of MGN is actually scanty. MicroRNA (miRNA) profiling in MGN and unaffected tissues was performed by TaqMan Low-Density Arrays. Expression of miRNAs and miRNA targets was evaluated in Real-Time polymerase chain reaction (PCR). In vitro transient silencing of miRNAs was achieved through transfection with miRNA inhibitors. Ten miRNAs (let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, miR-107, miR-129-3p, miR-423-5p, miR-516-3p, miR-532-3p, and miR-1275) were differentially expressed (DE) in MGN biopsies compared to unaffected controls. Interleukin 6 (IL6) and MYC messenger RNAs (mRNAs; targets of DE miRNAs) were significantly downregulated in biopsies from MGN patients, and upregulated in A498 cells following let-7a-5p or let-7c-5p transient silencing. Gene ontology analysis showed that DE miRNAs regulate pathways associated with MGN pathogenesis, including cell cycle, proliferation, and apoptosis. A significant correlation between DE miRNAs and mRNAs and clinical parameters (i.e., antiphospholipid antibodies, serum creatinine, estimated glomerular filtration, proteinuria, and serum cholesterol) has been detected. Based on our data, we propose that DE miRNAs and their downstream network may be involved in MGN pathogenesis and could be considered as potential diagnostic biomarkers of MGN.     

miRNA调控恶性黑色素瘤细胞上皮-间充质转化的分子网络及机制

黑色素瘤是一种极易发生转移的恶性皮肤肿瘤,具有高度的致死性。上皮-间充质细胞转化(Epithelial-mesenchymal transition, EMT)在胚胎发育过程中起到非常重要的作用,同时在肿瘤的发生和恶化过程中也扮演着重要的角色。miRNA具有广谱的调节能力,对于肿瘤发生和EMT形成都能产生不同程度的影响。本文整合黑色素瘤细胞系转录组和miRNA组测序数据,在转录组数据中筛选得到参与肿瘤EMT过程的基因,通过Mirsystem软件预测并从miRNA组数据中筛选出与之负相关的11个miRNA,包括miR-130a-3p、miR-130b-3p、miR-125a-5p、miR-30a-3p、miR-195-5p、miR-345-5p、miR-509-3-5p、miR-374a-5p、miR-509-5p、miR-148a-3p和miR-330-3p。经过生物信息学分析miRNA靶基因富集的分子网络和信号途径,发现了两个与细胞发育和细胞间相互作用密切相关的网络,以及多个参与调控EMT过程的信号通路。对11个miRNA进行分子生物学验证,发现miR-195-5p、miR-130a-3p、miR-509-5p和miR-509-3-5p共4个可以调节重要肿瘤基因的miRNA。本研究运用mRNA和miRNA两种转录组的测序数据筛选EMT相关miRNA的方法,为肿瘤多组学数据整合分析提供了新的研究思路,并以期能为肿瘤精准基因组学的发展发挥重要的推进作用。     

Circulating miR-23b-3p,miR-145-5p and miR-200b-3p are potential biomarkers to monitor acute pain associated with laminitis in horses

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several disorders and related pain. In equine practice, acute laminitis is a common disease characterised by intense pain that severely compromises horse welfare. Recently, the Horse Grimace Scale (HGS), a facial expression-based pain coding system, was shown to be a valid welfare indicator to identify pain linked to acute laminitis. The present study aimed to: determine whether miRNAs can be used as biomarkers for acute pain in horses (Equus caballus) affected by laminitis; integrate miRNAs to their target genes and to categorise target genes for biological processes; gather additional evidence on concurrent validity of HGS by investigating how it correlates to miRNAs. Nine horses presenting acute laminitis with no prior treatment were recruited. As control group, nine healthy horses were further included in the experimental design. Samples were collected from horses with laminitis at admission before any treatment (‘pre-treatment’) and 7 days after routine laminitis treatment (‘post-treatment’). The expression levels of nine circulating miRNAs, namely hsa-miR-532-3p, hsa-miR-219-5p, mmu-miR-134-5p, mmu-miR-124a-3p, hsa-miR-200b-3p, hsa-miR-146a-5p, hsa-miR-23b-3p, hsa-miR-145-5p and hsa-miR-181a-5p, were detected and assessed as potential biomarkers of pain by quantitative PCR using TaqMan® probes. The area under the receiver operating curve (AUC) was then used to evaluate the diagnostic performance of miRNAs. Molecular data were integrated with HGS scores assessed by one trained treatment and time point blind veterinarian. The comparative analysis demonstrated that the levels of miR-23b-3p (P=0.029), miR-145-5p (P=0.015) and miR-200b-3p (P=0.023) were significantly higher in pre-treatment and the AUCs were 0.854, 0.859 and 0.841, respectively. MiR-200b-3p decreased after routine laminitis treatment (P=0.043). Combining two miRNAs in a panel, namely miR-145-5p and miR-200b-3p, increased efficiency in distinguishing animals with acute pain from controls. In addition, deregulated miRNAs were positively correlated to HGS scores. Computational target prediction and functional enrichment identified common biological pathways between different miRNAs. In particular, the glutamatergic pathway was affected by all three miRNAs, suggesting a crucial role in the pathogenesis of pain. In conclusion, the dynamic expression of circulating miR-23b-3p, miR-145-5p and miR-200b-3p was detected in horses with acute laminitis and miRNAs can be considered potentially promising pain biomarkers. Further studies are needed in order to assess their relevancy in other painful conditions severely compromising horse welfare. An important implication would be the possibility to use them for the concurrent validation of non-invasive indicators of pain in horses.     

Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta

Scanning electron microscopy of the epicuticle of the tobacco hornworm, Manduca sexta, showed that pupae in diapause possess thicker wax layers than non-diapausing pupae. Extractions of the epicuticle with CHCl₃ established that diapausing pupae secreted over three times as much wax as non-diapausing pupae, and that the total period of wax secretion in diapausing pupae was two to three times longer than that of non-diapausing pupae. Apparently, the extra thickness of the wax layer of diapausing pupae protects the insect from desiccation during diapause. The deposition of additional wax may result from hormonal changes accompanying entry into diapause.     

Role of antioxidant defense during different stages of preadult life cycle in European corn borer (Ostrinia nubilalis, Hubn.): Diapause and metamorphosis

Antioxidant enzymes, total glutathione (GSH), and ascorbic acid (ASA) were determined in whole body homogenates of nondiapausing larvae, diapausing larvae during the diapausing period (October, December, and February), and in pupae emerged from both diapausing and nondiapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). The activities of catalase, selenium nondependent glutathione peroxidase (GPx), and glutathione-S-transferase (GST), as well as the content of GSH and ASA, were found to vary throughout the larval diapause. Compared to diapausing larvae, nondiapausing larvae were higher in levels of catalase, GPx, GST, and dehydroascorbate reductase (DHAR) activity. GSH content was also increased. However, nondiapausing larvae contained less ASA than diapausing ones. Pupae had higher GPx and GST activity and an increased ASA content compared to larvae. The pupae emerged from nondiapausing larvae had higher GST, glutathione reductase (GR), and DHAR activities, but lower GPx activity and ASA content than those emerged from diapausing larvae. Correlation analysis revealed differences in the way the antioxidant level is equilibrated for a particular stage and developmental pattern. The results suggest that cellular antioxidants are involved in both the protection of cells and the regulation of redox levels during the pre-adult stages of Ostrinia nubilalis. Arch. Insect Biochem. Physiol. 55:79-89, 2004.     

MicroRNA Profiling Implies New Markers of Chemoresistance of Triple-Negative Breast Cancer

Objective

Triple-negative breast cancer (TNBC) patients with truly chemosensitive disease still represent a minority among all TNBC patients. The aim of the present study is to identify microRNAs (miRNAs) that correlate with TNBC chemoresistance.

Methods

In this study, we conducted miRNAs profile comparison between triple-negative breast cancer (TNBCs) and normal breast tissues by microRNA array. Quantitative real-time PCR (qRT-PCR) was utilized to confirm the specific deregulated miRNAs change trend. We used starBase 2.1 and GOrilla to predict the potential targets of the specific miRNAs. Cells viability and apoptosis assays were employed to determine the effect of alteration of the specific miRNAs in TNBC cells on the chemosensitivity.

Results

We identified 11 specific deregulated miRNAs, including 5 up-regulated miRNAs (miR-155-5p, miR-21-3p, miR-181a-5p, miR-181b-5p, and miR-183-5p) and 6 down-regulated miRNAs (miR-10b-5p, miR-451a, miR-125b-5p, miR-31-5p, miR-195-5p and miR-130a-3p). Thereafter, this result was confirmed by qRT-PCR. We predicted the potential targets of the candidate miRNAs and found that they are involved in cancer-associated pathways. For the first time, we found that miR-130a-3p and miR-451a were down-regulated in TNBC. 9 of the 11 specific deregulated miRNAs were found to be associated with chemoresistance. In vitro assays, we found that up-regulation of either miR-130a-3p or miR-451a in MDA-MB-231 cells significantly changed the cells sensitivity to doxorubicin. The results suggest that TNBC chemotherapy might be affected by a cluster of miRNAs.

Conclusion

The abnormal expression miRNAs in TNBC are mainly chemoresistance related. This might be part of reason that TNBC likely to evade from chemotherapy resulting in early relapse and high risk of death. To alter their expression status might be a potential therapeutic strategy to improve the outcome of chemotherapy for TNBC patients.     

Aberrant expression of microRNA induced by high-fructose diet: implications in the pathogenesis of hyperlipidemia and hepatic insulin resistance

Fructose is a highly lipogenic sugar that can alter energy metabolism and trigger metabolic disorders. In the current study, microRNAs (miRNAs) altered by a high-fructose diet were comprehensively explored to elucidate their significance in the pathogenesis of chronic metabolic disorders. miRNA expression profiling using small noncoding RNA sequencing revealed that 19 miRNAs were significantly upregulated and 26 were downregulated in the livers of high-fructose-fed mice compared to chow-fed mice. Computational prediction and functional analysis identified 10 miRNAs, miR-19b-3p, miR-101a-3p, miR-30a-5p, miR-223-3p, miR-378a-3p, miR-33-5p, miR-145a-3p, miR-128-3p, miR-125b-5p and miR-582-3p, assembled as a regulatory network to potentially target key genes in lipid and lipoprotein metabolism and insulin signaling at multiple levels. qRT-PCR analysis of their potential target genes [IRS-1, FOXO1, SREBP-1c/2, ChREBP, insulin-induced gene-2 (Insig-2), microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (apoB)] demonstrated that fructose-induced alterations of miRNAs were also reflected in mRNA expression profiles of their target genes. Moreover, the miRNA profile induced by high-fructose diet differed from that induced by high-fat diet, indicating that miRNAs mediate distinct pathogenic mechanisms in dietary-induced metabolic disorders. This study presents a comprehensive analysis of a new set of hepatic miRNAs, which were altered by high-fructose diet and provides novel insights into the interaction between miRNAs and their target genes in the development of metabolic syndrome.     

Deregulated microRNAs in myotonic dystrophy type 2

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.     

Suppression of net transpiration by multiple mechanisms conserves water resources during pupal diapause in the corn earworm Helicoverpa zea

One critical aspect of an insect's ability to overwinter successfully is the effective management of its water resources. Maintenance of adequate water levels during winter is challenging because of the prevailing low relative humidity at that time of year and the short supply of environmental water that is not in the form of ice. These issues are further exacerbated for insects overwintering as pupae, comprising an immobile stage that is unable to move to new microhabitats if conditions deteriorate. The present study compares the water balance attributes of diapausing and nondiapausing pupae of the corn earworm Helicoverpa zea Boddie, aiming to identify the mechanisms used by diapausing pupae to maintain water balance during winter. Diapausing pupae are 10% larger than nondiapausing individuals. Water loss rates for nondiapausing pupae are low (0.21 mg h^?1) and are suppressed (0.01 mg h^?1) in diapausing pupae. Cuticular lipids, which serve to waterproof the cuticle and thus suppress cuticular water loss, are more than two‐fold more abundant on the surface of diapausing pupae, and oxygen consumption rates during diapause drop to almost one‐third the rate observed in nondiapausing pupae. Water gain can be accomplished only when atmospheric water content is near saturation or during contact with free water. At moderate relative humidities (20–40%), water loss rates are very low for diapausing pupae, suggesting that these moth pupae have robust mechanisms for combating water loss. The exceptional ability of H. zea to suppress water loss during diapause is probably a result of the combined effects of increased size, more abundant cuticular lipids and decreased metabolic rates.     

Cold hardiness and sugar content in photo‐insensitive individuals of the cotton bollworm Helicoverpa armigera

Abstract A proportion of Helicoverpa armigera collected from fields in Okayama Prefecture (Western Japan; 34.6°N, 134.1°E) does not enter diapause when reared under a short days at 20 °C during the larval stages. However, diapause in such photo‐insensitive individuals can be induced when they are reared at moderately low temperatures, such as 15 °C, regardless of photoperiod. To determine whether such photo‐insensitive individuals can survive overwintering in fields, the present study compares the cold hardiness and sugar content between nondiapausing and diapausing pupae of photo‐insensitive individuals selected over several generations at 20 °C under a short day photoperiod (LD 10 : 14 h). Diapausing and nondiapausing pupae are obtained under the short days by rearing at 15 and 20 °C, respectively, during larval and pupal stages. These pupae are stepwise acclimated at a reduction of 5 °C every 5 days to 0 °C. Maximum survival periods of nondiapausing and diapausing pupae at 0 °C are approximately 30 and 90 days, respectively. Trehalose content in diapausing pupae increases, reaches a maximum level (1.95 mg 100 mg^?1 in males and 2.1 mg 100 mg^?1 in females) 28 days after exposure to 0 °C and then decreases. On the other hand, glucose content in diapausing pupae increases (maximum level: 0.32 mg 100 mg^?1 in males and 0.21 mg 100 mg^?1 in females) with decreasing trehalose content 42 days after exposure to 0°C. The decrease in trehalose content and the increase in glucose content may be linked to termination of diapause in H. armigera. These results suggest that, in Japan, the photo‐insensitive individuals can only survive in the mild winters of southern regions, and not in the severe winters of northern regions.